Formulation of Personal Care Products With Bio-Surfactants

FORMULATION OF PERSONAL CARE PRODUCTS WITH BIO-
SURFACTANTS
A thesis submitted to The University of Manchester

for the degree of Doctor of Philosophy
in the Faculty of Science and Engineering
2021
Chi Zhang
School of Engineering
Department of Chemical Engineering & Analytical Science
Table of Contents
Chapter 1. Introduction ........................................................................................ 17

1.1 Research System ........................................................................................ 17
1.2 Research Motivation ................................................................................... 18
1.3 State-of-the-Art ............................................................................................ 19
1.4 Research Objectives and Aims .................................................................. 30
1.5 Overview of Thesis ..................................................................................... 30
1.6 Nomenclature .............................................................................................. 31
Chapter 2. Literature Review .............................................................................. 32
2.1 Surfactants................................................................................................... 32
2.1.1 Structure of Surfactants ...................................................................... 33
2.1.2 Classification of Surfactants ............................................................... 33
2.1.3 Surfactant Behaviour in Water Solution ............................................ 39
2.2 Bio-surfactants ............................................................................................ 44
2.2.1 Classification of Biosurfactants (BSs)................................................ 45
2.2.2 The Production and Extraction of Biosurfactants (BSs) ................... 46
2.2.3 Characterization of Biosurfactants (BSs) .......................................... 48
2.2.4 Application of Biosurfactants (BSs) in Various Fields ...................... 49
2.2.5 Potential Cosmetic-applicable Biosurfactants (BSs) ........................ 51
2.3 Emulsion ...................................................................................................... 65
2.3.1 Overview of Emulsion ......................................................................... 66
2.3.2 Emulsion Formation ............................................................................ 66
2.3.3 Mechanisms of Emulsion Instability ................................................... 73
2.4 Rheology ...................................................................................................... 75
2.4.1 Rheology of Emulsions ....................................................................... 75
2.4.2 Rheometry and Rheometers .............................................................. 77
Chapter 3. Materials and Methodology ............................................................. 81
3.1 Sophorolipids (SLs) Production ................................................................. 81
3.1.1 Producing Microorganisms ................................................................. 81
3.1.2 Chemicals ............................................................................................ 81
3.1.3 Production Strategies .......................................................................... 81
3.2 Mannosylerythritol Lipids (MELs) Production ........................................... 84
1
3.2.1 Producing Microorganisms ................................................................. 84
3.2.2 Chemicals ............................................................................................ 84
3.2.3 Production Strategies .......................................................................... 85
3.3 Preliminary Trials on Cream Formulation ................................................. 86
3.3.1 First Trial for Formulation of Cream without Sodium Lauryl Ether
Sulfate (SLES), Using a Homogenizer ............................................................. 86
3.3.2 Second Trial for Formulation of Cream with Sodium Lauryl Ether
Sulfate (SLES), Using an overhead stirrer ....................................................... 87
3.4 Modified and Standard Experimental Procedure for Cream Formulation .
...................................................................................................................... 90
3.4.1 Chemicals ............................................................................................ 90
3.4.2 Recipes ................................................................................................ 90
3.4.3 Apparatus and Configurations ............................................................ 95
3.4.4 Preparation Procedure for Standard Formulation............................ 96
3.5 Modification of Preparation Process .......................................................... 97
3.5.1 Formulation of Model Creams ............................................................ 98
3.5.2 Preparation Procedure with Different Mixing Time During Heating
Procedure ............................................................................................................ 98
3.5.3 Preparation Procedure with Different Mixing Speed During Heating
Procedure ............................................................................................................ 99
3.5.4 Preparation Procedure with Different Cooling Procedure .............. 100
3.6 Characterisation Methods ........................................................................ 100
3.6.1 Rheology ............................................................................................ 101
3.6.2 Differential Scanning Calorimetry (DSC) ......................................... 121
3.6.3 Droplet Size Distribution Analysis .................................................... 126
3.6.4 Microscopy ......................................................................................... 132
3.6.5 Surface and Interfacial Tension Measurement ............................... 132
3.6.6 Mass Spectrometry (MS) and Tandem Mass Spectrometry (MS-
MS) ............................................................................................................ 136
Chapter 4. Preliminary Characterisation of E45 Cream ....................... 139
4.1 Rheological Characterisation of E45 cream ........................................... 139
4.1.1 Preliminary Testing: Conditioning Step Determination................... 139
4.1.2 Rheological Characterisation on E45 Cream .................................. 146
4.2 Droplet Size Distribution (DSD) Analysis ................................................ 152
4.2.1 Experimental Procedure ................................................................... 152
4.2.2 Results and Conclusions .................................................................. 154
2
4.3 Differential Scanning Calorimetry (DSC) Analysis ................................. 155
4.3.1 Experimental Procedure ................................................................... 155
4.3.2 Results and Conclusions .................................................................. 156
4.4 Summary of Chapter 4.............................................................................. 156
Chapter 5. Variation of Mimic Creams Prepared with Different Emulsifying
System ............................................................................................................ 158
5.1 Explorer Formulation of Mimic Creams ................................................... 158
5.1.1 First Trial of Cream Formulation without Sodium Lauryl Ether
Sulfate (SLES), Using a Homogenizer ........................................................... 158
5.1.2 Second Trial of Cream Formulation with Sodium Lauryl Ether
Sulfate (SLES), Using an Overhead Stirrer .................................................... 159
5.2 Formulation_Ⅰ of Cream Formulation, Using a Simplified Configuration
.................................................................................................................... 161
5.2.1 Appearance of Mimic Creams in Formulation_Ⅰ ........................... 161
5.2.2 Rheological Characterisation of Mimic Creams in Formulation_Ⅰ....
............................................................................................................ 163
5.2.3 Droplet Size Distribution Analysis of mimic creams in
Formulation_Ⅰ ................................................................................................. 180
5.2.4 Thermodynamic Properties of Mimic Creams in Formulation_Ⅰ.. 182
5.3 Complementary Rheology Study of Creams Formulated in
Formulation_Ⅱ ..................................................................................................... 184
Chapter 6. Variation of Creams Prepared with Different Processes. 188
6.1 Effect of Mixing Time on Cream Formulation During Heating Procedure .
.................................................................................................................... 188
6.2 Effect of Mixing Speed on Cream Formulation During Heating Procedure
.................................................................................................................... 192
6.3 Effect of Cooling Procedure on Cream Formulation .............................. 193
Chapter 7. Production of Bio-surfactants ...................................................... 199
7.1 Sophorolipids (SLs)................................................................................... 199
7.1.1 Structural Analysis of Sophorolipids (SLs) ...................................... 201
7.1.2 Surface Tension Analysis of Sophorolipids (SLs)........................... 202
7.2 Mannosylerythritol Lipids (MELs)............................................................. 204
7.2.1 Structural Analysis of MELs.............................................................. 204
7.3 Thermodynamic Properties of Sophorolipids and MELs ....................... 207
3
Chapter 8. Production of bio-creams using Continuous Configuration
in Formulation_Ⅲ ................................................................................................. 209
8.1 Reformulation of Mimic Creams Using Continuous Configuration........ 209
8.2 Creams Formulated with Bio-surfactants in Mixed Paraffin Oils/Water
System................................................................................................................... 210
8.2.1 Appearance of Creams ..................................................................... 211
8.2.2 Rheological Properties of Creams ................................................... 211
8.2.3 Thermodynamic Properties of Creams ............................................ 223
8.3 Creams Formulated in Vegetable Oils/Water System ........................... 225
8.3.1 Appearance of Creams ..................................................................... 225
8.3.2 Rheological Properties of Creams ................................................... 227
8.3.3 Thermodynamic Properties of Creams ............................................ 241
Chapter 9. Conclusion and Future Work ........................................................ 245
References............................................................................................................... 249
4
List of Figures
Figure 2.1 Dependence of surface tension on the concentration of various solutes ....... 32
Figure 2.2 Schematic diagram of surfactant molecule .................................................... 33
Figure 2.3 schematic diagram of different types of surfactant molecules alignment at
water surface ................................................................................................................ 39
Figure 2.4 Dependence of structure and phase formation on the surfactant
concentration and temperature, adapted from Guo et al., 2018.................................... 42
Figure 2.5 General structure of sophorolipids (SLs) ........................................................ 55
Figure 2.6 General structure of mannosylerythritol lipids (MELs) ................................... 61
Figure 2.7 Instability phenomena of emulsions.............................................................. 74
Figure 3.1 Schematic diagram of simplified configuration and photo of overhead stirrer
..................................................................................................................................... 89
Figure 3.2 Schematic diagram of continuous configuration and corresponding container
parameters that applied in Formulation_Ⅲ .................................................................. 96
Figure 3.3 Flow behaviour of fluids plotted in shear stress-shear rate (left) and viscosity-
shear rate (right) diagram, according to Mezger, 2020 ................................................ 103
Figure 3.4 Schematic diagram of steady state shear and generated shear profile,
according to Mezger, 2020 .......................................................................................... 104
Figure 3.5 Qualitative S-shape rheological curve for typical shear thinning fluids and
corresponding model fitting range, according to Tatar et al., 2017 .............................. 105
Figure 3.6 Typical hysteresis loop of shear stress-shear rate behaviour for thixotropic
and rheopectic material, according to Maazouz, 2020 ................................................. 108
Figure 3.7 Schematic diagram of spring represents elastic behaviour (left) and dashpot
represent for viscous behaviour (right)........................................................................ 108
Figure 3.8 Creep and recovery test (a) and expected response of different materials:
response of linearly elastic material (b), response of viscous liquid (c) ......................... 109
Figure 3.9 Schematic diagram of Maxwell model ......................................................... 110
Figure 3.10 Creep and recovery test (a) and expected response of Maxwell model (d) 110
Figure 3.11 Schematic diagram of Kelvin-Voigt model ................................................. 111
Figure 3.12 Creep and recovery test (a) and expected response of Voigt model (b) ..... 112
Figure 3.13 Typical creep and recovery response (a) for Burgers model accompanied
with its schematic diagram (b)..................................................................................... 112
Figure 3.14 Response of viscous material and elastic material to creep test, expressed
with creep compliance with time in log-log plot .......................................................... 113
Figure 3.15 Two-plate model for oscillatory shear test and the applied oscillatory shear
profile ......................................................................................................................... 114
Figure 3.16 Typical frequency response of Maxwell model for a viscoelastic liquid (a) and
Voigt model for a viscoelastic solid (b)......................................................................... 117
Figure 3.17 Physical model of rheological measuring system ....................................... 118
Figure 3.18 Schematic diagram of cone and plate geometry ....................................... 119
Figure 3.19 Schematic diagram of power compensation DSC, adapted from Danley, 2002
................................................................................................................................... 122
Figure 3.20 Schematic diagram of heat flux DSC .......................................................... 123
Figure 3.21 Schematic diagram of Tzero measurement model for DSC ........................ 124
5
Figure 3.22 Schematic diagram of Laser diffraction when encountering different size of
particles ...................................................................................................................... 126
Figure 3.23 Diffraction patterns and the corresponding radial intensity for two spherical
particles 1 (a) and 2 (b) in different sizes ..................................................................... 127
Figure 3.24 Schematic diagram of laser diffraction particle size analyser ..................... 127
Figure 3.25 Droplet size distribution of a sample, and the corresponding illustration of
size classes .................................................................................................................. 128
Figure 3.26 Illustration of instrument Mastersizer 3000 connecting to the wet dispersion
unit ............................................................................................................................. 130
Figure 3.27 Schematic diagram of force that applied to increase the surface area, and
the surface tension is proportional to this measured force .......................................... 133
Figure 3.28 Physical model of tensiometer .................................................................. 134
Figure 3.29 Schematic illustration of Du Noüy ring method (left) and its cross-section
view (right).................................................................................................................. 135
Figure 3.30 Schematic diagram of the theory of a mass spectrometry ......................... 137
Figure 3.31 Schematic diagram of the theory of mass spectrometry ............................ 138
Figure 4.1 Exploratory flow characterisation of E45 cream for pre shear stress
determination, where viscosity varied as a function of shear stress ............................. 143
Figure 4.2 Oscillatory amplitude sweep of E45 cream for determination of oscillatory
stress within linear viscoelastic range .......................................................................... 144
Figure 4.3 Oscillatory time sweep of E45 cream for determination of equilibrium time
................................................................................................................................... 145
Figure 4.4 Steady state shear test on E45 cream, where viscosity varied as a function of
shear stress ranging from 10 Pa to 300 Pa ................................................................... 149
Figure 4.5 Shear ramp test on E45 cream for determination of hysteresis loop, where
shear stress ramped up and down as a function of shear rate ..................................... 151
Figure 4.6 Oscillatory frequency sweep on E45 cream, where G’ and G’’ varied as
function of angular frequency from 0.01 rad s-1 to 1000 rad s-1 at controlled oscillatory
stress of 4 Pa ............................................................................................................... 152
Figure 4.7 Comparison of the droplet size distribution curves between sample A of pre-
treated E45 cream, and sample B of pre-treated E45 cream with additional 2%wt of SLES
................................................................................................................................... 154
Figure 4.8 DSC thermogram of E45 cream (screen shot directly from the software) .... 156
Figure 5.1 Appearance of mimic cream prepared in the first trial, using CA as the sole
surfactant and a homogenizer for mixing .................................................................... 158
Figure 5.2 Appearance of mimic cream prepared in the second trial, using CA and SLES as
surfactants and a stirrer with pitched blade turbine for mixing.................................... 159
Figure 5.3 Comparison of representative flow behaviour between E45 cream and mimic
cream that emulsified by SLES and cetyl alcohol, where viscosity varied as a function of
shear stress ranging from 5 Pa to 300 Pa ..................................................................... 160
Figure 5.4 Appearances of mimic creams prepared in Formulation_Ⅰ ........................ 162
Figure 5.5 Flow profiles of 12 mimic creams prepared in the Formulation_Ⅰ, using
simplified configuration, where viscosity varied as a function of shear stress from 5 Pa to
300 Pa ......................................................................................................................... 164
6
Figure 5.6 Respective comparison of average of limit viscosity and corresponding yield
stress among mimic creams formulated with varied emulsifying system ..................... 166
Figure 5.7 Oscillatory strain sweep on mimic creams formulated with 6 wt% CA and 2
wt% GM with varied concentration of SLES, where G' and G'' varied as function
of %strain ranging from 0.01 to 100 ............................................................................ 169
Figure 5.8 Oscillatory frequency sweep on mimic creams formulated with 6 wt% CA and
2 wt% GM with varied concentration of SLES, where G', G'' and |η*| varied as a function
of frequency ranging from 0.01 Hz to 100 Hz ............................................................... 173
Figure 5.9 Comparison between steady shear viscosity and complex viscosity
respectively varied as a function of shear rate and angular frequency, for cream
containing 2 wt% SLES, 6 wt% CA and 2 wt% GM......................................................... 174
Figure 5.10 Comparison of storage and loss moduli among creams containing 6 wt% CA
and 2 wt% GM with varied concentration of SLES, where storage and loss moduli varied
as a function of frequency ranging from 0.01 Hz to 1000 Hz ........................................ 175
Figure 5.11 Comparison of dissipation factor among creams containing 6 wt% CA and 2
wt% GM with varied concentration of SLES, where dissipation factor varied as a function
of frequency ranging from 0.01 Hz to 1000 Hz ............................................................. 177
Figure 5.12 Comparison of the compliance (J) response among creams containing 6 wt%
CA and 2 wt% GM with varied concentration of SLES, where compliance varied as a
function of time. Curve illustrated with mean values, and standard deviations were
0.0002 1/Pa for 2 wt% SLES involved, 0.0002 1/Pa for 4 wt% SLES involved, 0.0003 1/Pa
for 6 wt% SLES involved .............................................................................................. 178
Figure 5.13 Typical plot of compliance varied as a function of time in a creep-recovery
test for a viscoelastic material ..................................................................................... 179
Figure 5.14 Mechanical model for interpretation of creep-recovery result .................. 179
Figure 5.15 Comparison of droplet size distribution among creams containing 6 wt% CA,
2 wt% GM with varied concentrations SLES, where volume density varied as a function
of diameter. Mean values are presented in curve for each cream................................ 180
Figure 5.16 Microscopic observation of mimic creams containing 6 wt% CA, 2 wt% GM
with varied concentrations of SLES .............................................................................. 181
Figure 5.17 DSC thermogram of cetyl alcohol and glycerol monostearate.................... 182
Figure 5.18 DSC thermogram of light liquid paraffin and white soft paraffin ................ 183
Figure 5.19 DSC thermogram of sodium lauryl ether sulfate (screen short directly from
software) .................................................................................................................... 183
Figure 5.20 Comparison of thermal behaviour among creams containing 6 wt% CA, 2
wt% GM with varied concentrations SLES, where heat flow varied as a function of
temperature ranging from 25 °C to 90 °C .................................................................... 184
Figure 6.1 Effect of varied mixing time during heating procedure on droplet size
distribution of creams containing 6 wt% CA and 2 wt% GM with varied concentrations of
SLES, at controlled mixing speed of 500 rpm ............................................................... 189
distribution of creams containing 6 wt% CA and 2 wt% GM with varied concentrations of
SLES, at controlled mixing speed of 700 rpm ............................................................... 190
7
Figure 6.3 Comparison of droplet size distribution among creams containing 6 wt% CA
and 2 wt% GM with 2 wt% SLES, respectively being mixed at 500 rpm, 700 rpm and 900
rpm for 3 minutes. Data presented as the mean value. ............................................... 192
Figure 6.4 Comparison of D [3, 2] values among creams containing 6 wt% CA and 2 wt%
GM with varied concentrations of SLES, respectively being mixed at 500 rpm, 700 rpm
and 900 rpm for 3 minutes. Data is presented with the standard deviation as error bar
................................................................................................................................... 193
Figure 6.5 Comparison of different cooling procedures on cream containing 6 wt% CA
and 2 wt% GM with 4 wt% SLES, where viscosity varied as a function of shear stress
ranging from 1 Pa to 300 Pa ........................................................................................ 195
Figure 6.6 Comparison of varied stirring speed during cooling procedure for 10 min on
cream containing 6 wt% CA and 2 wt% GM with 4 wt% SLES, where storage modulus
varied as a function of frequency ranging from 0.1 Hz to 100 Hz ................................. 197
Figure 6.7 Comparison of varied stirring duration during cooling procedure at controlled
stirring speed of 200 rpm on cream containing 6 wt% CA and 2 wt% GM with 4 wt%
SLES, where storage modulus varied as a function of frequency ranging from 0.1 Hz to
100 Hz ......................................................................................................................... 197
Figure 7.1 Phase separation of media broth of sophorolipids production. ................... 199
Figure 7.2 Appearance of extracted sophorolipids (a) right after rotary evaporation and
(b) after 24h dried in fume cupboard .......................................................................... 200
Figure 7.3 Result of HPLC measurement of sophorolipids ............................................ 201
Figure 7.4 Representative mass spectrum of sophorolipids obtained from mass
spectrometry .............................................................................................................. 202
Figure 7.5 Surface activity of SLs in water solution, where surface tension varied as a
function of the concentration of sophorolipids............................................................ 203
Figure 7.6 Appearance of MELs products from (a) batch fermentation and (b) fed-batch
fermentation............................................................................................................... 204
Figure 7.7 Results of mass spectrometry of mannosylerythritol lipids.......................... 205
Figure 7.8 Representative mass spectrum of mannosylerythritol lipids with m/z ranging
from 600 to 750 .......................................................................................................... 205
Figure 7.9 DSC thermogram of sophorolipids, where heat flow varied as function of
temperature ranging from -20 °C to 90 °C ................................................................... 207
Figure 7.10 DSC thermogram of mannosylerythritol lipids where heat flow varied as
function of temperature ranging from -20 °C to 90 °C ................................................. 207
Figure 8.1 Comparison of flow profile among creams formulated in Formulation_Ⅰ
using simplified configurations and that in Formulation_Ⅲ using the continuous one. 210
Figure 8.2 Appearance of bio-creams formulated with 6 wt% CA and 2 wt% GM
respectively incorporated with varied concentrations of SLs and MELs, in mixed paraffin
oils-water system ........................................................................................................ 211
Figure 8.3 Comparison of flow behaviour among creams containing 6 wt% CA and 2wt%
GM with varied concentrations of SLs in mixed paraffin oils-water system, where
viscosity varied as a function of shear stress ranging from 1 Pa to 300 Pa .................... 213
8
Figure 8.4 Comparison of flow behaviour among creams containing 6 wt% CA and 2wt%
GM with varied concentrations of MELs in mixed paraffin oils-water system, where
viscosity varied as a function of shear stress ranging from 1 Pa to 300 Pa .................... 214
Figure 8.5 Oscillatory strain sweep on bio-creams formulated with 6 wt% CA and 2 wt%
GM with varied concentration of SLs, where G' and G'' varied as function of %strain
ranging from 0.01 to 10 ............................................................................................... 216
Figure 8.6 Oscillatory frequency sweep on bio-creams containing 6 wt% CA and 2 wt%
GM with varied concentration of SLs, where G', G'' and |ƞ*|varied as function of
frequency ranging from 0.01 Hz to 100 Hz ................................................................... 218
Figure 8.7 Specific oscillatory frequency range of oscillatory frequency sweep test for
SLs-involved cream, including the range between 0.01 and 0.1 (left) and that between 10
and 100 (right), showing crossover of G' and G'' .......................................................... 219
Figure 8.8 Comparison of G' and G'' as function of frequency ranging from 0.01 Hz to 10
Hz among bio-creams containing 6 wt% CA and 2 wt% GM with varied concentrations of
SLs in mixed paraffins-water system............................................................................ 221
Hz among bio-creams containing 6 wt% CA and 2 wt% GM with varied concentrations of
MELs in mixed paraffins-water system ........................................................................ 221
Figure 8.10 Comparison of compliance as a function of time among bio-creams
containing 6 wt% CA and 2 wt% GM with varied concentrations of SLs in mixed paraffins-
water system .............................................................................................................. 222
containing 6 wt% CA and 2 wt% GM with varied concentrations of MELs in mixed
paraffins-water system ............................................................................................... 223
Figure 8.12 DSC thermograms of bio-creams formulated with varied concentrations of
SLs in mixed paraffins-water system............................................................................ 224
MELs in mixed paraffins-water system ........................................................................ 224
Figure 8.14 Appearance of mimic creams formulated involving SLES, respectively with
coconut oil and vegetable shortening in water, containing surfactant system of 6 wt%
cetyl alcohol and 2 wt% glycerol monostearate with varied concentrations of sodium
lauryl ether sulfate ...................................................................................................... 225
Figure 8.15 Appearance of bio-creams formulated involving SLs and MELs, respectively
with coconut oil in water, containing surfactant system of 6 wt% cetyl alcohol and 2 wt%
glycerol monostearate with varied concentrations of sodium lauryl ether sulfate ....... 226
with vegetable shortening in water ............................................................................. 227
Figure 8.17 Comparison of flow behaviour among mimic creams containing 6 wt% CA
and 2 wt% GM with varied concentrations of SLES in coconut oil-water system, where
viscosity varied as a function of shear stress ranging from 1 to 300 Pa ........................ 229
and 2 wt% GM with varied concentrations of SLES in vegetable shortening-water system,
where viscosity varied as a function of shear stress ranging from 1 to 300 Pa .............. 229
9
Figure 8.19 Comparison of flow behaviour among bio-creams containing 6 wt% CA and 2
wt% GM with varied concentrations of SLs in coconut oil-water system, where viscosity
varied as a function of shear stress ranging from 1 to 300 Pa ...................................... 230
wt% GM with varied concentrations of SLs in vegetable shortening-water system, where
wt% GM with varied concentrations of MELs in coconut oil-water system, where
wt% GM with varied concentrations of MELs in vegetable shortening-water system,
where viscosity varied as a function of shear stress ranging from 1 to 300 Pa .............. 233
Figure 8.23 Oscillatory strain sweep on mimic creams containing 6 wt% CA and 2 wt%
GM with 6 wt% SLES in coconut oil-water system, where G'and G'' varied as function of
strain% ranging from 0.01 to 100 ................................................................................ 234
Figure 8.24 Oscillatory strain sweep on bio-creams containing 6 wt% CA and 2 wt% GM
with 6 wt% MELs in coconut oil-water system, where G' and G'' varied as function of
strain% ranging from 0.01 to 100 ................................................................................ 234
Figure 8.25 Oscillatory frequency sweep on mimic creams containing 6 wt% CA and 2
wt% GM with varied concentrations of SLES in coconut oil-water system, where G', G''
and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz ......................... 235
wt% GM with varied concentrations of SLES in vegetable shortening-water system,
where G', G'' and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz .... 236
GM with varied concentrations of SLs in coconut oil-water system, where G', G'' and
|ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz ............................... 236
GM with varied concentrations of SLs in vegetable shortening-water system, where G',
G'' and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz .................... 237
GM with varied concentrations of MELs in coconut oil-water system, where G', G'' and
|ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz ............................... 237
GM with varied concentrations of MELs in vegetable shortening-water system, where G',
G'' and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz .................... 238
Figure 8.31 Comparison of compliance as a function of time among mimic creams
containing 6 wt% CA and 2 wt% GM with varied concentrations of SLES in vegetable
shortening-water system ............................................................................................ 240
containing 6 wt% CA and 2 wt% GM with varied concentrations of MELs in coconut oil-
water system .............................................................................................................. 240
containing 6 wt% CA and 2 wt% GM with varied concentrations of SLs in coconut oil-
water system .............................................................................................................. 241
10
Figure 8.34 DSC thermograms of mimic creams containing 6 wt% CA and 2 wt% GM with
varied concentrations of SLES in vegetable shortening-water system .......................... 242
Figure 8.35 DSC thermograms of bio-creams containing 6 wt% CA and 2 wt% GM with
varied concentrations of MELs in vegetable shortening-water system ......................... 242
varied concentrations of SLs in coconut oil -water system ........................................... 243
11
List of Tables
Table 1.1 Classification of ingredients formulated in E45 cream based on function ....... 18
Table 2.1 Examples of cationic surfactants and corresponding chemical structures ....... 34
Table 2.2 Examples of anionic surfactants and corresponding chemical structures ........ 35
Table 2.3 Example of non-ionic surfactants and corresponding chemical structures ...... 37
Table 2.4 Classification of bio-surfactants, adapted from Shoeb et al., 2013 ................. 45
Table 2.5 Typical shear rate ranges of emulsions and creams during different industrial
applications, adapted from Mezger, 2020 ..................................................................... 76
Table 2.6 Theoretical values of shear rate related to different processes of cream
application, adapted from Langenbucher and Lange, 1970 ............................................ 76
Table 3.1 Default settings for HPLC measurement for analysing SLs concentration
(Dolman et al., 2017) ..................................................................................................... 84
Table 3.2 Formulation of first trial cream, with cetyl alcohol as sole emulsifying agent .. 86
Table 3.3 Formulation of second trial cream, with cetyl alcohol and SLES as mixed
emulsifying system........................................................................................................ 88
Table 3.4 Classification of ingredients in the cream formulation .................................... 90
Table 3.5 Formulation_Ⅰ of mimic creams prepared with varied proportion of
surfactant system.......................................................................................................... 91
Table 3.6 Formulation_Ⅱ of mimic creams prepared with varied concentrations of fatty
alcohols ........................................................................................................................ 92
Table 3.7 Formulation_Ⅲ of mimic creams and bio creams with optimized surfactant
system .......................................................................................................................... 94
Table 3.8 Ingredients for cream preparation in Formulation_Ⅰand Formulation_Ⅱ..... 96
Table 3.9 Formulation of model creams used for studying the effect of different
manufacturing strategies on cream performance .......................................................... 98
Table 3.10 Parameters of different mixing durations applied for study the effect of
different mixing procedure on product performance .................................................... 99
Table 3.11 Specification of different mixing speeds during heating procedure, applied for
study the effect of different mixing procedure on product performance, modified from
Boxall et al., 2010 ........................................................................................................ 100
Table 3.12 Specification of different cooling procedures applied for study the effect of
different cooling procedures on product performance, adapted from Rønholt et al., 2014
................................................................................................................................... 100
Table 3.13 Classification of Non-newtonian fluids, according to Mezger, 2020 ............ 103
Table 3.14 Non-Newtonian models with constitutive equations, according to Mezger,
2020 ........................................................................................................................... 105
Table 3.15 Parameters for steady state shear test (SSS)............................................... 120
Table 3.16 Parameters for oscillatory strain sweep test (OSS)...................................... 120
Table 3.17 Parameters for oscillatory frequency sweep test (OFS) ............................... 120
Table 3.18 Parameters for creep and recovery test ..................................................... 121
Table 3.19 Details for SOP applied in droplet size analysis for mimic cream ................. 132
12
Table 4.1 Parameters of pre-shear stress determination for E45 cream characterisation
................................................................................................................................... 140
Table 4.2 Parameters for preliminary linear viscoelastic range (LVER) determination for
E45 cream characterisation ......................................................................................... 141
Table 4.3 Parameters for equilibrium time determination for E45 cream characterisation
................................................................................................................................... 142
Table 4.4 Parameters for steady state shear test on E45 cream ................................... 146
Table 4.5 Parameters for continuous shear stress ramp test on E45 cream ................. 147
Table 4.6 Parameters for new linear viscoelastic range (LVER) determination for E45
cream characterisation................................................................................................ 147
Table 4.7 Parameters for oscillatory frequency sweep on E45 cream........................... 148
Table 4.8 Details of SOP applied in droplet size analysis for E45 Cream ....................... 153
Table 5.1 Results of steady state shear measurement for E45 and mimic cream
containing SLES and CA ............................................................................................... 161
Table 5.2 Key parameters derived from viscosity profiles of creams containing 6 wt% CA
and 2 wt% GM with varied concentrations of SLES ...................................................... 166
Table 5.3 Key parameters derived from viscosity profiles of cream containing 2 wt% SLES
and 2 wt% GM with varied concentrations of CA ......................................................... 185
Table 5.4 Key parameters derived from viscosity profiles of cream containing 4 wt% SLES
and 2 wt% GM with varied concentrations of CA ......................................................... 186
Table 6.1 Sauter mean diameter D[3, 2] in cream containing 6 wt% CA and 2 wt% GM
with varied concentrations of SLES, being mixed at 500 rpm at ifferent mixing time. The
value is presented as mean value ± standard deviation ............................................... 189
Table 6.2 Sauter mean diameter D [3, 2] in cream containing 6 wt% CA and 2 wt% GM
with varied concentrations of SLES, respectively being mixed at 700rpm and 900rpm at
different mixing time. The value is presented as mean value ± standard deviation ...... 191
Table 6.3 Parameters for cooling process, where mixing speed and mixing time are
specified ..................................................................................................................... 194
Table 6.4 Key parameters derived from viscosity profile of cream containing 4 wt% SLES
with 6 wt% CA and 2 wt% GM formulated with different cooling procedure................ 195
Table 7.1 Prediction of MELs structure and corresponding possible fatty acid chains... 206
13
Abstract
Personal care products are necessities in people’s daily life, especially cosmetic
creams and lotions. Cosmetic creams are semi-solid emulsions, most of which are
normally at a thermodynamically metastable state, thus surfactants play a key role
in the formulation. Most industrially applied surfactants are chemically synthesized,
which are poorly biodegradable and biocompatible. With the increase in concern
for environment protection, considerable attention has been given to biosurfactants
due to their environmentally friendly merits and higher surface activity.
This project aims to study the preparation of cosmetic cream formulated with
biosurfactants, compared to a system of containing cetyl alcohol (CA), glycerol
monostearate (GM) and sodium lauryl ether sulfate (SLES) with paraffin in water.
Instead of applying the petroleum-based surfactants, the cream will be
reformulated with microbial-derived surfactants, e.g. sophorolipids (SLs) and
mannosylerythritol lipids (MELs). Key parameters for the performance of the cream
are analysed to allow understanding of the production process and the effect of
replacing the surfactant. Droplet size analysis was performed using a Mastersizer
3000. The d3,2 of the distributions were used to determine the dependencies of the
surfactant concentrations, the rotor speed, and the mixing time used to
manufacture the cream. Rheological properties of the cream were also examined,
e.g. shear stress sweep, and linked to the droplet size distributions. As a result,
structural mixture of SLs mainly consisting of diacylated acidic SLs of C18:1,
diacylated acidic SLs with C20:1 and diacylated lactonic SLs with C18:1, that
extracted from c. bombicola cultivation consuming glucose and rapeseed oil as
substrates, was successfully incorporated with fatty alcohols for cream formulation
in replacement of anionic surfactant SLES. In this study, bio cream with 6 wt% SLs
exhibited smooth texture with sufficient stiffness, reflecting as an average
maximum viscosity of approximate (2±0.7)×105 Pa·s. And a primary creep was
obtained from creep test, indicating a solid behaviour of the system. Also higher
concentration of SLs formulated in cream system led to better result with good
performance. Vegetable oils that formulated as alternatives to mixed paraffin oils
were well emulsified in water with surfactant system containing SLES and fatty
alcohols, especially coconut oil. In addition, 2 wt% MELs incorporating with cetyl
alcohol and glycerol monostearate formulated with coconut oil in water could
prepare cream with average maximum viscosity of (1.18±0.8)×105 Pa·s which is
comparable to that of system with 2 wt% SLES instead.
14
Declarations
No portion of the work referred to in the thesis has been submitted in support of
an application for another degree or qualification of this or any other university or
other institute of learning.
i. The author of this thesis (including any appendices and/or schedules to this
thesis) owns any copyright in it (the “Copyright”) and has given The
University of Manchester the rights to use such Copyright, including for any
administrative purposes.
ii. Copies of this thesis, either in full or in extracts and whether in hard or
electronic copy, may be made only in accordance with the Copyright,
Designs and Patents Act 1988 (as amended) and regulations issued under
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University has from time to time. This page must form part of any such
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iii. The ownership of certain Copyright, patents, designs, trademarks and other
intellectual property (the “Intellectual Property”) and any reproductions of
copyright works in the thesis, for example graphs and tables
(“Reproductions”), which may be described in this thesis, may not be owned
by the author and may be owned by third parties. Such Intellectual Property
and Reproductions cannot and must not be made available for use without
the prior written permission of the owner of the relevant Intellectual Property
and /or Reproductions.
iv. Further information on the conditions under which disclosure, publication

and commercialisation of this thesis, the Copyright and any Intellectual
Property and/or Reproductions described in it may take place is available
in the University IP Policy, in any relevant Thesis restriction declarations
deposited in the University Library, The University Library’s regulations and
in The University’s policy on Presentation of Theses.
15
Acknowledgements
I am very grateful to my supervisors Dr. Thomas Rogers and Dr. James Winterburn
for their careful guidance and useful advice throughout the project. Thanks to my
seniors who gave me care and support in both of life and study: to Ben Dolman for
his help with biosurfactant production; to Sergio Carrillo De Hert for his training on
rheometer and Mastersizer; to Sara Bages estopa for her training on surface
tension measurement. Appreciate for the support of Reynard Spiess with mass
spectrometry measurement. Thanks to University of Manchester for providing the
top educational resources for me.
Last but not least, I would like to sincerely express my appreciation to my parents
and my lovely fiancé for their understanding all along this PhD period, giving me
material and emotional support that are essential to rely on
16
Chapter 1. Introduction
1.1 Research System

Personal care and cosmetics include a wide variety of items that people commonly
get access to in their everyday life, including, for example, shampoos and soaps
for cleaning, skin creams and lotions for protecting and nourishing, foundation and
lipstick for beautifying. Occupying a large portion of market share around the world,
cosmetic creams are served as necessities that applied by people for various
purpose, which are multicomponent systems usually forming by two immiscible
liquids: oil and water, where one is dispersed in the other (Ying, 2010). As
thermodynamically unstable systems having tendency to demix into two liquids,
surfactants are usually applied in the formulation for facilitating emulsion formation
through adsorbing at the interface during homogenization and reducing the
interfacial tension to promote droplet dissociation (Khan et al., 2011). In addition,
as for the formulation of a cream, namely semisolid emulsions, mixed surfactant
system is largely applied instead of single surfactants, consisting of different types
of surfactants or emulsifiers such as ionic or non-ionic ones combined with fatty
amphiphiles. Researchers extensively studied the microstructure of oil in water
cream stabilized by a mixed surfactant system, finding a general four-phase-
system presented as (Colafemmina et al., 2020b):
a. Crystalline/Hydrophilic gel phase, consisting of bilayer of the mixed

emulsifier system and intralamellarly fixed water
b. Lipophilic gel phase, consisting of the superfluous co-emulsifiers which is
not aligned in the mixed emulsifier system.
c. Bulk water
d. Dispersed oil phase which is immobilized by the lipophilic gel phase.
The microstructure of multicomponent emulsion system is macro-reflected by its

flow property. When the balance between thermal and interparticle forces reaches
an equilibrium, the system is correspondingly in various states, from the liquid-like
viscous microstructures with low resistant to external force to the semisolid-like
viscoelastic dispersions with three-dimensionally self-bodying structure exhibiting
as yield stress or storage moduli (Ha et al., 2015). The original structure and
relevant properties will be altered and rebuilt when the system subject to an
external driving force, where the introduced hydrodynamic forces interact with
thermal and interparticle forces, leading to a sophisticated microstructure involved
17
melting or deforming and so on. As one of the most significant characteristics of a
cream during production and application processes, flow property is closely related
to the quality, stability and efficacy of product. Rheology is a subject that studies
the behaviour of flow and deformation of materials. Being as a useful method for
cream production and improvement, rheological characterisation help understand
the nature of system, select raw materials and control manufacturing processes
(Tatar et al., 2017). In addition, the end use of creams could be predicted by
conducting rheological measurements, from removing from the container to
applying on the skin. As the success or failure of final products is greatly
determined by their flow properties, rheological study is significant for the
improvement of manufacturing process and the development of customer-satisfied
products.
In this project, cream E45 was used as a standard model cream, purchased from
Boots. Sourcing from product label, the ingredients of E45 shown in Table 1.1 were
classified based on their functions, where the weight concentration of three key
components are specified according to its product introduction.
Table 1.1 Classification of ingredients formulated in E45 cream based on function
Weight
ingredients concentration function
(wt%)
White soft paraffin 14.5
Light liquid paraffin 12.6
Hypoallergenic anhydrous Emollient, skin lubricant, moisturizer
1.0
lanolin
Glyceryl monostearate
Surface active compounds (emulsifiers,
Cetyl Alcohol
surfactants)
Sodium Cetostearyl Sulphate
Sodium Hydroxide Neutralizing agents, adjust acid/base
Citric Acid Monohydrate balance
Carbomer Thickener/viscosity enhancer/stabilizer
Methyl Hydroxybenzoate
Propyl Hydroxybenzoate Anti-fungal agent, preservative
Purified water
1.2 Research Motivation

Surfactant system generally accounts for 10~20 wt% of cream, playing significant
roles in the production, with which a three-dimensional gel structure will be formed.
Traditional surfactants that widely applied in commercial cosmetic creams are
chemically synthesized and petroleum derived, which have been suggested to be
18
harmful to both of marine or land environment and human body due to their
hazardous origin and poor biodegradability (Mujumdar et al., 2017). It has been
reported that petrochemical surfactants destroy the external mucous layer of
aquatic animals and cause damage to the gill of fishes. Moreover, some of them
will accumulate in the food chain, which indirectly cause threat to human health
(Sajna et al., 2015). In addition, synthetic surfactants have great potential of
causing skin irritation as their close contact. They denature proteins and strip lipids
in stratum corneum (SC). By penetrating through the SC layer, synthetic
surfactants further pose a threat to cells in deeper skin layers and interfere with the
function of the cell membrane (Seweryn, 2018). Especially ionic surfactants, which
strongly bind to proteins due to electrostatic interactions, exhibit more sever skin
irritation compared to non-ionic surfactants which interact with protein via weak
forces of hydrogen and van der Waals bonds (Mulligan, 2005). As the increasing
of people’s eco-friendly awareness, surfactants that widely applied in industries are
expected to be “greener” for the sake of environment and human beings. Based
on this, microorganism-derived biosurfactants are gradually drawn attention from
both of the academia and the industry, for replacing those petroleum-derived
surfactants in products directly linked to human health such as food,
pharmaceuticals and personal cares.
1.3 State-of-the-Art
Surfactant is generally known as surface active ingredient which has been widely
studied and commercially applied since very long before. With the development of
economy, a sharp increase was witnessed in the production of surfactants since
early 20th century. Up to today, surfactants are already not simply applied for
cleansing, but are multifunctional substances used for emulsifying, dispersing,
solubilizing, defoaming and wetting in various fields such as petroleum industry,
detergent industry, environmental pollution treatment, food industry, personal care
industry and so on (Awad et al., 2011). Owing a polar head group showing affinity
to water and a non-polar tail group having opposite affinity, surfactant molecule
behaves amphiphilicity and functions at interfaces of water/oil or water/air to modify
the properties of the interface.
For the surfactants’ application in oil industry, more recent studies focused on
surfactant flooding technique for tertiary phase of oil recovery, known as enhanced
oil recovery (EOR). With combined mechanisms of surface activities, including
interfacial tension reduction, reservoir rock wettability alteration, foam generation
19
and water-oil emulsification, the optimised surfactant formulation was injected into
specific reservoir, therefore minimizing capillary forces presented in oil production
and improving the overall oil displacement efficiency (Alsinan et al., 2019). Those
mechanisms of different types of surfactants have been widely investigated. The
interfacial tension reduction by non-ionic surfactants, anionic surfactants,
zwitterion surfactants, and polymeric surfactants on oil-water interface were
assessed to be capable for their application for EOR. More recently, researchers
started to look at the possibility of using natural surfactants in EOR applications,
for eco-friendly purposes. Eslahati et al. found that 4 wt% of Saponin solution
helped increase the total oil recovery by 19.2% using spontaneous imbibition (A et
al., 2020). And in another study, during the tertiary oil recovery phase, 52.1% of
original oil in place (OOIP) in reservoirs was recovered with 5g L-1 Saponin solution
added. In the study of Dashtaki et al., a natural surfactant was developed from
Vitagnus plant extract, which obtained the OOPI recovery of 10.6% when 3000
ppm applied (Dashtaki et al., 2020). In order to bypass the problem of alkalis
involvement when single surfactant applied, mixed surfactant system was also
designed for EOR. Surfactant-polymer system was formulated and helped achieve
recovery of 24.5%~34.8% OOIP without alkali involved (Han et al., 2019), also the
anionic and zwitterionic surfactant mixtures lowered oil-water interfacial tension
below 0.001 dynes cm-1 leading to a displacement of 63~75% of residual oil, which
could not be achieved by single surfactants (Han et al., 2019).
In the field of pollution abatement, surfactants are capable of dealing with

contaminated soil, through mobilizing or solubilizing organic pollutants, petroleum
hydrocarbons and heavy metals, and enhancing the degradation of organic
contaminants, known as chemical surfactant flushing technique which could be
carried out both in situ and ex situ (Ali et al., 2017). The principles for the viability
of the technique, focused on solubilisation of hydrophobic substances by
surfactants (Zhu, 2011, García-Cervilla et al., 2020), behaviour of surfactants in
aqueous solution (Xia et al., 2020, Jardak et al., 2016, Li et al., 2017), interactions
between different types of surfactants and pollutants (Sharma et al., 2017,
Katarzyna et al., 2017), and for the improvement of the technique, focused on
increasing surfactant efficiency (Naghash and Nezamzadeh-Ejhieh, 2015, Hailu et
al., 2017, Bankole et al., 2017), optimizing the formulation of surfactant flushing
solutions, have been extensively studied. From the perspective of cost saving and
environmental protection, more scientific researchers have found cheaper
alternatives for surfactant solutions in flushing processes such as surfactant foam
20
(Bertin et al., 2017, Wang and Peng, 2015, Karthick et al., 2019, Li et al., 2020),
colloidal gas aphron (Mukhopadhyay et al., 2015, Zhang et al., 2019b, Aiza et al.,
2019) and so forth. But this new subject is still need more studies to support its
perfect implementation in contaminated soil treatment.
The study of application of surfactants in food, pharmaceutical and cosmetic

industry has been extensively studied, most of them focused on formulating high-
performance and innovative products through both theoretically and
experimentally analysing the roles of different types of surfactants on product
systems (Wang and Marangoni, 2016, Drakontis and Amin, 2020b). Still, the
unique molecular structures endow surfactants with their ability to adsorb to the
interfaces, self-assemble into micelles and further various structures of liquid
crystallines, therefore playing significant roles in the formulation (McClements and
Gumus, 2016). Emulsion-based products are ubiquitous in above mentioned
industries, the system of which usually contains multiple components such as oil,
water, fragrances, preservatives, active ingredients and surfactants. Thus it is
obvious to notice that the microstructure and interaction between those
components should be well designed in order to achieve a perfect product that
meets their required standards such as consistency, texture, appearance and
stability. Researchers have already made efforts to clarify unique amphiphilicity-
based properties of surfactants that lays foundation for their potential applications
in actual product development, including solubility, micellization, cloud point, krafft
point, adsorptivity and so on (Bnyan et al., 2018, Song et al., 2018, Pengon et al.,
2018, Shibaev et al., 2019, Kirby et al., 2017, Tao et al., 2017, Tummino et al.,
2018). Also, the synergistic effects of using mixed surfactant system, surfactant-
polymer mixed system and surfactant-nanoparticle system have also been
characterised in some literature papers (Bera et al., 2013, Kumari et al., 2018,
Sintang et al., 2017, Kumar et al., 2016, A et al., Agneta et al., 2019, Zhou et al.,
2019, Qian et al., 2020, Fuzhen et al., 2018, Ren et al., 2019, Wang et al., 2018d).
As for formulation technology, more recent studies utilize the combination of
experiments and computer-aided tools, such as simulations, modelling and
thermodynamics, to provide guidance and achieve optimal results when studying
the properties and phase behaviours of surfactants in specific systems, instead of
traditional model-based and trial-and-error methods (Preux et al., 2020, Chen et
al., 2017b, Ali et al., 2018).
The large market share of surfactants directly demonstrates their widely industrial
application. According to the report, the global surfactant market revenue
21
generation was $41.3 billion in 2019, and is projected to reach $58.5 billion by 2027,
growing at a CAGR of 5.3% from 2020 to 2027 (Pooja et al., 2018). Similarly,
another statistic analysis indicated that the global surfactants market is expected
to reach $52.4 billion by 2025 from $42.1 billion in 2020, at a CAGR of 4.5% from
2020 to 2025 (Markets and Markets, 2020). Nowadays, the surfactant market is
dominant by chemically synthesized surfactants which are mostly petroleum
derived. It is the large scale usage of surfactants in industries that researchers
gradually pay more attention to their safety study. Scientists found that the
presence of corrosive elements in the structure of synthetic surfactants and long
hydrophobic part consisting of C-C and -CH leads to their toxicity and unstable in
product systems (Lukic et al., 2016). Sodium dodecyl sulfate (SDS) has been found
to have side effect on gastrointestinal tract. And the presence of sulphur in SDS
boosts corrosive; the existence of quaternary ammonium component in CTAB
inhibits the enzymatic activity; the accumulation of hydrophobic moiety in Tween
20 destabilize the air and inhibits formation of stable foam (Guzman et al., 2016,
Lin et al., 2016b) .
At the same time, the concept of “green chemistry” always drives scientists and
engineers to seek for novel formulations that are more sustainable, eco-friendly
and safer for both human and environment. Microbial-based surfactants, generally
known as microbial biosurfactants, are the emerging sustainable alternatives for
their chemical synthetic counterparts. It should be pointed out that, in this thesis,
“microbial biosurfactant” will be simplified as “biosurfactants” representing for those
surfactants that obtained through microorganisms metabolism or synthesis , as
researchers indicated that the term of “biosurfactants” has to be clarified because
some plant-based surfactants such as saponin are also named as biosurfactants
(Ahmadi-Ashtiani et al., 2020).
Actually, studies related to biosurfactants began in the 1960s and they are
gradually applied into industries in recent times. Researchers have carried out
extensive investigations on biosurfactants in the aspect of detecting and screening
potential production microorganisms, structural analysis, physicochemical
properties characterisation, media optimization for increasing the yield,
improvements and innovation of fermentation and downstream technology, and
their potentially industrial application (Spina et al., 2018, Schultz and Rosado,
2020).
22
Biosurfactants are promising as their high biodegradability, low toxicity, low
environmental impact, structural diversity and high activity at extreme conditions,
especially their human-friendly and eco-friendly natures (Schultz and Rosado,
2020). Early in the 1990s, rhamnolipids secreted by Pseudomonas aeruginosa had
been shown low toxicity compared to chemically synthesized ones (Kuyukina et al.,
2015). When comparing to synthetic surfactant “Marlon A-350”, rhamnolipids
exhibited nontoxicity and non-mutagenicity (Irfan-Maqsood and Seddiq-Shams,
2014). Gein et al. found that glycolipid biosurfactant derived from Rhodococcus
ruber is non-cytotoxic towards human lymphocytes (Gein et al., 2011). In a study
of Kim et al., no inactivation of mouse fibroblast L929 cells was witnessed after 48-
hour exposure to a biosurfactant mannosylerythritol lipid (MEL-SY16). And
Pseudozyma spp.-produced mannosylerythritol lipids (MELs) exhibited protective
effect on skin through activating the fibroblast and papilla cells (Kim et al., 2002).
Vollbrecht et al. carried out the irritation test on trehalose tetraester that produced
by Phodococcus spp. 51 T7 and the chemically synthesized sodium dodecyl
sulfate (SDS), indicating less irritation of trehalose lipids against keratinocytes and
fibroblasts compared to chemical surfactant SDS (Kügler et al., 2014, Makkar et
al., 2011). In the same aspect, sophorolipids were also studied and displayed low
cytotoxicity on human keratinocytes (Lydon et al., 2017). In addition, 5%~10% of
MELs (MEL-A solutions) have potential ability to moisturize human skin cells
suffering damage of a chemically synthesized surfactant. The biodegradability
tests of biosurfactants have already been extensively conducted. Rhamnolipids
were proved to be biodegradable under anaerobic and aerobic conditions, showing
greater ability compared to Triton X-100 which only partially biodegrade under
aerobic conditions (Reddy et al., 2018). In the study of Chrzanowski et al.,
biodegradability of rhamnolipids when being cultivated in different types of soils
were studied, where the final results indicated degradability of 92% of total amount
of rhamnolipids in all soils after seven-days incubation (Liu et al., 2018,
Chrzanowski et al., 2012). Candida bombicola-produced sophorolipids even
exhibited almost instant degradation after the production of the compound by
cultivating the strain (Goswami et al., 2020, Minucelli et al., 2017). Similarly, in the
biodegradation study of MELs, Candida antarctica-produced biosurfactant were
productively biodegraded by activated sludge microorganisms in five minutes or so
(Wada et al., 2020, Saika et al., 2017).
Over the past decades, the commercial-scale of products that incorporated with
biosurfactants have been developed in a few companies. A Belgian manufacturer,
23
Ecover Eco-Surfactant, formulated multi-purpose cleansing products using
sophorolipids that originated from Evonik (Germany) (Tang et al., 2020). Soliance
(France), SyntheZyme LLC. (USA), Kaneka Ltd. (Japan), and Saraya (Japan) have
also applied sophorolipids for their application in detergents, cosmetics and other
products (Hilares et al., 2018). Kanebo Cosmetics Inc. (Japan) have produced
Mannosylerythritol lipid B (MEL-B) applying in cosmetic industry (Adu et al., 2020).
Rhamnolipids are widely produced in a range of companies, such as Jeneil
biosurfactant (USA), Paradigm Biomedical Inc. (USA), AGAE technologies Ltd.
(USA), TeeGene Biotech Ltd. (UK), Urumqi Unite Bio-Technology Co., Ltd. (China),
Rhamnolipids Companies, Inc. (USA) (Araújo, 2018). Nevertheless, comparing to
the global production of surfactants which is expected to reach more than 24 million
tons annually by 2020 (Hrůzová et al., 2020), statistic research estimated the
biosurfactant production to be only around 462 kilo tons per year by then (Souza
et al., 2018), indicating about 2%~3% occupation in the annually global surfactant
production. In addition, market share of microbial-derived biosurfactants only
account for less than 0.1% of the global market, despite some chemically
synthesized biosurfactants such as alkyl polyglycosides (APGs) and plant-based
biosurfactants take up 4% of the total (Roelants et al., 2019b).
The commercialisation of microbial-derived biosurfactants is promising but also

need to expand by breaking through the bottleneck. As reported, the impediments
to the large-scale application of biosurfactants are mainly ascribed to their highly
money-consuming production process and sometimes low yield (Olasanmi and
Thring, 2018). The price of biosurfactants is approximate 20% higher than
chemically synthesized surfactants (Silva et al., 2019a), where 10% to 30%~50%
of the total cost of biosurfactants refers to feedstock and substrates, and 60% to
70~80% of that arises from production aspect including biotechnology processes
and downstream strategies (Drakontis and Amin, 2020a, de Almeida et al., 2019,
Hrůzová et al., 2020). Thus, more recent studies in this field aim to improve their
cost performance by investigating low-cost substrates which are from either
renewable or waste materials, optimizing processes, and selecting novel strains
for production enhancement. Utilization of renewable substrates for biosurfactant
production was review by Banat et al (Thavasi and Banat, 2019b). Cheap and non-
conventional substrates for strain cultivation were highlighted in their studies,
including those from agro-industrial wastes and crop residues (Bran, beet
molasses, cassava, rice, hull of soy, corn and sugar cane molasses), animal fat
wastes, coffee processing residues (coffee pulp and coffee husks), plant oils (palm
24
oil and soybean oil), distillery wastes, oil-containing wastes (coconut cake, peanut
cake, olive oil wastes, soapstock and lubricating oil waste), food processing by-
products (frying edible oils, olive oil and potato peels rape seed oil), fruit processing
by-products (pine apple, carrot industrial waste and banana waste) (Borah et al.,
2019, Pele et al., 2019, Devaraj et al., 2019, Lima et al., 2020, Verma et al., 2020,
Kezrane et al., 2020, Louhasakul et al., 2020, Das and Kumar, 2019). Vecino et al.
carried out biosurfactant production using vineyard pruning waste (VPW ) as low
cost substrates, where lingnocellulosic wastes were applied as carbon sources for
L. paracasei consumption, achieving two types of biosurfactants. When growing
on glucose-based medium from VPW, L.paracasei produced glycolipopeptide,
while glycoprotein was achieved when the strain consuming lactose instead
(Thavasi and Banat, 2019a). However, researcher suggested that lignocellulose
feedstock is needed pre-treatment using fractionation strategy for enabling
cellulose saccharification (Wang et al., 2020a, Mota et al., 2019). In another study,
wood hydrolysates from birch and spruce woodchips were applied as glucose
source for rhamnolipids production by cultivating P. aeruginosa DBM 3774,
although the yield of rhamnolipids when applying renewable sources (2.31±0.10 ~
2.34±0.17 g L-1) was only about half of that when pure glucose (4.18±0.19 g L-1)
was used as a carbon source (Hrůzová et al., 2020).
Animal fat combined with corn steep liquor was applied as carbon source for
glycolipid biosurfactant production by cultivating yeast Candida lipolytica UCP0988,
where a maximum yield was achieved when comparing to other applied substrates
(Souza et al., 2016). Whey, the by-product of food processing, is full of lactose (75%
of dry mass), protein, organic acids, minerals and vitamins. When growing
Streptococcus thermophiles, Lactobacillus acidophilus and Lactobacillus
rhamnsus on medium of whey wastes, biosurfactants were produced and exhibited
emulsifying, inhibitory and antiadhesive properties (Soukoulis et al., 2017, Santos
et al., 2019, Jiang et al., 2016). In the study of Kaur et al., sophorolipids were
secreted by yeast Starmerella bombicola when consuming restaurant leftover food
waste as substrates, and the yield was comparable to traditional cultivation (Kaur
et al., 2019, Wang et al., 2020b). In the study of Jadhav et al, sunflower acid oil
refinery waste was applied as substrates for sophorolipids production using S.
bombicola as production strain (Jadhav et al., 2019). Also, another report claimed
sophorolipid production by cultivating strain on residual oil cake medium (Jiménez‐
Peñalver et al., 2020). Both of above two investigations determined the effective
emulsification ability of biosurfactants. Very recently, a biosurfactant extract was
25
obtained from waste stream of corn wet-milling industry, showing capability for
increasing the stability of vitamin C in aqueous solution for cosmetic application
(Rincón-Fontán et al., 2020).
From another aspect, researchers also looked at various methods for increasing
the production of biosurfactants to further maximize their profit, such as optimizing
media components and growth conditions, applying modified strains through
metabolic engineering or altering their composition, and the emerging recombinant
DNA technology (Jimoh and Lin, 2019a). This technology refers to construct and
develop recombinant or mutilative hyperproducing microorganisms for increasing
biosurfactant yield also producing associated effective bio-products (Kandasamy
et al., 2019). A bio-dispersant originated from a mutant defective Acinetobacter
calcoaceticus A2 was produced in a higher level, and its further downstream
treatments including purification, recovery and application were relieved due to the
less protein involved in the product (Sáenz-Marta et al., 2015). Researches on
biosurfactants biosynthetic genes and enzymes are significant. The heterologous
expression of surfactin synthetase genes was depicted from B. licheniformis NIOT-
06 in the study of Anburajan et al., and the modified strain can synthesize
biosurfactant at high rates (Anburajan et al., 2015). Bunet et al. proposed that the
polyketide synthases, non-ribosomal peptide synthases and fatty acid synthases
could be activated by the cloned Sfp-type phophopantetheinyl transferases for bio-
synthesizing fatty acids and antibiotics (Bunet et al., 2014). Similarly, Jimoh and
Lin reported lipopeptide production through cloning of biosurfactant genes from B.
subtilis SK320 and Paenibacillus sp. D9 (Jimoh and Lin, 2019c). In addition to that,
they also studied the optimization of medium and growth conditions for lipopeptide
production using Paenibacillus sp. D9, where effect of carbon, nitrogen, carbon to
nitrogen ratio, metals supplementation, pH, temperature and inoculum size on the
production have been thoroughly investigated (Jimoh and Lin, 2019b). Earlier than
that, another study was carried out by Parthipan et al., analysing similar conditions
for B. subtilis A1 cultivation to produce lipopeptide (Parthipan et al., 2017). Except
experimental path, Kiran et al. carried out statistical model based optimization of
media components in order to obtain lipopeptide through cultivating Brevibacterium
aureum MSA13, where full-factorial central composite design was applied (Kiran
et al., 2010). Mnif et al. applied statistical model of Box-Behnken design for media
components optimization, where B. subtilis SPB1 was cultivated to produce a
biosurfactant (Mnif et al., 2013). The glycolipid, mannosylerythritol lipids was
26
secreted by P. aphidis ZJUDM34 growing on a medium that optimised using
statistical model (53).
As for downstream processes, complex mixtures of biosurfactant after

manufacture and molecular variants of microbial-derived surfactants could make it
harder if specific species is required. Organic solvent extraction was proved to
achieve high yield of biosurfactant, but hazard and toxic chemicals harming human
and environment health is inevitable compensated for this strategy. More recent
studies focused on applying new biosurfactant recovery method for the production,
such as gravity separation (Dolman et al., 2017, Dolman et al., 2019), foam
fractionation (Bages-Estopa et al., 2018, Najmi et al., 2018). On top of that, some
novel biotechnologies supported energy-saving production processes. Perfumo et
al. suggested the production of low-temperature biosurfactants through cultivating
cold-adapted microorganisms, where no heat was required during the cultivation,
therefore introducing a low-energy-demand process of biosurfactant production
(Perfumo et al., 2018).
Properties characterisations of biosurfactants along with their potential application

have been extensively studied, which provides its high possibility for their
commercialisation. In personal care industry, the demand for biosurfactants in
personal care is expected to reach 50.7 kilo tons by 2020, accounting for more
than 10% of total biosurfactant market which is in the second place just after 44.6%
occupation of the market by household detergent, growing at a CAGR of 4.5% from
2014 to 2020 (Pham et al., 2018). Bezerraa et al. studied the comparison of
emulsifying properties between vegetable-based (Chenpodium quinoa) and
microorganism-derived (Pseudomonas aeruginosa) biosurfactants for their
application in cosmetic industry (Bezerraa et al., 2020). As a result, higher
emulsification index of oils when biosurfactant originated from P. aeruginosa was
used as emulsifier, which reached 71% (oil of rosemary), whereas C. quinoa-
derived biosurfactant maximally led to 51% emulsification index of coconut oil. In
addition, both of biosurfactants were stable until the temperature was up to 100°C,
and their resistance to pH variation was also studied, where vegetable-based
biosurfactant remained stable within pH rang of 4~8 and that for microorganism-
based biosurfactant was within pH range of 6~10. Also, another research was
carried out introducing the potential application of biosurfactant in cosmetic
industry, where a biosurfactant extract combined with Tween 80 in a shampoo
formulation was applied for the stabilization of Zn pyrithione in tea tree oil with
27
water emulsion. An optimal formulation was proposed giving the emulsion good
stability of 91% after 30 days, achieving highest solubility of Zn pyrithione of 59%
(Lukic et al., 2016).
Very recently, a lip gloss of water-in-oil emulsion was formulated using different
concentrations of rhamnolipids and sophorolipids as stabilizer, showing a stable
product via rheological analysis. However, silica particles were involved in the
formulation for building up the viscosity in the continuous phase, and larger
diameter size of silica particle imparted a more rigid network (Drakontis and Amin,
2020b). Resende et al. studied the formulation of toothpastes incorporating
biosurfactants that produced by P. aeruginosa, Bacillus methylotrophicus, and
C.bombicola combined with chitosan that extracted from fungus Mucorales, where
properties of toothpastes were analysed, including pH, foamability, cytotoxicity,
and antimicrobial action, and the results showed comparable to commercial
products (Resende et al., 2019). Similarly, another mouthwash formulation
involving biosurfactants also presented lower toxicity comparing to commercial
ones (Farias et al., 2019). Some researchers found the possibility of formulating
Lactobacillus paracasei derived biosurfactants in essential oils and natural
antioxidant emulsified in water, for enhancing the stability of the emulsion (Ferreira
et al., 2017, Vecino et al., 2016), therefore providing new eco-friendly cosmetic
formulations.
The application of biosurfactants in pharmaceutical industry mainly focused on

drug delivery improvements, and their abilities of antimicrobial, anti-adhesive,
antiviral, anticancer, anti-inflammatory and immunomodulatory (Rodríguez-López
et al., 2019, Sandeep and Rajasree, 2017, Janek et al., 2019, Adu et al., 2020). It
has been suggested that sophorolipids with amino acids presented antibacterial
activities against gram-positive and gram-negative organisms, anti-HIV, and anti-
spermicidal activities (Xu et al., 2019). Also, sophorolipids have been proved to
help in wound healing and dermatological care, through binding to silk fibroin
protein therefore accelerating its gelation (Maxwell et al., 2020). Lactoacilli spp.-
and marine bacteria-produced biosurfactants all exhibited effective anti-biofilm
activity against S. aureus CCM 3953 and P. mirabilis CCM 7188 (Englerová et al.,
2018). In food industry, researchers recently proposed the application of glycolipids
as food additives and preservatives in formulations, due to their anti-biofilm and
antioxidant activities (Merghni et al., 2017, Nataraj et al., 2020). A glycolipid
produced via cultivating Saccharomyces cerevisiae URM 6670 in a medium
containing agricultural by-product was incorporated into the cookie dough
28
formulation as the substitute for egg yolk, presenting an excellent thermal stability
and comparable properties of firmness and elasticity to standard formulation
(Ribeiro et al., 2020). From another aspect, by-products in food industry could be
converted to high value substances during biosurfactants synthesis (Satpute et al.,
2017), realizing the same goal as growing microorganisms on waste or renewable
substrates for biosurfactant production. Kiran et al. found a biosurfactant producing
strain, which was isolated Nesterenkonia sp. from a marine sponge Fasciospongia
cavernosa, and proposed the biosurfactant as a potential food addictive (Kiran et
al., 2017). In a recent study, rhamnolipids were investigated in terms of their
activities in different conditions, showing their antibacterial ability in food usage by
controlling the growth of pathogens, but pH alteration and basic conditions may
hinder its application (de Freitas Ferreira et al., 2019). Another glycolipid,
sophorolipids, that extracted from C.albicans and C. glabrata, exhibiting excellent
antibacterial activities against B. subtilis and E. coli. This providing their potential
as emulsifiers and antibacterial agents applying in food industry (Gaur et al., 2019).
Through the mechanisms including increasing substrate bioavailability for

microorganisms, interacting with the cell surface to increase cell surface
hydrophobicity for easily associating hydrophobic substrates with bacterial cells,
biosurfactants are capable of applying in environmental bioremediation (Karlapudi
et al., 2018). Researchers have found the application of biosurfactants for
removing heavy metal contaminants (Tang et al., 2017, da Rocha Junior et al.,
2019, Chen et al., 2017a, Lal et al., 2018, Sun et al., 2020), treating wastewater
(Bhosale et al., 2019, Ndlovu et al., 2016, Damasceno et al., 2018, Guo and Gao,
2020), cleaning up oil spill and other aspects (Shah et al., 2019, Patel et al., 2019,
De Souza et al., 2018). It has been reported that adding rhamnolipids with
concentration higher than CMC value enhanced solubilisation of petroleum
components, leading to an increase of biomass growth from 1000 to 2500 mg L -1
and 40%~100% of diesel biodegradation (Mostafa et al., 2019). In addition, a few
marine bacterial strains were reported to have the potential application for
biosurfactant production when consuming hydrocarbons (Xu et al., 2020), thus
proving the possibility of using biosurfactants in marine environment abatement.
For soil bioremediation, Pseudomonas aeruginosa W10 secreted biosurfactant
W10 effectively biodegraded polycyclic aromatic hydrocarbons (PHAs) including
phenanthrene and fluoranthene (Chebbi et al., 2017). Similarly, glycolipids
obtained from Pseudomonas sp. MZ01 has been applied for PHAs elimination
through electrokinetic-microbial remediation (EMR) method (Lin et al., 2016a).
29
Another research was conducted using lipopeptide (Paenibacillus dendritiformis
CN5-derived) for removing PHA, indicating that higher concentration of lipopeptide
(600 mg L-1) enhancing the biodegradation of pyrene (Hanano et al., 2017).
Bacillus, Acinetobacter, Sphingobium, Rhodococcus, and Pseudomonas Spp.
isolated from polluted soil all presented total petroleum hydrocarbons removal
ability (up to 50%) after seven-days incubation in peptone medium from beef
extract (Wang et al., 2020c). The application of biosurfactant for oil recovery is
highly promising where crude product or even the whole cell broth could be used
due to no requirements for the purity, thereby economizing on downstream
processing. Nocardia rhodochrous produced trehalose lipids increased total oil
recovery from underground sandstone by 30% (Le Roes-Hill et al., 2019).
Traditional EOR could be enhanced through involving biosurfactants production
process resulting in microbial enhanced oil recovery (MEOR) technique, Specific
microbes tailored to oil reservoir are involved in MEOR, experiencing metabolic
events and facilitating biosurfactants synthesis, therefore enhancing oil recovery
(Purwasena et al., 2019).
1.4 Research Objectives and Aims

This project primarily aims to provide information for formulation design of personal
care creams incorporating with biosurfactants, with understanding of the
production process and the effect of replacing the surfactant. As standard models
for comparison, lab-made mimic creams formulated with simplified surfactant
system that modified from commercially available E45 cream would be helpful.
The objectives of the project are:
1. to produce biousurfactants using fermentation technology and characterise

their structure
2. to formulate mimic creams and bio-creams with the system of respective
containing chemically synthesized surfactants and biosurfactants with mixed
paraffin oils in water, for understanding the effect of surfactant alteration on
cream performance.
1.5 Overview of Thesis

Chapter 1 described the project background, aims and objectives. Chapter 2 serve
as literature reviews related to the concepts involved in this project. Chapter 3
30
illustrated the methodology and corresponding theories that has been used in the
project. Chapter 4 and Chapter 5 respectively described the characterisation of
commercial E45 cream and production of mimic creams containing different
concentrations of SLES. Chapter 6 discussed the effect of manufacturing process
on the performance of creams. Chapter 7 presented the results of biosurfactants
production and their structural analysis. The final chapter 8 exhibited the
production of bio-creams that formulated with biosurfactants, and discrepancies
between bio-creams and mimic creams in terms of their property variations.
1.6 Nomenclature
Specific nomenclatures that applied in this thesis are indicated in the text. For
supplementary, some of frequently used nomenclatures are listed here.
Sodium lauryl ether sulfate SLES
Cetyl alcohol CA
Glycerol monostearate GM
Sophorolipids SLs
Mannosylerythritol lipids MELs
Biosurfactants BSs
31
Chapter 2. Literature Review
In this chapter, concepts relating to the project are introduced in details, including
chemically-synthesized and bio-derived surfactants, cream formulation and
rheology.
2.1 Surfactants
Surfactants are known as surface active agents that reduces the surface or
interfacial tension of a solvent and changes interfacial condition of the system,
thereby playing a key role in wetting, emulsifying, foaming, solubilizing, dispersing
and so on. Due to these functions, surfactants are wildly used in households,
personal cares, foods, pharmaceuticals and various fields (Kumari et al., 2018).
It has been studied that the surface tension of aqueous solutions will be changed
with the variation of solution concentrations, presenting three type of dependence,
as shown in Figure 2.1 (Hiemenz, 1986). Most organic solutes lower the surface
tension at water-air interface by adsorbing at the surface, resulting in exhibition of
attracted forces between molecules at surface due to weaker intermolecular forces
of organic solute (compared to that of water) and larger intermolecular distance of
molecules at surface (compared to that in bulk liquid), while inorganic electrolytes
remaining in bulk solution tend to slightly increase the surface tension because the
interaction between attractive ion and water molecules in the bulk leads to
destabilize water interaction at surface (Boyer et al., 2017).
inorganic electrolytes
surface tensioin
surfactant solutes
concentration of component
Figure 2.1 Dependence of surface tension on the concentration of various solutes
Among organic solutes, surfactants (Green curve in Figure 2.1) are able to sharply
reduce surface tension within low concentration range before the concentration
32
reaching a critical value, and there is no further reduction afterwards (Mittal and
Shah, 2013).
2.1.1 Structure of Surfactants
The surfactant molecule consists of a water-favouring hydrophilic head group

comprising charged ion group or uncharged polar group, mainly determining
different types of surfactants, and an oil-favouring hydrophobic tail moiety which is
usually an alkyl chain with or without side chain (Mitru et al., 2020). This unique
amphiphilic structure of surfactant molecules determines its ability in reducing the
surface and interfacial tension of different phases. Figure 2.2 shows the general
diagram of a surfactant molecule.
Hydrophilic head
(Polar)
Hydrophobic tail (Non polar)

Figure 2.2 Schematic diagram of surfactant molecule
2.1.2 Classification of Surfactants

Based on the molecular mass, surfactants are classified into low molecular mass
surfactants and polymeric surfactants. In respect to low molecular mass
surfactants, differences of “tail” moieties between different surfactants are not
significant, but hydrophilic “head” group is of great varieties. Anionic, cationic, non-
ionic and amphoteric surfactants are four main categories of petroleum-derived
surfactants which are classified according to the nature of their head groups (Peffly
et al., 2016).
a) Cationic surfactants
The hydrophilic head group of cationic surfactant molecules dissociates cations in

aqueous solutions. Most commercially valued cationic surfactants are the
derivatives of organic nitrogen compound, having positive ion charge carried by
nitrogen atom, such as amine salt cationic surfactant and quaternary ammonium
cationic surfactant (Ozkan et al., 2020). Some examples of quanternary
33
ammonium coumpounds (QAC) and corresponding chemical structures are listed
in Table 2.1.
Table 2.1 Examples of cationic surfactants and corresponding chemical structures
Name and Structure
Stearalkonium
Chloride
Cetrimonium
Chloride
Dicetyldimonium
Chloride
In personal care industry, QACs are one of the most effective classes of cationic
surfactants (Falbe, 2012). Due to carried positive charge, QACs have an
advantage in antistatic applications. Based on this, they are wildly used in hair care
products for softening hair and making it easy to rinse (Pati and Arnold, 2020). A
research (Ran et al., 2009) has been done to investigate the adsorption kinetics of
dimethylpabamidopropyl laurdimonium tosylate (DDABDT) onto the corneum of
scalp, in which the wettability of hair fibers changed from hydrophobic to
hydrophilic with the concentration of DDABDT only increasing from 0.05 mmol L-1
to 0.15 mmol L-1. Also, the formation of bilayer structure is responsible for the
enhancement of the wettability application.
Besides, QACs are also frequently used as antibacterial agents. In the study of
Nakata et al (Nakata et al., 2011), after treating the bacterial Escherichia. Coli cell
with cetyltrimethylammonium bromide (CTAB), a state of superoxide and hydrogen
peroxide generation was witnessed. This indicates that the generation of
superoxide in the cell becomes the main reason for the antibacterial function of
cationic surfactant. But it has not made clear that how superoxide and hydrogen
peroxide generated in the cell treated by CTAB. Regarding to stearalkonium
chloride and cetrimonium chloride, a patent has claimed that the combination of
these two QACs in the formula offers an advantageous of minimizing the total
34
amount of usage of QACs, thus the manufacturing cost of personal care products
will be decreased (Verboom and Bauer, 2003).
b) Anionic surfactants
In slightly acidic, neutral or alkaline aqueous solutions, the hydrophilic ‘‘head’’

groups of anionic surfactants are negative charged, for example, carboxylates
(alkane carboxylate salts), alkane sulfate esters, sulfonates (alkane-aromatic
sulfonic acid salts) and phosphoric acid esters. In aqueous solutions, anion head
group forms a structure with counter ions such as Na +, or K+ (Caracciolo et al.,
2017). Examples of anionic surfactants are listed in Table 2.2, including most
frequently used functional groups of anionic surfactants and the corresponding
representatives.
Table 2.2 Examples of anionic surfactants and corresponding chemical structures
Type Name and Structure

Carboxylates Sodium Stearate
(-COOM) C17H35-COO--Na+
Sulfonates Sodium Dodecyl Benzene Sulfonate (SDBS)

(-SO3M) C18H29-SO3--Na+
Sulfate Sodium Cetostearyl Sulphate

esters C16H33-O-SO3--2Na+
(-OSO3M)
By ionization, anionic surfactants increase the negative potential of the interface

between substance and granular dirt; enhances the repulsive force between
substance and dirt (Li and Ishiguro, 2016). Therefore, they have good effects on
removing granular dirt and preventing it from redepositing. It has been reported
that anionic surfactants, such as linear alkylbenzene sulfonates and alkyl sulfates,
35
are normally used in heavy duty detergents (Tai et al., 2018). Besides, it can also
be used as an emulsifier in different types of cosmetic creams, food industry and
pharmaceutical fields, such as Triethanolamine salt of dodecyl benzene sulfonic
acid (TDS), which showed the ability to stabilize the oil in water emulsion (Zhang
et al., 2017b).
Carboxylated salts are a subgroup of carboxylates, generally applied as cleansing

agents for hand wash, skin cleansers, shaving products and so on. The typical
product is soap which is metal fatty acid (Sharma, 2014). Sodium stearate, a very
common carboxylate anionic surfactant, is used in various commercial products
such as the brand LUSH and other brands’ soap product.
Sulfate surfactants (R-SO3M) are soluble in water and also have a good effect on
cleaning, emulsifying and foaming. The most common used products are alkyl
sulfates, alkyl ether sulfates, amide ether sulfates, and alkyl glyceride sulfates
(Tiwari et al., 2018). Properties of alkyl sulfates depend on their chain length and
the degree of branching of the hydrocarbon chain. Although presenting excellent
foaming properties and widely being applied in cosmetics, shampoos and skin
cleansers, relatively sever irritation of alkyl sulfates to human skin is nonnegligible
(Seweryn, 2018). Thus, even though alkyl sulfates are the most commonly used
type of anionic surfactants in various personal care products, their safety still
remains controversial. From this aspect, amide ether sulfates with magnesium
salts are promising alternatives showing good skin compatibility also with perfect
foaming ability, providing a potential surfactant for mild personal care cleansing
formulation (Ananthapadmanabhan, 2019). Compared to sulfate compounds,
sulfonates are suggested as anionic surfactants with less irritation. The linear alkyl
benzene sulfonate (LAS) is one of the most common used sulfonates (Tai et al.,
2018). Due to its better solubility, stronger decontamination and lower cost, LAS
plays an important role in detergent industry (Metian et al., 2019, Zígolo et al., 2020)
c) Non-Ionic Surfactants Surfactants
Non-ionic surfactants do not dissociate into ions in an aqueous solution. Their

hydrophilic moieties are made up of a number of oxygen-containing groups such
as ether group or hydroxyl group, which can form hydrogen bonds with water to
implement dissolution (Porter, 2013). The classification of non-ionic surfactants
depends on the type of their hydrophilic moiety. Common types are fatty alcohols,
ethoxylated fatty alcohols, alkylphenol ethoxylates, alkyl polygycosides,
36
ethoxylated fatty acids, alkyl carbohydrate esters, amine oxides and so on (van Os
et al., 2012).
Compared to ionic surfactants, non-ionic ones have a higher stability, which is not
susceptible to the existence of strong electrolyte inorganic salt (Deyab, 2019).
Thus, they are capable of being used in hard water due to the invulnerability of
Mg2+ and Ca2+. In addition, they exhibit excellent effect on emulsifying and
solubilizing, such as alcohols and esters that are commonly applied in personal
care industry. Another significant characteristic of non-ionic surfactants is their
good skin compatibility, maintaining their dominant application in products for
sensitive skin or baby skin. However, as weak foaming ability, non-ionic surfactants
are generally applied as emulsifier combing with ionic surfactants or other
stabilizers in formulations (Shubair et al., 2020, Zhang et al., 2018a).
In the formula of cosmetic cream, cetyl alcohol, stearyl alcohol, and glycerol
monostearate are normally used to help emulsify and stabilize the product. Besides,
Spans and Tweens are two common non-ionic surfactants that are reported to
perform much better than ionic surfactants (Koneva et al., 2017). Table 2.3
presents chemical structures of representative non-ionic surfactants.
Table 2.3 Example of non-ionic surfactants and corresponding chemical structures
Name and Structure

Cetyl
alcohol
Glycerol
mono-
stearate
Sorbian
mono-
stearate
(Span 60)
Polyethylen
e glycol
sorbian
mono-
stearate
(Tween 60)
37
d) Amphoteric surfactants
The hydrophilic group of amphoteric (zwitterionic) surfactants carry both of positive

and negative charge, such as RN+(CH3)2CH2COO-. They dissociate into anions
and cations based on the pH in aqueous solution (Guzmán et al., 2020, Ren et al.,
2017), thus neither like ionic surfactants that only adsorb on a positively charged
surface followed by changing it into cationic surface, nor the cationic ones that only
adsorb on a negatively charged surface and change it into positive one, amphoteric
surfactants are capable of adsorbing on both positively and negatively charged
surfaces without alter surface charge (Yarveicy and Haghtalab, 2018). Due to their
versatile properties, amphoteric surfactants are gradually applied in various
industries as an alternative to other type of surfactants. In recent, amino sulfonate
amphoteric surfactants attract attention among researchers due to their different
properties from conventional amphoteric surfactants that endowed by their unique
molecular structure consisting of one or more latent cationic centres and a small
range of isoelectric points (Ren et al., 2017). Ren et al. studied the mixed surfactant
system consisting of an amino sulfonate amphoteric surfactant (C12AS) that
carried two positive charges on its hydrophilic head group and a non-ionic
surfactant (OP-n), providing an agreement between critical micelle concentration
value of the system predicted using molecular-thermodynamic method and that
obtained from experimental work, with deviation due to hydrophilicity of the
micellization of nonionic surfactant (Ren, 2017). Also, different co-solvents are
applied to study the micelllization. More recently, a study carried out micellization
and interfacial properties analysis of system consisting of C12AS and different
types of alcohols of 70 g L-1, and further explained the electronic delocalization
structure of C12AS molecule presented at air-liquid interface or in bulk phase,
laying theoretical fundamental for their industrial applications (Huang and Ren,
2020).
38
2.1.3 Surfactant Behaviour in Water Solution
When surfactant molecules dissolve in aqueous solutions, surfactants experience
the process of self-assembly, and different structures are gradually formed, from
the initial monomers, to micelles and then liquid crystals.
2.1.3.1 Monomers
When dissolving in water, surfactant molecules align at the surfaces or interfaces

and form monolayers (Saad et al., 2019). Figure 2.3 shows diagram of the
alignment of different types of surfactant molecules at water surface.
Surfactants exhibits various surface or interfacial activities, where surface tension

reduction is the basic representative for identification of the presence of a
surfactant in the solution. Through surfactant molecules adsorbing and
accumulating at surfaces, some of water molecules in the surface are replaced by
surfactant molecules, and forces of attraction between surfactant and water
molecules are less than those between two water molecules, thus the contraction
force is reduced, leading to the reduction in the surface tension (Hantal et al., 2019).
From another aspect, the alignment of surfactant monomers at the surface reduces
the increased system free energy that caused by the dissolution of single
surfactant molecule in water, thereby maintaining the stability of the system
(Rehman et al., 2017).
air
a) cationic
water
b) anionic
c) Non-ionic
(Take Spans as an example)
d) Amphoteric
Figure 2.3 schematic diagram of different types of surfactant molecules alignment at

water surface
After monomolecular film at surface is saturated, surfactant molecules begin to
migrate into bulk liquid. The individual surfactant molecule that presented in the
39
volume phase of solution is known as monomer, which is in constant motion. Thus
the consistent exchange between monomers in solution and that aligned at the
surface help minimize interactions between water molecules and hydrophobic
groups of monomers in solution (Saad et al., 2019). Surfactant monomers are also
directly associated with the occurrence of skin irritation, through adsorbing on the
skin surface, interacting with the stratum corneum’s keratin protein, causing
denaturation of its α-helix structure (Morris et al., 2019b). Rhein et al. presented
the work showing that the severity of skin irritation was high during skin exposure
to surfactant solution before critical micelle concentration was achieved where
surfactants in volume phase are in the form of monomers (Rhein, 2017).
2.1.3.2 Micelles
Further increasing surfactant concentration in the solution results in the self-

assemble and aggregation of monomers. After a specific concentration, known as
critical micelle concentration (CMC) is exceeded, the aggregate structures, namely
micelles, are formed (Kelleppan et al., 2018). The value of CMC varies depending
on different surfactant types. The formation of micelles in solution is caused by
hydrophobic effect of surfactants interacting with water molecules with their
hydrophobic groups, displaying molecule clusters with hydrophilic groups towards
solvent molecules to protect hydrophobic moieties in the core from contacting with
solution (Ramadan et al., 2018).
The size of the micelle (micellar weight) is usually measured using light-scattering
method, and the number of associated molecules in the micelle could be calculated
by dividing micellar weight with surfactant molecular weight, which is determined
by surfactant molecular shape (Ritter et al., 2016). Within low concentration range,
the number of molecules only depend on the environment conditions. It has been
reported that, higher temperature leads to larger micelles of non-ionic surfactants,
whereas when the concentration of counter ions increases in solution, ionic
surfactant forms larger micelles (Hu et al., 2019).
Simple surfactant molecules with a single alkyl chain boned to a large polar head
group generally form spherical or oval micelles, with a packing factor (V/l·S) of less
than 1/3 (V represents for the volume of a single surfactant molecule, l indicates
molecular length and S is the surface area occupied by a molecule) (Manohar and
Narayanan, 2012). Change in concentration results in a micellar shape difference.
Take sodium dodecyle sulfate (SDS) as an example, when the concentration of its
40
solution reaches CMC (0.008 mol L-1), spherical micelles forms; when the solution
concentration increases to 10 times of CMC, rod-shaped micelles forms. Further
increasing the concentration of SDS solution will aggregate rod-like micelles
together to form hexagonally packed rod micelles, eventually forming palisade
layer micelles (Bang et al., 2010).
Depend on different type and structure of surfactants, the shape of micelles that
they form varies. Cylindrical micelles, showing packing factor of 1/3~1/2, are
formed by one-chained surfactants with a smaller polar group or ionic surfactants
in the presence of electrolyte (Xu et al., 2018). While, double-chain surfactants with
a large hydrophilic head group and flexible chains tend to form vesicles or
bimolecular structures (V/l·S = 1/2~1.0), and when a small head group is boned to
two chains that are stiff, planar or stretched micelles (V/l · S = 1.0) are formed
instead. Reverse micelles (V/l · S > 1.0) are formed if two-chained surfactants
connected with a small polar head group and large non-polar head group
((Faramarzi et al., 2017, Manohar and Narayanan, 2012).
2.1.3.3 Liquid Crystals
Liquid crystalline phases are usually involved in the surfactant system formulated
in structured fluids, where concentration of surfactant is high enough and micelles
aggregate together forming distinct structures (Jing et al., 2016). The shape,
structures and optical properties of liquid crystrals (LCs) are different from micelles.
As seen in Figure 2.4, where schematically presents the change of phase
conditions in the surfactant solution depending on the temperature and
concentration, surfactants of concentration higher than CMC are preliminary
crystal hydrates (insoluble) when temperature is below the phase transition
temperature Tc. Increasing the temperature over Tc leads to molecular soluble
phase gradually changing from spherical micelles to rodlike micelles with
concentration increased, further forming lyotropic LCs with the relocation and
aggregation of micelles (Guo et al., 2018).
41
Liquid-liquid
phase Separation
Cloud Point
boundary
Micelle Solution Liquid Crystal Formation
Area
Solid
Phase
Area
Temperature High
Molecular
Rodlike Middle Phase Cubic Lamellar
soluble phase Spheric Phase
(Hexagon Phase
Micelle Micelle
s form)
s
Krafft
Point
Tc boundary
Critical
Micelle Hydrated Solid (Lamellar Structure)
Concentrati
on (CMC)
Surfactant concentration High
Figure 2.4 Dependence of structure and phase formation on the surfactant
concentration and temperature, adapted from Guo et al., 2018
Liquid crystals (LCs) are matters in mesomorphic state which show the properties
of both liquid and solid (Guo et al., 2010). Phases of LCs that usually formed are
hexagonal LCs (H1 and H2), cubic LCs, nematic LCs, and gel phase (Lβ),
intermediate phase, lamellar phase (Lα) LCs.
Lamellar phase (Lα) lays fundamention for other structured phases, which involves
bilayers of surfactant molecules trapping abundant interlamellar water in between.
Lamellar phase is originated from coagels which is in a bilayer structure (trans-
zigzag) of hydrated solids at low temperature, then through a gel phase (Lβ) where
the temperature is over Tgel (gel phase transition temperature) but below Tc. Almost
no water exists between hydrophilic groups of coagels, while Lβ behaves the same
trans-zigzag structure but involves plenty of water in between. No alignment of
hydrocarbon chains is found in Lα, imparting lamellar phase more flexible and
easier to move, thus the viscosity in lamellar phase is lower than that in gel phase.
This property is applied in the formulation of cream products, where cooling helps
transfer Lα to Lβ achieving a more rigid product (Kim et al., 2020a).
LCs that self-assembled from surfactant molecules have been wildly used in food,
cosmetic, oil exploration and many other aspects related to people’s daily life,
which should be given more attention in the following research. Some researchers
have proved that the liquid crystalline phase in the cosmetic emulsion exerts the
42
advantage of stabling the emulsion and increasing its viscosity, through
surrounding dispersed droplets and acting as barriers to prevent coalescence, or
structuring the three-dimensional network in continuous phase to inhibit the
mobility of droplets (Racheva et al., 2018, Terescenco et al., 2018a, Chellapa et
al., 2016). LCs in emulsions are capable of combining with water, oil or other active
ingredients (Kulkarni, 2016), where combined water is generally in two forms when
LCs exist in an emulsion: interlamellarly fixed water (bound water) and bulk water
(free water). Bound water in emulsions tends to improve the moisturising properties
of the product, due to the low evaporation rate of interlamellarly fixed water (Savic
et al., 2005). Through analysing an alkyl polyglycoside stabilized emulsion, it has
been suggested that LCs were formed during the cooling stage and the lamellar
liquid crystal structure provided a good spreadability to the product (Terescenco et
al., 2018b). Besides, it has been reported that increasing the liquid crystal structure
in an emulsion helps reduce the transepidermal water loss, indicating the hydrating
effect of LCs on the emulsion (Zhang and Liu, 2013).
43
2.2 Bio-surfactants
Bio-surfactants (BSs), natural surface active agents, are synthesized by a range of
microorganisms. Possessing the similar structure as chemically synthesized
surfactants, their molecules also consist of both hydrophilic part which comprise
an acid, peptide cations or anions, mono-, di- or polysaccharides and hydrophobic
portion which comprise unsaturated or saturated hydrocarbon chains or fatty acids
(Silva et al., 2019c). Although most BSs are regarded as secondary metabolites,
they play a significant role in promoting microbial growth. BSs are secreted by
microorganisms, which in turn have the ability to enhance the consumption of
nonpolar and undissolved hydrocarbon substrates by microorganisms, through
adjusting the hydrophobicity of microbial cell surface (Yang et al., 2012).
BSs possess advantages over chemically synthesized surfactants in terms of low

toxicity, high biodegradability, high resistance to extreme environment and
excellent surface activity (Singh et al., 2019). Many BSs are claimed with
bactericidal activity, and this advantage is exerted in the activity of bacteria gliding
through interface and during the metabolic process tolerating environmental
extremes (Sana et al., 2018). The aggregate forming capacity, generally presented
with critical micelle concentration (CMC), is an indicator for surfactant efficiency.
Specifically, lower CMC value endows a surfactant powerful surface activity. To
some extent, CMC value of BSs are proved to be much lower than that of a few
chemically synthesized surfactants. In the study of Bharali et al., CMC of the BS
secreted by P. aeruginosa JBKI was around 540 mg L-1, and produced by strain
S5 was 96.5 mg L-1 (Bharali et al., 2014), which were lower than CMC value of
chemically synthesized surfactants such as sodium dodecyl sulphate (SDS) with
CMC of 2010 mg L-1 (Wang et al., 2018c), tetradecyl trimethyl ammonium bromide
(TTAB) with CMC of around 2000 mg L-1 (Whang et al., 2008), cetyltrimethyl
ammonium bromide (CTMAB) with CMC of 322 mg L-1 , Triton X-100 with CMC of
181 mg L-1 (Liang et al., 2014). B. subtilis ATCC 21332 produced surfactin was
capable of reducing surface tension to 27.9 mN m-1 with CMC value of 45 mg L-1
(Silva et al., 2010). Similarly, lipopeptides from Bacillus sp. ZG0427 showed high
surface activity by lowering surface tension of water to 24.6 mN m-1 with CMC of
50 mg L-1 (Hentati et al., 2019). Both of them are powerful than chemical synthesis
surfactant sodium lauryl sulfate which was reported as decreasing surface tension
to 56.5 mN m-1 (Hamed et al., 2020, Bhattachar et al., 2011). In addition,
researchers found the surface activity of BSs has close relationship with their
purification process (Silva et al., 2010). It has been studied that, crude
44
biosurfactants that produced by strain FLU5 decreased surface tension of ultra-
pure water from 72 to 34 mN m-1, while purified lipopeptides further lowered the
value to 28 mN m-1 (Hentati et al., 2019).
2.2.1 Classification of Biosurfactants (BSs)
Biosurfactants (BSs) are classified according to their microbial sources, chemical

structure, production method and applications. Basically five categories are
identified based on different structures: neutral lipids, glycolipids, lipopeptides,
phospholipids and polymetric bio-surfactants (Sobrinho et al., 2013, Shah et al.,
2016).
In addition, according to molecular weight, Rosenberg and Ron (Rosenberg and

Ron, 1999) divided the microbial surface active compounds into BSs (low
molecular weight) and bio-emulsifiers (high molecular weight). The low-molecular-
weight BSs, such as glycolipids, phospholipids and lipopeptides, are applied for
lowering the surface and interfacial tension, while the bio-emulsifiers, such as
polysaccharides, lipopolysaccharides proteins, are more capable of stabilizing
emulsions (Satpute et al., 2010). In Table 2.4, representative BSs examples are
listed (Shoeb et al., 2013).
Table 2.4 Classification of bio-surfactants, adapted from Shoeb et al., 2013
Type of BSs Examples

Rhamnolipids, Sophorolipids,
Glycolipids Mannosylerythritol lipids,
Trehalolipids
Low mass BSs Lipopeptides and Surfactin, Gramicidin S,
lipoprotein Polymyxin
Phospholipids, fatty acids,
Phosphatidyleth-anolamine
and Neutral lipids
Emulsan, Bio-dispersan,
Polymeric BSs
Liposan, mannoprotein
High mass BSs
Vesicles and fimbriae,
Particulate BSs
Wholecells
Glycolipids are one of BSs that have been deeply studied. Regarding to their
structure, long-chain fatty acid is linked by a covalent bond to carbohydrates,
where alkyl of fatty acid constitutes the hydrophobic group and saccharide makes
45
up the hydrophilic group (Caffalette et al., 2020). Not only possessing excellent
surface activities, glycolipids also have various functions such as antioxidant,
emulsification, foaming, washing, dispersion and antistatic, which makes them as
a promising alternative to chemically synthesized surfactants in various fields such
as food, pharmaceutical and cosmetic industries (Onwosi et al., 2020).
2.2.2 The Production and Extraction of Biosurfactants (BSs)

BSs can be produced via three methods: microbial fermentation, enzymatic
synthesis and natural biological extraction. Most biological surface active
compounds are secreted by bacterial, yeast or fungus. Different microorganisms
produce different types of BSs under different conditions, and researches have
screened different types of microorganisms that are capable of producing BSs with
various structures (Nayarisseri et al., 2018, Wang et al., 2017, Hassan et al., 2018,
Кайырманова et al., 2020). Compared to microbial fermentation, enzymatic
synthesis is an organic reaction where exogenous enzymes are used to catalyse
bio-surfactant synthesis. Through this production process, BSs of simplified
structures and single varieties are produced due to the selectivity of enzyme
(Enayati et al., 2018, Marcelino et al., 2020, Torres et al., 2020). Natural biological
extraction refers to the extraction of effective BSs from natural bio-ingredients. To
exemplify this, phospholipid and lecithin are also BSs that derived from egg yolks
and a soybean. However, due to the limitation of raw materials, this method is
hardly applied in a large scale production (Wan et al., 2017).
At present, mainly due to high cost of production and purification of BSs, it cannot
deny that the replacement of chemically synthesized surfactants by microbial BSs
that produced through fermentation for commercial use is still difficult, although the
efficacy of BSs in lab-scale and small-volume production has been extensively
manifested. It has been reported that the high yield of rhamnolipids is greatly
determined by the usage of hydrophobic substrates which is relatively more
expensive than those hydrophilic ones (Varjani and Upasani, 2017), indicating the
high cost of raw materials for their large-scaled production. Thus as stated
previously, more recent researchers started to carried out fermentation with
renewable and inexpensive substrates for strain cultivation (Dalili et al., 2015). In
addition to that, downstream process contributes the most to the higher operational
cost of BSs production, due to the sometimes their low concentration and unique
amphiphilic nature with various structure making it difficult for separation them from
medium broth (Moutinho et al., 2020). Chemical solvent extraction and
46
vaporization are the most widely used technique that reported to help reach the
maximum BSs recovery rate, but this conventional method is high-priced and
energy-intensive, also with a tendency to cause irreversible damage to producing
cell (Dolman et al., 2017). In addition, chemical solvent extraction is not feasible
for the commercial-scale production of BSs due to the large productivity
benchmark of no less than 2 g L-1h-1 is required (Roelants et al., 2019b). As an
alternative path to that, a reverse extraction was recently proposed for
rhamnolipids separation where alkaline aqueous solution (equimolar NaOH to
rhamnolipids) was used for their back extraction, achieving 97% of total
rhamnolipids recovery in aqueous phases (Invally et al., 2019). Integrated
separation methods are of great interests as their ability for higher productivity and
yield, such as gravity separation, foam fraction and membrane separation.
Gravity–based integrated separation method is emerging that help overcome
drawbacks of low production and costly extraction process. As suggested in the
study of Dolman et al., where a fermentation of highly viscous sophorolipids
production yielded volumetric productivity of 0.62 g L -1h-1, the integrated recovery
method controlled oxygen limitation during production and alleviated inhibition for
genes biosynthesis caused by continuously produced sophorolipids with high
viscosity, thereby enhancing productivity and yield (Dolman et al., 2017). Moreover,
the technique was successfully applied in a pilot scale working volume of
fermentation (30 L), indicating the possibility of wider application of in situ gravity
separation method in BSs extraction process. Compared to this, a two-stage
separation system was proposed by Zhang et al., where applying a novel
bioreactor with dual ventilation pipes and dual sieve-plates in the fermentation
achieved higher productivity of 1.59 g L-1h-1, but this configuration obviously
increased the cost (Zhang et al., 2018b). Other methods such as crystallization
and precipitation, combining flotation, standing, rotary vacuum filtration and
centrifugation to remove cell pellet are also reported in literatures. Acid
precipitation is frequently used for rhamnolipids recovery from broth medium,
followed by solvent extraction and chemical evaporation. As stated in a study,
applying alcohol precipitation for biopolymer removal prior to normal acid
precipitation for rhamnolipid extraction increase the purity from 66% to 87% before
further extraction process (Invally et al., 2019). More recently, the integrated foam
fractionation method wass widely studied especially for rhamonolipid extraction
(Jiang et al., 2020), as the technology has the ability to alleviate foaming problem
specifically liquid foam during fermentation process by continuous isolating
rhamnolipids from broth medium (Heyd et al., 2011), which could be promoted by
47
introducing foam breaker with perforated plates for further enhancing foam
destabilization (Liu et al., 2013). But more efforts are needed for its large-scale
application due to the complexity of the configuration. However, extraction
methods are established specific to the type and nature of BSs. For example,
flotation and standing are not applicable for separating BSs that produced by
bacterial cell (Daverey and Pakshirajan, 2010). Regarding to new technologies for
BSs extraction, ultra-filtration is one of the most effective ones. Using ultrafiltration
membrane with molecular weight cut-off (MWCO) of 10000 (YM210) to extract
rhamnolipids, the yield reaches 92%. Also the yield of 80% and 58% was obtained
when using ultrafiltration membrane with MWCO of 30000 (YM230) and 50000
(YM250) respectively (Pereira et al., 2012).
2.2.3 Characterization of Biosurfactants (BSs)

BSs could be characterized by several conventional methods, such as thin layer
chromatography (TLC), mass spectrometry (MS) and high performance liquid
chromatography (HPLC), in order to study their structures and properties (Ndlovu
et al., 2017, Ankulkar and Chavan, 2019, Ong, 2017).
Mass spectrometry is usually applied to identify the structure of different BSs. The
principle of this technology is that the chemical species are ionized and then the
ions are classified according to the mass-to-charge ratio. Conducting mass
spectrometry measurement, the structure of dirhamnolipids (Rha-Rha-C10-C10)
was identified from the Rhamnolipid, where the rhamnolipid was extracted using
2:1 chloroform/methanol solvent mixture (Rahman et al., 2002). High performance
liquid chromatography (HPLC) is proved to be an effective method used for the
detection of BSs and even their separation. This measurement system is made up
of mobile phase, stationary phase and a detector. The commonly used detectors
are evaporating light scattering detectors, UV, refractive index and so on. During
the measurement, the sample is carried by mobile phase flowing over the
stationary phase which is a solid, where components are separated and pass
through the detector successively. Then the detector records the data and gives
the response in terms of each peak on a chromatogram. For determining
rhamonolipids structure, HPLC measurement was carried out, where the
Supelcosil LC-18 column was used with a CH3CN/THF (55/45 v/v%) mobile phase
at the flow rate of 0.75 ml min-1. The result was detected through a UV detector at
the wavelength of 225 nm. The following anthrone analysis compensated for the
48
inaccurate result from HPLC, before which Rhamnolipids were acid hydrolyzed to
avoid the presence of carbon substrates (Chayabutra and Ju, 2001).
2.2.4 Application of Biosurfactants (BSs) in Various Fields

BSs have a great potential in application in a wide range of fields, such as
petroleum exploitation, pharmaceuticals industry, cosmetic industry, food industry
and agriculture (Kiran et al., 2017, Patowary et al., 2018, Santos et al., 2017,
Ribeiro et al., 2019, Adu et al., 2020, Xu and Amin, 2019, Bai and McClements,
2016).
In the field of oil recovery, microbial- enhance oil recovery (MEOR) is proposed as
a cost-effective and eco-friendly technique, in replacement of conventional
enhanced oil recovery (EOR) that heavily consumes chemical synthesized
surfactants resulting in relatively high cost (Ribeiro et al., 2020). MEOR is
implemented by introducing indigenous or exogenous microorganisms in
reservoirs for the production of metabolites (BSs) that are capable of demulsifying
and separating oil-water mixed system, in order to optimize oil production from
existing reservoirs and recycle waste crude oil for reprocessing or energy recovery
in petroleum industry (Yang et al., 2020). Cultivating strain Azotobacter vinelandii
AV01 was reported to produce BSs which showed ability of emulsifying the crude
oil up to 90%, leading to a 15% increase in the recovery efficiency of crude oil
(Helmy et al 2010). Similarly, Salehizadeh et al. have done another research and
found that the BSs produced by Alcaligenes faecalis MS103 showed 10.7%
increase of the crude oil recovery efficiency (Salehizadeh and Mohammadizad,
2009). More recently, rhamnolipid secreted by different microorganisms showed
excellent performance in oil recovery application. The efficacy of MEOR by
rhamnolipids was evaluated, through cultivating Pseudomonas aeruginosa that
isolated from artificially contaminated soil with crude oil, achieving an optimal result
that rhamnolipids with concentration of 100% (higher than its CMC which is 127
mg L-1) effectively recovered 11.91 ±0.39% of oil with API gravity of 21.90 (Câmara
et al., 2019).
Although lots of efforts have been made to screen aerobic functional

microorganisms for their ex situ application in MEOR and investigate the oil
recovery efficiency of ex situ production of BSs (Haloi et al., 2020, Saravanan et
al., 2020), where BSs are externally produced and then injected into oil reservoir,
in situ application of BSs in MEOR is proposed to be more beneficial compared to
that, for their cost effective without transportation and complex configurations for
49
BSs production (Du et al., 2019). But this process is relatively disadvantageous if
aerobic microorganisms are used, due to additional air pumping in the reservoirs
leading to higher cost, poorer operation and lower safety (Zhao et al., 2015). Thus
microorganisms that are capable of producing BSs under anoxic conditions are
required. Zhao et al. identified Pseudomonas aeruginosa SG that isolated from
Xinjiang oil field as a promising strain, that could produce rhamnolipid under anoxic
condition by consuming various type of organic substrates. In their study, an extra
8.33% of original crude oil in the core was extracted through in situ production of
rhamnolipid by the strain (Zhao et al., 2015), but the production was inhibited by
H2S which is produced from sulfate-reducing bacteria (SRB) widely existing in the
petroleum industry. Thus introducing a recombinant Pseudomonas stutzeri Rhl
helped effectively remove H2S and at the same time produce rhamnolipids under
S2- stress below 33.3 mg L-1 (Zhao et al., 2016).
Glycolipids possess strong medicinal activity, which can be used to prepare tablets
including semi-synthetic penicillin and macrolide antibiotics. This can increase the
load of drug in blood per unit time, thereby facilitating the drug absorptivity of
digestive system (Nguyen et al., 2010). BSs also plays an important role in
bioremediation. The contamination of industrial waste water, solid wastes,
pesticides, heavy metal and other pollution sources has become increasingly sever
to water body and soil, and BSs produced by microorganisms help improve the
hydrophilicity and bio-accessibility of hydrophobic compounds, which displacing
pollutants into environment with continuously degradation (Kreling et al., 2020).
In food industry, BSs favours for their application as antimicrobial and anti-biofilm
agents, foaming agents, wetting agents, emulsifiers, food additives and so forth
(Rai et al., 2019). The emulsifying activity of BSs has been extensively evaluated,
with different oils or hydrocarbons. In a study of sophorolipids production from
yeast strain Candida albicans SC5314 and Candida galabrata CBS138, their
emulsifying ability was determined against castor oil with the emulsification index
of 51% and 53% separately for C. albicans and C. glabratag, providing their ability
as food emulsifiers. In addition, the stability of sophorolipids were confirmed within
a wide range of pH (2~10) and temperature (4~120 °C) as well as salt
concentration (2~14%) (Gaur et al., 2019). In addition, lipopeptide BSs and
rhamnolipids were confirmed to form stable emulsions with various oils such as
soybean oil, coconut fat, and linseed oil (Nitschke and Pastore, 2006), showing
high potential of application in food industry. Similarly, a glycolipid that produced
by cultivating marine bacteria Kluyveromyces marxianus FRR1586 on lactose-
50
based medium was able to emulsify corn oil in water and stabilize the system at
pH varying from 3 to 11 and salt concentration varying from 2 to 50 g NaCl L-1
(Fonseca et al., 2008). Marine strain, Enterobacter cloacae was identified for
producing bioemulsifier which showed excellent ability to enhance viscosity of
acidic solution, confirming its application in food industry (Dubey et al., 2012). In
addition to emulsify and stabilize the system, BSs could be food additives for
improving the texture and consistency of dairy products by preventing
aggregations of fat droplets. In the study of Mnif et al., more cohesive texture of
dough was obtained when adding a lipopeptide BS in the formulation than that
formulated with soy lecithin, resulting in a higher quality of bread (Mnif et al., 2013).
Similar result was also achieved when incorporating sophorolipids in bread
formulation, where the bread volume was increased and desirable appearance
was presented. Owning antibacterial ability, BSs are capable of keeping food safe
to use. Lipopeptide BSs, including lichenysin, pumilacidin, iturin, gramicidin S and
polymyxins that produced by Bacillus sp., were proposed in large amount of
studies for their application in foods (Coronel León et al., 2016, Saggese et al.,
2018, Kim et al., 2020c, Wenzel et al., 2018, Nirosha et al., 2016).
Apart from above mentioned functions of BSs in food industry, surfactants of

microbial origin could be alternatives for chemical surfactants in the formulation of
nano-sized delivery system (Nirosha et al., 2016), the molecules of that self-
aggregate to form unique structures trapping hydrophobic or hydrophilic
compounds within the structural core, thereby forming microemulsions,
nanoparticles and liposomes. It has been studied that sophorolipids and
rhamnolipids were capable of forming biocompatible microemulsions when mixing
with lecithins in system (Nguyen et al., 2010). Rhamnolipids was demonstrated to
facilitate partition of ω-3 polyunsaturated fatty acids for preparing emulsion-based
fish oil delivery system (Liu et al., 2016). In another study, for developing drug
delivery system of vitamin E, a self-emulsifying system of high quality was
established when having surfactin in the system, showing higher emulsification
efficiency, dissociation rate and oral bioavailability (Nirosha et al., 2016), which
indicates the merits and potential of applying BSs in food industry.
2.2.5 Potential Cosmetic-applicable Biosurfactants (BSs)

The application of surfactants is significant in cosmetic industry, especially for
biosurfactants, owing to their low toxicity, antibacterial property, moisturising
capacity to skin. The mechanisms of interaction between surfactants and skin have
51
been studied. When surfactant monomers damage the secondary and tertiary
structure of stratum corneum (SC) through adsorbing on skin surface, SC may
expose sites for binding water molecules and become swelling. Also SC keratin
protein may be degraded and washed from the skin, as well as solubilizing lipid of
the intercellular cement within the SC. Longer-term interaction may lead to
penetration of surrounding stimulus such as chemical compounds and pathogens
to deeper SC layers for inducing living cells’ immune response, showing as topical
red on skin or itching (Seweryn, 2018). Researchers found that both surfactant
monomers and micelles exhibited irritation to skin, as the irritation activity was
detected when the CMC was exceeded. Some of them attributed this to the
disintegration of micelles into monomers after contacting with skin, while other
researchers claimed it may because smaller-sized submicelles were formed
(Morris et al., 2019b). Also, when the surfactant concentration was over CMC,
significant increase of skin irritation effect caused by sodium dodecyl sulfate (SDS)
was witnessed, where micelles that formed were small to easily penetrate into hair
follicle orifices, while the lower increase was presented when ethoxylated sodium
dodecyl sulfate was involved (Cohen et al., 2016).
However, opposite to those synthetic surfactants, BSs of natural origin comprising

of sugars, lipids and proteins that are compatible with skin cells membrane. Thus
they are not only pose no threat to living organisms, but they generally have
antioxidant and antibacterial effects on skin, exhibiting promising efficacy for
application in skin care products. BSs of plant origin, such as phospholipids, have
various benefits in cosmetic product, such as improving the dispersibility of
cosmetics, maintaining skin moist and adjusting acidity of skin (van Hoogevest and
Fahr, 2019). And sucrose ester takes advantage in improving washing property of
cosmetics, increasing skin smooth and tender (Laville et al., 2020). As for microbial
BSs, Vecino et al. evaluated the antimicrobial and anti-adhesive activities of
glycolipopeptide that produced by lactic acid bacteria (“Generally Recognized As
Safe” by the American Food and Drug Administration), showing that approximately
50 mg mL-1 glycolipopeptides exhibited antimicrobial activities against
Pseudomonas aeruginosa, streptococcus agalactiae, Staphylococcus aureus,
Escherichia coli, Streptococcus pyogenes and Candida albicans (Vecino et al.,
2017). Similarly, another study also investigated the cell-bound glycoprotein that
produced by Lactobacillus agilis CCUG31450, 5 mg mL-1 of which inhibited growth
of Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus
agalactiae (Gudiña et al., 2015). In addition to that, the antimycotic activity of
52
sophorolipids, that obtained from Rhodotorula babjevae strain YS3, against
dermatophytes was in vitro and in vivo evaluated, indicating that the biosurfactant
effectively treated dermatophyte by interacting with the cell membrane of pathogen
and disturbing the membrane integrity, although only one resistance strain, T.
mentagrophytes, was investigated (Sen et al., 2020).
Glycolipids may be the most frequently used type of biosurfactants in the

formulation of personal care products, due to their multifunctional properties.
Generally, they consist of aliphatic acids or hydro-xyaliphatic acids and a
carbohydrate group (Lukic et al., 2016). Two attractive glycolipids, sophorolipids
(SLs) and mannosylerythritol lipids (MELs), that has potential in skin care products
formulation will be introduced in details.
2.2.5.1 Sophorolipids (SLs)
SLs are non-ionic biosurfactants (BSs) that having various effects on personal care
products, such as emulsifying, detergency, wetting, defoaming and most
significantly biocompatible to human with low toxicity, exhibiting high potential of
application in cosmetic industry. Sophorolipids (SLs) is suggested to be affinitive
with human skin, which is capable of acing as a humectant to keep skin moist; also
it can be used in the manufacture of detergent. It has been reported that SLs of 1
mol L-1 are highly affinity with skin which can be used as an excellent moisturizer
(Pekin et al., 2005). A Japanese company has applied SLs in various cosmetic
products, such as conditioner, emulsion and lipstick, as a moisturizer, using Sofina
as its trade name. Also, the fermentation procedure of SLs has been studied by
this company and industrialized (Mujumdar et al., 2017). From another Japanese
company Saraya, SLs have also been commercially produced and used as
cleaning agents in cosmetics, catering and dry cleaners (Kim et al., 2020b). In
addition, SLs also play a role in the production of baby skin care products by a
France company named Soliance (Baccile, Nassif et al. 2010).
2.2.5.1.1 Structures and Properties of Sophorolipids (SLs)
The SLs is mainly produced by yeasts, which is naturally a mixture of SLs

molecules with different structures. These SLs molecules all consist of hydrophobic
and hydrophilic moieties. Among them, hydrophilic part is sophorose which is the
diglucose combined with belta-1, 2 glycosidic bond, and hydrophobic group is
made up of saturated or unsaturated long chain omega- (or omega-1) hydroxylated
fatty acid (Gaur et al., 2019). These two parts is connected by belta-glycosidic bond.
53
The structures of SLs molecules are mainly varied in two aspects, which are
acetylation and lactonization (Figure 2.5). The diglucose hydrophilic part of SLs
molecules may either contain acetyl groups at the 6’ and/or 6’’ positions or not; the
carboxylic end of fatty acid of hydrophobic group can either be free acidic form
(open form) or internally esterified (closed ring) at the position of 4’’, 6’ or 6’’
(carboxylic group of fatty acid esterified reacts with hydroxyl group at the 4’’, 6’ or
6’’). Other differences of structures are the hydrophobic group, including length of
carbon chains (generally contain 16 or 18 carbon atoms), saturation and the
position of hydroxylation (Kim et al., 2020b). SLs with various structures show
different physicochemical properties. Lactonic SLs possess better surface
properties and antibacterial activities, while acidic forms show better foamability
and solubility. The lactonization decreases the atomic free rotation angle, thereby
easily forming the transparent crystal. However, acidic SLs tends to exist in the
form of viscous oil. (Van Bogaert et al., 2011). Besides, although the introduction
of acetyl groups decreases the solubility of SLs, the antiviral property will be
enhanced.
Lactonic Sophorolipid
54
Acidic Sophorolipid
R1=R2=Ac Diacylated SLs

R1=R2=H Non-acylated SLs
R1=H, R2=Ac; R1=Ac,R2=H Monoacylated SLs
Figure 2.5 General structure of sophorolipids (SLs)
It has been reported that the surface tension in water can be reduced from 73 mN
m-1 to 30~40 mN m-1 by SLs, and the CMC value was 40~100 mg L-1. In addition,
CMC value of SLs has a correlation to carbon chain length of fatty acid. Specifically,
the longer carbon chains the SLs had, the lower the CMC value it presented
(Minucelli et al., 2017). In the study by Zhang et al, where SLs akyl (methyl, ethyl
and butyl) esters were synthesized by chemically modification of SLs, CMC value
was reduced by halving the introduction of one –CH2 to the akyl group of SLs akyl
ester. This also manifests that the biodegradability is enhanced with the increase
of carbon chain length of molecules of SLs derivatives (Zhang et al., 2004). Shin
et al. also found that the SLs methyl ester containing oleic acid (C18) is more
difficult to biodegrade than that containing erucic acid (C22) (Shin et al., 2010).
2.2.5.1.2 Production of Sophorolipids (SLs)
When cells enter stationary phase, SLs begin to form, generally after being
inoculated 24~48 hours. During stationary phase of cells, SLs are well produced.
It has been reported that 10 days is an optimal value for the whole process for SLs
production (Van Bogaert et al., 2011). As extracellular glycolipids, SLs are
produced by a number of microorganisms, includes Candida apicola, Starmerella
bombicola, Torulopsis bombicola, Candida bombicola, Candida Batistae, Candida
stellate, Candida riodocensis, where Candida. bombicola is the most wildly applied
which produces SLs of the highest yield (Konishi et al., 2018). Researchers have
also discovered novel producing strains, such as Candida keroseneae GBME-
55
IAUF-2, Issatchenkia orientalis, Meyerozyma guilliermondii YK32 and Candida
rugose, for SLs production (Roelants et al., 2019a, Ganji et al., 2020), through
screening surface active ingredients in environmental isolates using different
methods such as haemolytic activity, drop-collapse assays, and mostly applied
biochemical data analysis. But misidentification occurred of producing strains when
assigning names of novel described BSs producers only according to biochemical
data. As reported, a novel SLs producer named Wickerhamiella domercqiae var.
SL in the study of Chen et al. was identified based on BIOLOG analysis, showing
excellent SLs productivity, while it was realized that no dissimilarity of their whole
genome sequences compared to previously described S. bombicola sequences
(Ma et al., 2014, Li et al., 2016). Apart from that, it was suggested that molecular
techniques can applied for yeast species identification (Silva et al., 2019b). For
instance, Nwaguma et al. isolated BSs producing yeast from oil palm and Raphi
palm, identifying six promising producers as Candida haemulois SA2, Pichia
kudriavzevii SA5, SB3, SB5, SB6, and SB8, using molecular and phylogenetic
evolutionary methods (Nwaguma et al., 2019).
a) Substrates
Two types of substrates are needed in the production of SLs: hydrophilic (glucose
or sugar-rich molasses) and lipophilic substrates (oil, alkanes, fatty acids or fatty
esters), but SLs can still be produced if both substrates are not simultaneously
contained in the medium, even though the combination results in the highest yield
(Van Bogaert et al., 2014). As found in a study, the production from the media
containing both glucose and Turkish corn oil (40 g L-1) was higher than that
containing Turkish corn oil as the sole carbon source (30 g L-1) (Pekin et al., 2005).
Also, when the concentration of carbon source decreased, the SLs may be
decomposed to supplement the strain with carbon source. For instance, S.
bombicola restarted produce fatty acids for SLs production, consuming more time
and energy compared to the process where hydrophobic substrates initially added
(Shah et al., 2017). Based on this, controlling the concentration of hydrophilic and
hydrophobic carbon sources has a crucial effect on improving the SLs yield.
Glucose of 100 g L-1 is generally used as hydrophilic carbon source in the
fermentation medium for SLs production, which is also suggested as the best value.
Less SLs were produced when cultivating cells on 200 g L-1 or 300 g L-1 glucose
(Joshi-Navare et al., 2013). Some hydrophilic carbon sources have been tried,
such as sucrose, galactose and lactose, deproteinized whey as the replacement
56
of glucose, but the yield of SLs was relatively lower than that with glucose (Jadhav
et al., 2019).
The hydrophobic carbon source can be alkane, fatty acid or oil. Through comparing
the influence of different hydrophobic carbon sources to SLs production, fatty acid
methyl esters or ethyl esters that derived from vegetable oils were superior to the
corresponding vegetable oils, and both of them had an advantage over alkanes
(Shah et al., 2017, Ma et al., 2020). Oleic acid is a kind of free fatty acid with
specific carbon length, which can achieve a relatively high SLs yield (Solaiman et
al., 2007). Due to vegetable oil containing the oleic acid which is the most suitable
for SLs formation, it can facilitate the production. Rapeseed oil is an ideal vegetable
oil substrate (Kim et al., 2009). The effect of alkanes on SLs production depends
on their carbon length. When using hexadecane (C16), heptadecane (C17) or
octadecane (C18) to cultivate stains, the production of SLs is higher than using
other hydrophobic carbon sources. The possible reason for this may be that, they
can directly transform into hydroxyl fatty acid and then integrated into SLs
molecules (Ma et al., 2020, Habibi and Babaei, 2017, Ashby and Solaiman, 2019).
This direct conversion mode of alkanes obviously affects the composition of fatty
acid chain in SLs mixtures. Hydrophobic substrates also have an influence on the
SLs composition. There is an equilibrium of the proportions of lactonic and acidic
forms in SLs mixture, which is affected by substrate species, especially the type of
hydrophobic carbon sources (Shah et al., 2017, Konishi et al., 2018). To exemplify
this, 85% of lactonic forms SLs was produced when using n-hexadecane as the
substrates, while only 50% of that was produced when soybean oil was used
(Callaghan et al., 2016). Also, when using fatty acid esters or the by-product of
biodiesel as the substrates, more acidic SLs were produced
Nitrogen source is also required for the production, where the yeast extract of 1~5
g L-1 is often used. However, that the time for entering the stationary phase should
be determined by the limitation of nitrogen, for instance, higher carbon nitrogen
ratio (C/N ratio) ensured the SLs formation by specific strains (Callow et al., 2016,
Da Costa et al., 2017, Sanchuki et al., 2017). Other compositions in medium, such
as non-essential nutritional source, citric acid, buffer substances and inorganic ions
(Mg2+, Fe3+ and Na+), are sometimes included in the medium for strain cultivation,
and appropriate amount help enhance SLs production.
b) Biosynthesis Pathway
57
In the biosynthesis pathway of SLs production, glycolipid and fatty acid chain are
mainly involved. Target yeasts begin to synthesize SLs from the hydroxylation of
fatty acid. Fatty acid is obtained either directly from media or from hydrolysis of
triglyceride or fatty acid methyl ester by extracellular lipase (Ma et al., 2020).
Another indirect method to achieve fatty acid is cultivating yeast cells with a
medium containing alkane. Candia. bombicola is able to growth in the media that
has alkane as the only carbon source. This means that, intracellular enzyme that
catalyses the terminal oxygenation of alkane stepped oxidizes alkane to
corresponding fatty acid (Yang et al., 2019). When no hydrophobic carbon source
is provided in the media, fatty acid will be formed through de novo synthesis which
starts from acetyl-coenzyme A (COA) derived from glycolysis pathway. The de
novo synthesis has been confirmed by the related research about Cerulenin which
is the inhibitor of fatty acid synthesis (Van Bogaert et al., 2008).
After fatty acid transfers to hydroxyl fatty acid, two active UDP-glucose molecules
are added to the hydroxyl fatty acid consecutively. Glucose in medium is not
directly used for SLs production, but only go through glycolysis path to complete
gluconeogenesis which is necessary in the formation of SLs (Minucelli et al., 2017).
This explains that the head group of SLs will not be altered by changing the
provided different types of saccharides; also SLs can still be produced even if
under the condition of no glucose or other polysaccharide involved that can
degrade to glycolipid (Saerens et al., 2011).
c) Fermentation Parameters
The production of SLs is affected by various fermentation parameters. Generally,

the optimal growth temperature of C. bombicola is 28.8 ºC. However, 21 ºC was
determined to be the optimal temperature (Elshafie et al., 2015, Göbbert et al.,
1984). Most widely used temperature in literatures ranging from 25 ºC to 30 ºC,
and no big difference of SLs yield was witnessed. Nevertheless, the biomass
increment is lower and the utilization of glucose is higher when cultivating the cell
at 25 ºC (Pulate et al., 2013).
Different pH value in broth can influence the type of SLs that produced. It has been
found that, when the pH value is 3.5, lactonic SLs was the major product from C.
bombicola cultivation (Ciesielska et al., 2016). In addition, it has been discovered
that C. apicola mainly produced acidic form of SLs when the pH value was lower
than 2.0, and when adjusting the pH value to 3.0, more lactonic SLs were formed
(Konishi et al., 2018). The pH value of fermentation broth decreases sharply during
58
exponential phase. In order to maintain the cell growth and increase SLs yield,
NaOH solution frequently added into the broth for maintaining pH value at 3.5
(Delbeke et al., 2016). In addition, lower pH values that maintained during
fermentation process can reduce the potential of bacterial contamination.
Dissolved oxygen is an important factor that will influence SLs production. Due to
the highly viscous of SLs that continuously produced during fermentation tending
to hinder oxygen dissolving and inhibit cell growth, much longer time will induce
lower production effectiveness for a single batch of fermentation. Apart from that,
the cell growth during exponential phase and the biosynthesis of SLs will be
affected, where low oxygen supply has potential for limiting biological activity, but
no effect on fermentation if a threshold was exceeded (Almeira et al., 2015). A
study manifested that the optimal oxygen supply was between 50 to 80 mM O2 L-1
H-1 in terms of oxygen transfer rate (Guilmanov et al., 2002). In the study of Pedro
et al., the optimal aeration rate was investigated as 0.30 L kg -1 min-1, achieving an
optimized solid-state fermentation process for SLs production by cultivating
Starmerella bombicola on a residual oil cake substrate, also no further increase of
SLs yield with higher aeration rate supplied as the threshold of oxygen air flow was
exceeded (Jiménez-Peñalver et al., 2016). SLs containing saturated fatty acid will
be mainly achieved when lower oxygen dissolved in the broth (Elshikh et al., 2017).
d) Extraction and Purification
Due to the density difference between SLs and the media; it can be preliminary
separated from media by decanting after natural sedimentation or centrifugation,
as proposed gravity-based separation method in the study of Dolmann et al
(Dolman et al., 2017). Solvent extraction is a frequently used method for SLs
further purification, usually with the help of ethyl acetate, followed by vacuum rotary
evaporation to get rid of the solvent in the product (Ma et al., 2011). Many methods
have been conducted to separate SLs into different specific structures according
to their physiochemical properties. As reported, lactonic SLs was soluble in ethanol
and the solubility was increased with the temperature rising, while the acidic SLs
was slightly soluble in ethanol and the solubility may not change with temperature
(Ashby and Solaiman, 2019). Thus the lactonic SLs can be extracted firstly by
dissolving the SL product in ethanol at high temperature, and then cooling down
the solution to crystallize lactonic forms. But this method has the potential of losing
lactonic SLs in ethanol. Based on different solubility in water, especially high pH
water, where acidic form is soluble and lactonic SLs is insoluble, Hu et al.
separated acidic and lactonic forms in phthalates and phosphate buffers. This
59
method has the advantages such as no use of organic solvent and relatively high
recovery (Hu and Ju, 2001).
2.2.5.2 Mannosylerythritol lipids (MELs)
Mannosylerythritol lipids (MELs) not only has favourable emulsifying capacity,

biodegradability and other high surface activity, it also has antimicrobial activities
such as inducing cell differentiation and cytometaplasia, and strong coordinate
ability with glycoprotein (Banat et al., 2010). Thus it has great potential for applying
in the field of cosmetics, food and pharmaceutical industry.
2.2.5.2.1 Structures and properties of MELs
MELs generally contain 4-O-β-D-mannopyranosyl-erythritol as the hydrophilic

head group, attaching to fatty acid chains as hydrophobic group. There are four
different structures of MELs, according to the number and position of the acetyl
group on mannose or erythritol, it can be classified as MEL-A (diacetylated), MEL-
B/MEL-C (monoacetylated at C6 position and C4 position respectively) and MEL-
D (deacetylated) (Niu et al., 2017). The structure of MELs is schematically shown
in Figure 2.6. The structure includes three moieties: mannopyranosyl (in red circle),
erythritol (in orange circle) and acyl chain (in blue circle) (Niu et al., 2019).
60
CH
3
CH
2
H C OH
CH
3
1
OR H C OH
2
CH
2
R O
O O
MEL-A: R1=R2=Ac (CH3CO); MEL-B: R1=Ac, R2=H; MEL-C: R1=H,

R2=H; MEL-D: R1=R2=H
Figure 2.6 General structure of mannosylerythritol lipids (MELs)
Different strains tend to produce MELs with different structures. Ustilago maydis
DSM4500 mainly produce MEL-A. Pseudozyma antarctica tends to produce the
mixture of MEL-A, MEL-B and MEL-C, where MEL-A dominate the product
accounting for 70% (Saika et al., 2018a). In addition, cultivating same strain under
different fermentation conditions leads to the synthesis of different types of MELs.
Pseudozyma parantarctica, Pseudozyma Antarctica and Pseudozyma rugulosa
produced MELs (including MEL-A, MEL-B and MEL-C), while when consuming 4
wt% olive oil and 4 wt% mannose as carbon source, a new surfactant was
synthesized named MML (Morita et al., 2009a).
Due to the difference in chirality of carbon atom in erythrityl, a variety of diversity

of MELs structures exist including many kinds of diastereoisomers. A new type of
extracellular MELs diastereomer has been reported by Fukuoka et al. through
cultivating Pseudozyma crassa. In the study, the structure of the new MELs is
similar to that of MEL-A, MEL-B and MEL-C, but the stereostructure of erythritol is
totally different which is 4-O- β-D-mannopyranose-(2R,3S)-erythritol. Also,
compared to the general medium resulted fatty acid chain, partial short fatty acid
chain (C2 or C4) and long fatty acid chain (C14, C16 or C18) are attached to
mannosyl moiety, leading to different properties of the product (Fukuoka et al.,
2008). By cultivating Pseudozyma antarctica and Pseudozyma rugulosa in the
consumption of soybean oil as carbon source, Kitamoto et al. produced MELs with
high hydrophobic property, containing three acetyl group (Morita et al., 2013).
61
Similar structure was also reported, where Pseudozyma churashimaensis that
separated from sugarcane was used as the producing strain (Morita et al., 2011a).
Researchers have studied the properties of MELs of various structures. Takahashi

et al. investigated the DPPH radical- and superoxide anion- scavenging activities
of MEL-A, -B, and –C, indicating that all MEL derivatives exhibited anioxidant
activities, although most of them were less effective than arbutin. Especially for
MEL-C, that secreted by P. hubeiensis KM-59 using soybean oil as carbon source,
highest DPPH radical scavenging activity of 50.3% at 10 mg mL-1 and highest
superoxide anion-scavenging activity of 60% at 2 mg mL-1 were showed. In
addition to that, it has been found that the activity was stronger as increasing the
concentration of MELs, and MEL-A with higher unsaturated ratio (55.7%) exhibited
higher activities when compared to MEL-A with that ratio of 41.2% (Lukic et al.,
2016). Yamamoto et al. applied MEL derivatives on skin that pre-treated with
sodium dodecyl sulfate, and found that MELs worked similar as natural ceramide
to recover the viability of skin cells at a high recovery rate of over 80% (Yamamoto
et al., 2012). In addition to their moisturising effects on skin, Morita et al. also found
the healing power of MELs on damaged hair, where the cracks on damaged
artificial hairs were repaired by treating with MEL-A, and –B and the tensile strength
was also increased. The inhibition of increase of the average friction coefficient,
from 0.126±0.003 of damaged hair to 0.108±0.002 when MEL-A was applied and
to 0.107±0.003 when MEL-B was applied, which indicated the ability of MEL
derivatives to smooth hair (Morita et al., 2010). Antibacterial capacity of MELs was
studied by Shu and the group, where MELs of at a minimum concentration of 0.625
mg mL-1 secreted by Pseudozyma aphidis (80% MEL-A dominated) showed
significant inhibitory effects against approximately 80% Gram-positive Bacillus
cereus spores germinated and grew into vegetative cells through disrupting the
formation of cell membrane (Shu et al., 2019). It has been demonstrated that this
antibacterial activity against Gram-positive bacteria was affected by the alkyl
chains and pattern of CH3CO group on the mannopyranosyl moiety of MELs
(Nashida et al., 2018). More recently, MEL-A was evaluated to show antibacterial
activity against another Gram-positive bacteria Listeria monocytogenes that bear
in food, indicating their promising application as food preservatives (Liu et al.,
2020).
62
2.2.5.2.2 Production of MELs
Many researches have successfully produced MELs using strains of the genus
Ustilago and Pseudozyma which obtained from rotten fruit (Morita et al., 2011b),
factory wastewater (De Andrade et al., 2017) and so on. Different microorganisms
utilize different carbon sources and synthesize MELs with different structures. It
has been found that MELs containing unsaturated fatty acids were greatly
produced when the microorganisms consuming vegetable oil as carbon source
(Lukic et al., 2016). Soybean oil, sunflower oil and olive oil are reported to be
suitable carbon sources for the cultivation of P. rugulosa NRBC 10877 and P.
parantarcitica JCM11752 (Yu et al., 2015, Morita et al., 2013, Recke et al., 2013).
Using oily substrates as carbon source normally leads to a higher production of
MELs. For example, Rau obtained 165 g L-1 MELs by cultivating P.aphidis DSM
14930 in the consumption of soybean oil (Rau et al., 2005a). However, difficulties
are heavily induced for the downstream process of product purification. Based on
this, some researchers suggested that water-soluble carbon sources such as
glucose, glycerol and cane sugar are good alternatives (Faria et al., 2014, Yu et
al., 2015, Saika et al., 2018b, Madihalli et al., 2020, Kinjo et al., 2019). In the
cultivation of Ustilago scitaminea NBRC 32730 in the medium containing
sugarcane juice (22.4 wt% sugars) as sole carbon source, Morito reported a yield
of 12.7 g L-1 MELs in the form of MEL-B (Morita et al., 2009b). Also, Pseudozyma
Antarctica T-34 was reported to produce MELs when consuming glucose as sole
carbon source (Morita et al., 2015). Although the utilization of water-soluble carbon
source for strain cultivation results in relatively lower production of MELs, and only
a few strains grow well when consuming water-soluble substrates as single carbon
source, it can help reduce the cost and is in favour of downstream purification.
2.2.5.2.3 Separation and Purification of MELs
Similar as other BSs, organic solvent extraction is the most widely used purification
method for MELs isolation, where equal volume ethyl acetate is frequently used for
the extraction (Shen et al., 2019, De Andrade et al., 2017, Wada et al., 2020, Shu
et al., 2020), followed by a rotary evaporation to get rid of organic solvent or silica
gel column chromatography. Solvent extraction method is simple and easy to carry
out. But due to large consumption of solvents resulting in higher cost and
contamination to environment, development of new technologies for MELs
isolation are uninterrupted. Rau et al. combined adsorption method with solvent
extraction in the separation of MELs, obtaining good separation effect. In the study,
ion-exchange resin adsorption, organic solvent extraction and heating up media
63
broth to 100~121 °C were carried out. During the heat treatment, MELs transferred
to solid state continually, achieving a recovery of MELs of 93% and the purity of
87% (Rau et al., 2005b). With only hydrophilic carbon source, cassava wastewater,
applied in medium, cultivating P. tsukubaensis for MEL-B production was proposed
by Andrade et al., using a novel separation strategy where the overflow was
integrated with ultrafiltration. As a result, for small scale configuration of 20 mL
centrifugal device, 80% of MEL-B was isolated in one step using 100 kDa MWCO
membranes, also scaling up to ultrafiltration of 500 mL is feasible where similar
result was obtained (De Andrade et al., 2017).
In order to get rid of residual oils and fatty acids in the crude MELs product, n-
hexane is typically applied. Some studies suggested the usage of chemical
mixtures combing hexane, methanol and water in various compositions. Rau et al.,
proposed hexane/methanol/water at a ratio of 1:6:3 (pH=5.5) for lipid removal (Rau
et al., 2005b), and recently Shen et al. developed an extraction method for oil and
free fatty acids removal, using the solvent system containing n-
hexane/methanol/water at a ratio of 1:2:1 (pH=2) for MELs extraction as the first
step, achieving a recovery of MELs of 80%, followed by extraction with solvent
mixture at a ratio of 1:3:1 which isolated 14% of MELs, and after the last step where
equal volume of n-hexane and methanol was mixed for purification, over 90%
MELs were extracted (Shen et al., 2019). The combination of hexane and methanol
should realize a better removal, due to the reason that hexane is non-polar solvent
which is only used for extract lipid of low polarity (neutral lipid); while methanol is
polar solvent which is miscible with medium to high polar lipid.
2.2.5.2.4 Phase Behaviour of MELs in water
As being synthesized from fatty alcohols and sugars, MELs are able to self-
assemble into vesicles, self-assembled monolayer, sponge phase, bicontinuous
cubic phase and three-dimensional ordered lyotropic liquid crystral phase that is
stabled by hydrogen-bond between glycosyl, van der waals force and interaction
between molecules (Imura et al., 2007). Moreover, the thermal stability is
influenced by the chirality of carbon atom. The liquid crystal structure endows
MELs with excellent wetting properties. It has been reported that presence of multi-
lamellar vesicles facilitated the fusion of MELs and membrane, favouring for the
effect of active substance on cell and the enhancement of gene transfection
efficiency (Worakitkanchanakul et al., 2008, Coelho et al., 2020, Kitamoto et al.,
2009). Different structures of MELs tend to self-assemble into different structures.
64
MEL-A was suggested to form sponge phase (L 3 phase) when the concentration
is higher than 1 mM (Imura et al., 2007). The structure morphology was interpreted
as coacervates that derived from bilayer structure. Besides, MEL-A is a natural
compound which can spontaneously form this structure without the aids of other
co-surfactants (Morita et al., 2013, Niu et al., 2019). In terms of MEL-B and MEL-
C, due to the lack of 4’-O-acetyl group or 6’-O-acetyl group, causing self-bend
during self-assembling process to change coacervates to vesicles, and they can
form vesicles with large diameter over 10 μm (Konishi and Makino, 2018, Fan et
al., 2018). When the bend curvature becomes zero, lamella phase (Lα) is formed.
Thus, MEL-B and MEL-C can form Lα phase which is stabled by hydrogen-bond
between hydroxyl in C-4’ or C-6’ (Worakitkanchanakul et al., 2009, Fukuoka et al.,
2011, Fukuoka et al., 2012).
The phase behaviour of ternary system of MELs in water has been studied by
Worakitkanchanakul et al., where MEL-A/water/n-decane and MEL-B/water/n-
decane systems were analysed. When using n-decane as oil phase, diacetylated
MEL-A formed single phase system, namely microemulsion (W/O). And MEL-A
formed L3, V2 and Lα phase. While monoacetylated MEL-B only formed one phase
and bicontinuous microemulsion (Worakitkanchanakul et al., 2009). Noticeably,
Lα+oil region of O/W emulsion in the system of MEL-B/water/n-decane was easily
to be formed, which helped stable emulsion for over a month (Saika et al., 2018c,
Saika et al., 2018b). As the amphiphilic molecules of MELs are different from
traditional ones, the study of liquid phase may help reveal the relationship between
MELs structure and its function (Madihalli and Doble, 2019, Ohadi et al., 2020,
Beck et al., 2019).
2.3 Emulsion
Cosmetic creams and emulsions can be used as the skin protector, which prevents
skin from the environmental damage such as windy, dusty, chilly, dryness and
humidity, and moisturizes the outermost layer of the skin, namely, stratum corneum,
providing oily components to the skin. Apart from that, emulsions are also good
carriers of active ingredients and drug, making them easy to be absorbed by skin,
thereby nourishing and regulating the skin (Aswal et al., 2013, Banerjee et al.,
2019).
65
2.3.1 Overview of Emulsion
An emulsion is a multiphase colloid system, consisting of one or more liquid

dispersing as small droplets in another immiscible liquid. Generally, emulsions can
be classified as simple emulsions and multiple emulsions, where simple emulsion
refers to the system of one liquid dispersing (dispersed phase) as droplets in
another immiscible liquid phase (continuous phase) (Zhu et al., 2018). Oil-in-water
emulsions (oil droplets dispersed in continuous water phase), O/W, and water-in-
oil emulsions (water droplets dispersed in oil phase), W/O, are two common types
of simple emulsion. In comparison, the system of multiple emulsions is more
complex, where one or more droplets exist in multiple emulsion globule, forming
oil-in-water-in-oil (O/W/O) multiple emulsions or water-in-oil-in-water emulsions
(W/O/W) (David et al., 2019, Bonnin, 2019). Microemulsions are isotropic and
thermodynamically stable system with dispersed droplets sizing from 1 to 100 nm.
While for macroemulsions with droplet size of larger than 200 nm and
nanoemulsions with that less than 200 nm are thermodynamic instable systems,
as the generated two-phase boundary (interface) is large and the energy of the
system is relatively high. On account of this, emulsifiers are usually added in the
formulation to stable the emulsion system (Patel and Joshi, 2012).
2.3.2 Emulsion Formation
Emulsions are generally formed through either low- or high- energy technologies.
Low-energy method refers to spontaneous emulsification, where no external
energy is required and the emulsion system that internally changed in a specific
way under the environment or composition alteration provides stored chemical
energy for itself. Researchers proposed transitional inversion, where hydrophile-
lipophile balance (HLB) was affected by changing factors such as temperature or
electrolyte concentration, and catastrophic inversion methods where volume
fraction of the disperse phase is increased, for emulsion preparation (Solans et al.,
2016, Perazzo et al., 2015). However, most of accessible surfactants or emulsifiers
are not capable of involving in this type of methods, especially those natural
surfactants, thus at present, high energy emulsification (dispersion) is commonly
applied for commercial use, where four main elements are generally required in
the preparation of emulsions: water phase, oil phase, surfactants and energy
(external force) (Cantero del Castillo, 2019, Caritá et al., 2020).
66
2.3.2.1 Mechanism of high energy emulsification
The change in free energy of emulsification can be expressed according to the

Equation 2.1 (Leal-Calderon et al., 2007):
∆G = ∆Aγ − T∆S 2.1
Where, T is the temperature, ΔS is the change of entropy of dispersion, γ is the
interfacial tension between oil and water, ΔA is the increase of interfacial area of
oil and water after the formation of emulsion.
Generally, during the process of emulsification, Δ A γ is no smaller than T Δ S,
namely, free energy is always positive. If the component in the system is unable to
acquire energy from their own, the emulsification process is non-spontaneous,
where the energy input is needed. Typically, mechanical applications such as
homogenizers and mixers are applied for providing energy, in order to fragment
dispersed phase into small droplets and intermingle two immiscible phases.
Noticeably, large energy is needed to generate disruptive forces for overcoming
the Laplace pressure (ΔPL) of the droplets, thereby realising fine droplets disruption
(Wang et al., 2018b).
4𝛾
∆𝑃𝐿 = 2.2
𝑑
Where, ΔPL is the Laplace pressure, γ is the interfacial tension between oil and
water, d is the droplet diameter.
From Laplace equation (Equation 2.2), when destructive force is higher than
Laplace pressure, smaller droplets are obtained. In another aspect, lowering down
the interfacial tension and maintaining energy input at a certain level can also
produce smaller droplets. Thus from this aspect, surfactants or emulsifiers involved
in the formulation for emulsification could help facilitate the fragmentation of
dispersed phase into fine droplets, through adsorbing onto the droplet surfaces
and reducing the interfacial tension (Lian et al., 2019). But this is only worked when
the surfactant adsorbing rate to interface is faster than the droplet disruption rate,
for ensuring that the droplets are fully covered by surfactant molecules before they
break down (Agrawal et al., 2017). Different types of surfactants or emulsifiers
showing various surface activities help generate droplets in different sizes. It has
67
been indicated that, biopolymers do not effectively active water and oil interface
(surface tension≈15~25 mJ m-2) when compared to small molecular surfactants
(surface tension <5 mJ m-2) so that they help form larger droplets during mixing
(Zembyla et al., 2020, Xie et al., 2017, Hantal et al., 2019). Another role of
surfactants or emulsifiers play in emulsification is their ability of inhibition of droplet
coalescence, for stabilizing the system (Dao et al., 2018).
2.3.2.2 Surfactants in Formulation
Actually, instead of using single surface active agent, blending of different types of
surfactants in the formulation is more advantageous (Hantal et al., 2019, Patil et
al., 2015). Mixed emulsifier system containing two or more types of surfactants or
emulsifiers could exhibit better emulsification effect (Vilasau et al., 2011b). On the
contrary, the interfacial film that formed by highly pure surfactant may not be
closely packed, thus the mechanical strength is low. It has been found that liquid
paraffin with cholesterol dispersed into sodium hexadecyl sulphate solution will
produce stable oil in water emulsion, while only use cholesterol or sodium
hexadecyl sulphase will form an instable one (Ahmadi et al., 2020).
Generally, mixtures of ionic surfactants and non-ionic surfactants in the formulation

combining both of steric and electrostatic forces could significantly inhibit instability
of the product and present the favourable synergistic effects (Vilasau et al., 2011a).
Take Sorbitan esters (Spans) and Polyoxyethylene sorbitol fatty acid esters
(Tweens) mixed surfactant system as an example, because the derivative of
polyoxyethylated sorbitol has strong interaction with water phase, its hydrophobic
group stretches more into water phase than non-ethoxylated sorbitol, thus the
hydrophobic groups of them got closer to each other at the interface. Based on this,
the interaction between the molecules of two types of surfactants was stronger
than using alone, thereby forming an interfacial film with higher strength (Koneva
et al., 2017, Posocco et al., 2016, Yoo et al., 2020). Also, the mixed emulsifier
system containing sodium dodecyl sulphate (sodium lauryl sulphate/SLS) and
lauryl alcohol can effectively help stable the emulsion (Ade-Browne et al., 2020,
Morris et al., 2019a, Penkina et al., 2020). In the study of Mandal et al., in
comparison with single surfactant-water-oil system, the synergistic effect of
combined anionic surfactant (sodium dodecylbenzenesulfonate/SDBS) and non-
ionic surfactant (Tween 80) system on the modification of wettability of a reservoir
rock was studied, with a ration of SDBS:Tween 80 at 1:1 wt%. And optimal results
68
were obtained from mixed surfactant system, showing that the contact angle of
quartz substrate was dramatically decreased with time for realising the complete
alteration of quartz from oil wet to water wet under ambient conditions (Mandal et
al., 2016). In the study of surface adsorbed film of surfactant solution, polar organic
compounds such as fatty alcohol in the film will greatly increase the surface activity
and the film strength. Because fatty alcohols have relatively small hydrophilic head
group (-OH), it can effectively adsorb at the interface and insert into the adsorption
layer of adjacent surfactant molecules, thereby causing large surface excess and
low interfacial tension (Falbe, 2012). Ibrahim et al. studied the formulation of palm
methyl ester-in-water system with different mixed non-ionic surfactants, indicating
that the hydrophilic moiety of the non-ionic surfactants affected the stability of
emulsions. And an optimal combination of fatty alcohol POE (25 EO) with DLS1
(HLB 11±1) was obtained with highest stability, where the stable zeta potential was
ranged from -37.91 mV to -40.8 mV and low surface tension value was
31.186~32.865 mN m-1 (Ibrahim et al., 2015).
Moreover, the concentration of surfactants is important for emulsion formation.

When adding surfactants or emulsifiers in the system, surfactant molecules adsorb
at the interface forming interfacial film which has certain strength. This film protects
dispersed droplets to prevent coalescence when crashing into each other
(Marquez et al., 2018). Sufficient surfactants in the system, namely higher
concentration of surfactants, are likely to form interfacial film of strong strength
consisting of tightly arranged surfactant molecules, resulting in stronger resistance
to the coalescence of droplets, and the emulsion will be formed easily and remain
stable (Kanouni et al., 2002). In a study where an emulsion system containing non-
ionic surfactant with oil in water, increasing the concentration of surfactant from 2%
to 6% led to formation of an emulsion with narrower droplet size distribution
microstructure with enhanced stability (Feng et al., 2018). This is also proved by
the theory of composite membrane, indicating that only when the molecules of
emulsifier closely aligned to form condensed film can the emulsion be stable
(Poerwadi et al., 2020). However, the addition of co-emulsifiers may also cause
too high viscosity or even phase separation, which directly results in a way too rigid
cream and crystallisation precipitation during the storage (Ballmann and Müeller,
2008). Thus appropriate concentration of surfactants in the formulation is required.
Hydrophile-lipophile balance (HLB) is a key factor that affects the choice of

surfactants and the performance of emulsion system especially for its stability.
69
Generally, more hydrophobic surfactants with HLB value ranging from 3 to 6 are
suitable for emulsifying W/O emulsion, and O/W emulsion is generally prepared
using the HLB value ranged from 8 to 18 (Tadros, 2009). Feng et al. studied the
effect of different HLB values of surfactants on the polyoxyethylene castor oil ether
(non-ionic surfactant)/oil+lambda-cyhalothrin/water (at ratio of 6%/5%+5%/84%)
emulsion preparation for pesticide appliations. It showed that increasing HLB value
of surfactants from 10.5 to 15.5 resulted in larger droplets in the system (sized from
0.44μm to 4.27μm) and wider droplet distribution, thereby resulting in the instability
of the system (Feng et al., 2018). However, the value of HLB for selected
surfactants and/or emulsifiers should be similar to the value that required by the
emulsion system (Hong et al., 2018). In another study from Hong et al., the effect
of HLB value of a mixed non-ionic surfactant system on the formation and stability
of the O/W emulsion was investigated. Two mixed surfactant systems, MS-01 and
MS-02 respectively containing different concentrations of Span 60&Tween 60 and
Span 80&Tween 80 were studied in the formulation of the emulsion with required
HLB value of 10.85. The minimum droplets and highest zeta-potential value,
standing for a more stable emulsion system, for MS-01 involved emulsion were
observed at HLB=10.8, and that for MS-02 incorporated emulsion were at
HLB=10.7, both of the HLB values were close to the required HLB of the system.
Also the cream index further provided similar results, indicating more stable system
obtained with a HLB value of surfactants similar to the required value of emulsion
system (Hong et al., 2018).
2.3.2.3 Process of Formulation
The preparation of emulsion refers to dispersing one liquid in forms of droplets into
another immiscible liquid. Theoretically, an emulsion can be formed by simply
mixing two immiscible liquids together and then giving it thoroughly shaking, but
the resulted emulsion will be super unstable. Thus a more rational method is
suggested as, firstly dissolving emulsifiers into the phase in which it is most soluble,
following by the adding of another phase. Then a high speed mixing or vigorous
agitation is used to shear the mixture (Tadros, 2013). Apart from that, the addition
sequence of organic/aqueous phases and initial location of emulsifiers may also
affect the performance of emulsions. Feng et al. studied the effect of changing
addition sequence of beta-cypermethrin/aqueous phase and different types of
emulsifiers on the nanoemulsions using low-energy emulsifying process, finding
that the emulsion prepared by adding aqueous phase into organic phase with
70
emulsifiers exhibited the highest stability, compared to other sequences (Feng et
al., 2016).
Mixing provides external shear force for the fraction of dispersed phase into small
droplets, facilitating formation of emulsions. Liquid-liquid mixing is often under
turbulent condition, where the interaction between two phases exists (Naeeni and
Pakzad, 2019). The turbulent fluctuation in continuous phase facilitates the
breakage of dispersed droplets, resulting in the formation of smaller droplets and
big contacting area (Boxall et al., 2012). On the contrary, dispersed phase has a
damping effect on the turbulence of continuous phase, which may reduce its
strength. Thus breaking mechanism of dispersed droplets is significant for liquid-
liquid heterogeneous intensive mixing (Theron et al., 2010). Research showed that
there were two main factors of droplet breaking in hydraulics: 1) viscous shear
stress caused by velocity gradient; 2) instant shear stress and local pressure
fluctuation (Reynolds shear stress) caused by turbulence (Liu et al., 2010).
Podgorska et al. studied the breaking mechanism of silicon oil droplet in a stirred
tank equipped with Rushton agitator and four baffles, indicating that droplets
breaking happened mainly around stirring blade due to high system average
energy dissipation rate in this region. Besides, high viscosity of dispersed phase
helped stabilize droplets in pressure pulse, thus having adverse influence on the
deformation and breaking of droplets (Podgórska, 2006).
In the system of liquid-liquid dispersion, droplets collide, followed by coalescence

or separation is based on velocity pulse. The collision course can be seen as the
process of film drainage of continuous phase between two droplets, and
coalescence time and contact time of droplets determine whether collided droplets
merge immediately or separate apart. Namely, two droplets will coalesce when the
contact time is longer than coalescence time. In the study of modelling droplets
coalescence in liquid-liquid dispersions in flow through fibrous media, where a
model formulation named coalescence efficiency was used in order to estimate the
tangible effect of coalescence, a simplified model of Coulaloglou was applied
(Krasinski, 2013).
𝑡𝑑
𝜂𝑐𝑜𝑎𝑙 = exp (− ) 2.3
𝑡𝑐
Where, td is the drainage time (referred to coalescence time), tc is the contact time.
The coalescence time is required for thinning the film between two droplets to a
71
certain value (critical thickness). Ban et al. studied the coalescence behaviour of
the system with methylbenzene droplets in water, suggesting that concentration of
acetone in methylbenzene, direction of mass transfer, contact time of droplets and
flow velocity of continuous phase have influence on the coalescence of
methylbenzene droplets. Among them, the concentration of acetone and direction
of mass transfer determined the duration of coalescence time. When acetone
transferred from dispersed phase to continuous phase, average coalescence time
decreased with the concentration of acetone increases; in the opposite direction,
the coalescence of droplet was easily be blocked (Ban et al., 2000).
During the mixing process, droplet coalescence and breakage is in a dynamic

equilibrium. The minimum stable droplet size dmin is a judgement for whether
droplets coalesce or not. When droplet size is smaller than dmin, droplets are
instability and easily coalesce. According to the analysis of isotropic turbulent
dispersed system, Liu proposed a model for calculating dmin (Liu and Li, 1999):
3.11 𝛾1.38 𝐵0.46

𝑑𝑚𝑖𝑛 = 2.4
0.0272𝜇𝑐 𝜌𝑐0.84 𝜀 0.89
Where, dmin is the minimum stable droplet size, γ is the interfacial tension, B is the
van der Waals constant, μc is the viscosity of continuous phase, ρc is the density
of continuous phase, ε is the energy dissipation. The equation directly reflects the
relationship between minimum droplet diameter and physical properties of system.
In order to achieve homogeneously mixed products, the mixing equipment should

allow the fluid system either flow entirely to avoid any stagnation area or under
high shear or high flow mixing to break the inhomogeneity (Gao et al., 2016)
Mechanical devices that wildly used for mixing are mixing stirrers, colloid mills,
homogenizers and ultrasound generators. Mixing stirrers are generally divided into
high speed stirrers and low speed ones, which refers to agitating liquid under a
turbulent flow and viscous flow respectively (Vikhansky, 2020). The former ones
(such as blade, propeller, and turbine type) are applicable for mixing low viscous
liquid and the latter ones (such as anchor) are normally used for high viscous and
non-Newtonian fluid (Uhl, 2012). Homogenizers consist of a rotor-stator system,
creating shearing behaviour between the gap of rotor and stator, which is usually
applied for liquid emulsification and solid-liquid material crush, dispersing and
mixing (Castellano et al., 2019, Farzad et al., 2018).
72
Some parameters should also be taken into account for cream preparation, such
as emulsification temperature, time and the agitation speed. Generally, the
temperature of oil and liquid phase should be controlled between 75˚C and 85˚C
for semi-solids production. During the cooling stage, although higher cooling rates
will generate smaller droplets, too high cooling rate may also lead to materials with
high melting point or low solubility crystalize, thereby bringing poor emulsification
effect (Moens et al., 2019). For the same system and dispersion method, the
droplets size will decrease as increasing the emulsification time. But it will reach
an equilibrium, that is to say, when the droplets become small enough, further
emulsification will not change its size. Thus the emulsification time should be
controlled to a rational value in case of meaningless economic loss (Pivsa-Art et
al., 2019). The agitating speed also has significant effects on the emulsification.
Too fast speed will entrap air into the system which tends to make the emulsion
unstable. Thus, as a general rule, higher speed agitating is helpful at the beginning
of emulsification, when the process enters cooling stage, medium or lower speed
of mixing is preferred for the purpose of minimize the trapping of air (Colafemmina
et al., 2020a, Chizawa et al., 2019, Santos et al., 2016).
2.3.3 Mechanisms of Emulsion Instability
As mentioned above, the emulsification process is generally non-spontaneous. In

the opposite, when the droplets coalesce, interfacial area of system will decrease,
namely, the free energy of system (G) decreases. This is a spontaneous process.
Therefore, emulsion system is thermodynamic instable, where the
physicochemical properties will change with time. Four phenomena of emulsion
instability have been reported: coalescence, flocculation, creaming and breaking,
which are illustrated in figure 2.7 (Khan et al., 2011).
Flocculation is a process where two or more small emulsion droplets associate

together to form large aggregates, which is reversible because each droplet still
remains its individual integrity. Some researchers made a statement that the
reason for this process is due to the depletion effect when excess surfactant exists
in the continuous phase of an emulsion system (Huck-Iriart et al., 2016). In detail,
excess surfactant will form micelles flowing around in the bulk liquid. If two droplets
are very close to each other (droplets distance smaller than the diameter of the
micelles), there may be low concentration of micelles in the inter space between
two droplets (Koroleva et al., 2015). As a result, the osmotic pressure difference
73
drives micelles flow out of the gap between the droplets and induces the
aggregation of them (Dickinson, 2019).
Flocculation Coalescence
Good emulsion
Creaming Breaking
Figure 2.7 Instability phenomena of emulsions
Creaming phenomenon is happened when the dispersed phase separates and

then forms a layer upon the continuous phase. Christopher and Dawn pointed out
that the increase of the viscosity of continuous phase will help inhibit this
phenomenon, which is also proved by Stoke’s law (Langley and Belcher, 2012):
𝐷2 (𝜌𝑆 − 𝜌𝑂 )𝑔
V= 2.5
18𝜂
Where, V is the creaming rate, D is the diameter of dispersed droplets, ρs is the
density of dispersed phase, ρ o is the density of continuous phase, η is the

continuous phase viscosity and g is gravitational acceleration (Shinoda and
Uchimura, 2018). Over time, when the droplets merged together to form a large
droplet, a new process occurred which is known as coalescence, followed by the
breaking of emulsions (Trujillo-Cayado et al., 2016). Factors that influence the
stability of emulsions normally can be divided into two aspects: internal factors and
external factors. The internal factors include the interfacial tension, the intensive of
interfacial film, effect of interfacial charge, droplet size distribution, and phase
volume ratio and so on (Marquez et al., 2018, Neumann et al., 2018, Sun et al.,
2017). As for external factors, mixing temperature, mixing speed and time will affect
the stability of emulsion (Wang et al., 2018a).
74
2.4 Rheology
Flow properties of cosmetic materials directly associate with the quality of final
products and people’s preference, which could be characterised with the help of
rheology (Colo et al., 2004). Cream products applied by consumers for end-use
undergo sampling, rubbing to after-feeling. Sampling refers to the process when
consumer taking the cream out from the container with the fingertip, where
appropriate thickness and consistency of the cream is expected. The physical and
chemical parameters related to this stage are hardness, cohesiveness, springiness
and adhesiveness. During rubbing, the cream is expected to exhibit good
spreadability and absorbency. After spreading the cream on the skin, the
consistency of cream without any granular sensation is expected, after which
appropriate amount of greasy leftovers on the skin are also key factors determining
customers’ satisfaction (Moravkova and Stern, 2011) .
2.4.1 Rheology of Emulsions
Some cosmetic products, such as toothpastes, lipsticks, foundations, anhydrous

cream, parts of emulsions, are plastic fluids. When the system is at rest, particles
form three-dimensional space structure (Brummer, 2013). The existence of yield
value is due to the strong three-dimensional space force which makes the system
possess the property of the solid-like and have relatively high viscosity during low
shear range. Once the extra shear stress surpasses this critical value, the structure
will be collapsed, and then fluid begins to flow. When this external stress is
removed, the structure of the system will gradually recover to some extent (Akbari
and Nour, 2018). In real practice, semi-solid creams show both viscosity and
elasticity responses to external force, thus these substances are known as
viscoelastic materials (Tschoegl, 2012). In this type of fluid system, after the
external force is removed, part of deformation energy is used to return to its original
state and part of that is converted to heat and lost, thereby performing like both
viscous liquid and elastic solid.
Most cosmetic emulsions and creams possess sophisticated shear related and
time related flow characteristics. Thus, from the blending process to filling process,
then until any time during consumers use, the viscosity of the cosmetic changes
with applied shear rate or stress. Table 2.5 presents typical shear rate ranges of
emulsions and creams occurring in different industrial applications (Mezger, 2020).
75
Table 2.5 Typical shear rate ranges of emulsions and creams during different industrial
applications, adapted from Mezger, 2020
Process Shear rate range ṙ (s-1)

Sedimentation of particles 10-6 to 10-3
Mixing or stirring 10 to 104
Rubbing the cream on the skin 103 to 105
However, Sherman suggested that, when consumers dispensed and rubbed

creams on hand or face, the shear rate is in a certain range (Sherman, 1968). The
choice of the measurement range of rheological behaviour aims to provide the
information of properties that related to the product at rest or during the usage of
consumers (Salehiyan et al., 2018). Applying the Equation 2.6 which defines the
shear rate ṙ, along with some assumptions, specific shear rate values for different
processes are calculated by Langenbucher et al. (Langenbucher and Lange, 1970).
V
ṙ= 2.6
h
Where, V refers to the speed of rubbing by hand, h refers to the thickness of cream
layer on skin surface. Table 2.6 shows calculation values of shear rate occurring
in different applications of creams under certain assumptions (Langenbucher and
Lange, 1970):
Table 2.6 Theoretical values of shear rate related to different processes of cream
application, adapted from Langenbucher and Lange, 1970
Process Assumptions in Calculation

calculation values of shear
rate
ṙ (s-1)
Taking cream from the jar Layer thickness: 2cm 1
Velocity: 2cm/s
Rubbing on Layer thickness: 0.2cm 120
the skin Velocity of dispensing and
extending: 24 cm/s
primary stage Layer thickness: 0.1cm 100
Velocity of dispensing and
extending: 10 cm/s
intermediate stage Layer thickness: 0.01cm 103
extending: 10 cm/s
ending stage Layer thickness: 0.001cm 104
extending: 10 cm/s
76
2.4.2 Rheometry and Rheometers
Rheometry is the technology which is used to measure rheological behaviour of
the flow and determine the corresponding rheological data with the help of a
rheometer, where the flow phenomena are studied allowing the materials subject
to various external forces (Coussot, 2005, Salehiyan et al., 2018). Typically, two
main measurements are normally carried out to investigate flow properties: steady
state test and dynamic oscillatory test. The steady state tests are non-linear, which
is used to characterize the viscous behaviour. Within a range of shear stresses
and shear rates, the viscosity is measured as a function of the imposed parameters
(Malkin, 2013). There are two modes in rotational tests: tests with controlled shear
rate (CSR), that usually applied for the investigation of liquid presenting self-
levelling behaviour, and tests with controlled shear stress (CSS), where the shear
stress or torque is pre-set and controlled by the rheometer (Zhao et al., 2013, Li et
al., 2012). CSS method is generally used to determine yield points of dispersions
or gels, and more viable for determining rheological behaviours of non-Newtonian
flows especially with semi-solid properties compared to CSR. (Coussot, 2005,
Kukla et al., 2016, Ahmed, 2019).
Dynamic oscillatory test refers to adding oscillatory stress or stain to the

viscoelastic materials to measure the generated shear strain that related to time.
Generally, a function of frequency or time will be measured, including measuring
parameters such as storage and loss moduli (G’ and G’’), phase lag, complex
modulus (G*) and viscosity (η*). These properties are normally confined to a
specific range of strains or stresses where no visually movement of the material is
observed. This range is known as linear viscoelastic range, where the storage and
loss moduli are independent with oscillatory strain or stress (Pan et al., 2018,
Kaspchak et al., 2017, Sanz et al., 2017, Zhang et al., 2019a).
Rheological studies were carried out in order to understand flow properties and
viscosity profiles of emulsions and surfactant solutions that applied in emulsion
formulation. The rheological behaviour of systems where cetyltrimethylammonium
chloride (CTAC), behenyltrimethylammonium chloride (BTAC),
CTAC/hydroxyethyl cellulose (HEC) respectively mixed with fatty alcohols (FAs)
were studied, showing that higher concentration of FA increased the storage
moduli, the yield stresses and the zero-shear-rate viscosity in CTAC/FA and
BTAC/FA emulsions (Nakarapanich et al., 2001). This behaviour was also
investigated by Ade-Browne et al., where the increase the amount of lauryl alcohol
77
in sodium lauryl sulfate with different degrees of ethoxylation enhanced the system
viscosity and the formation of a gel (Ade-Browne et al., 2020). Similar, higher
concentration of an individual alcohol, cetyl alcohol, in the system of sodium
dodecyl sulfate (SDS) facilitated the formation of stronger gel with higher storage
modulus (Grewe et al., 2015). The mechanisms of solubility limits of fatty alcohols
(FAs) in sodium laureth sulfate (SLES)/cocoamidopropyl betaine (CAPB) mixed
micellar solutions were studied, indicating that the solubility limits were positively
associated with the surfactant concentration and negatively related to the alcohol
chain length (Tzocheva et al., 2015). Mitrinova et al. studied rheological impacts of
co-surfactants of various structures on mixed surfactant solutions containing
sodium laureth sulfate (SLES) and zwitterionic cocoamidopropyl betaine (CAPB).
They revealed that viscoelasticity of SLES/CAPB system was affected by the
chain-length and head-group size of cosurfactants. In addition to that, the head-
group charge gave priority to govern this behaviour (Mitrinova et al., 2018).
Rheological behaviour of mixed surfactant solutions of sulfonated methyl esters
(SME) and cocamidopropyl betaine (CAPB) were also investigated, which
exhibited a higher viscosity compared to the system containing sodium dodecyl
sulfate (SDS) and CAPB. It also showed that further addition of the fatty alcohol,
1-Dodecanol, exceeding their concentration limit led to the decrease in viscosity,
and precipitation was witnessed due to giant micelles transforming into drops or
crystallites. However, the addition of the non-ionic surfactant cocamide
monoethanolamine (CMEA) as thickener only promoted the growth of micelle and
increase of system, without causing precipitation (Yavrukova et al., 2020). CMEA-
SLES binary mixtures were investigated by Pandya et al., revealing that CMEA
solubilized in SLES solution facilitated the micellar transition from sphere-like to
rod-like and the increase in viscosity (Pandya et al., 2020). Some studies also
investigated systems that stabilised by biosurfactants. A concentrated emulsion
containing 50 wt% oil that emulsified by rhamnolipids were formulated in the study
of Li et al., and shear-thinning behaviour and low consistency coefficient of the
emulsion were determined (Li et al., 2018). In addition to that, ternary system of
sodium laureth sulfate (SLES)/ zwitterionic cocamidopropyl etaine
(CAPB)/rhamnolipids (mono/dirhamnolipids mixture) was characterised with the
help of rheology. It was found that the addition of rhamnolipids biosurfactant on
SLES/CAPB system led to a decrease in viscosity, providing rheological
understanding of surfactants/biosurfactants ternary system for bio-based product
formulation (Xu and Amin, 2019).
78
In order to obtain relatively accurate rheological result, different measuring
systems are used based on the natures of materials. The most common measuring
systems are: concentric cylinder measuring system, cone and plate system and
parallel plate system (Song et al., 2017). In the rheological measurements for a
cream system containing water, oil and sorbitan monoester as surfactant, a
rheometer equipped with a concentric cylinder system (diameter of 15 mm) was
applied. The LVR was obtained using the oscillatory stress sweep at the constant
frequency of 1Hz, where the oscillatory stress increased from 0.06 to 100 Pa. The
end point of LVR was determined in terms of oscillatory stress when the storage
modulus value was decreased by 10% from the linear plateau. After that, a value
within LVR was selected using in a creep recovery test, where the sample was
imposed the stress for 120 s and then the recovery was set to 360 s. As a result,
the creep compliance J changed depending on time was obtained. This can also
be used to indicate the elastic and viscous structure of the cream (Korhonen et al.,
2002).
When using cone and plate geometry, much less sample is required than using
concentric cylinder. Normally, the angle between the surface of the cone and the
plate is of the order of 1°, and the cone is rotated and the force on the cone is
measured (Maazouz, 2020). This type of measuring system is more suitable to
measure samples with medium and high viscosity (Kulik and Boiko, 2018). In order
to study the influence of different polymers in an O/W emulsion, Gilbert et al.
applied rheological measurements in the study, where the flow properties of natural,
natural modified, and chemically synthetic polymers of 1 wt% that respectively
formulated in an emulsion were tested. Continuous flow test was conducted using
a rheometer equipped with cone-plate geometry (an angle of almost 1°, diameter
of 40 mm). The gap between cone and plate was set to be 27 μm. The viscosity
was recorded under the imposed shear rate ranging from 0.01 to 1000 S-1 for 150
s. From the result, it was obtained that all the emulsions showed shear thinning
behaviour. Also three emulsions exhibited a yield stress (Gilbert et al., 2013).
During the viscoelastic properties study, oscillatory measurements were carried

out using a cone and plate with an angle of 4°(diameter of 40mm) and the gap
was changed to 130 μm. An oscillatory strain sweep was conducted from the strain
ranging from 0.01% to 100%, at the frequency of 1 rad s-1, to obtain the linear
viscoelastic region (LVR). Besides, a time sweep and a creep-recovery test were
also carried out to characterize the viscoelastic properties of each emulsion with
different polymers (Gilbert et al., 2013). Another study was conducted rheological
79
measurement on cosmetic emulsions using rheometer equipped with a cone and
plate sensor system (2°for measuring body lotions and facial creams; 1°for sun
lotions and eye creams). Through carrying out a steady state shear with shear rate
increasing from 0 to 600 S-1, the fluid type of each cosmetic emulsion was obtained.
Also the yield stress was obtained for some types of emulsions. By comparing the
rheological analysis and sensory assessment, the former was proved to be more
applicable in the evaluation of stability of cosmetic emulsions (Moravkova and
Stern, 2011).
However, the cone and plate measuring system is not applicable to measure
dispersion system with large particles, as the particles in the cone angle area are
needed to be forced out to contact with cone plate. The normal forced is required
to measure the radicle flow of sample in the gap. If the sample has very high yield
stress, the radicle squeezing flow will be hindered. Sometimes, radicle secondary
flow will happen, which has the opposite effect on the annular main flow. This can
influence the laminar condition of main flow (Moravkova and Stern, 2011). Thus,
parallel plate measuring system seems to be a good substitute for cone and plate
one, which uses an upper plate to replace the cone plate. This design avoids the
problem of radicle secondary flow; thus it is suitable to measure materials with
large particles (Mezger, 2006). However, if the viscosity of measured material
greatly depend on shear rate, the constant shear rate cannot be obtained under
the given spinner speed. Thus, the results from parallel-plates measurement are
required to be corrected using Weissenberg-Rabinowitsch corrections (Stan et al.,
2017, Morillas and de Vicente, 2019). Another study of the application condition of
cream and lotion was conducted, using a rheometer equipped with parallel plate
system (diameter of 25 mm, gap of 2 mm). The steady state shear test was carried
out at the temperature of 35 ˚C, with the shear rate ranging from 0.01 to 625 S -1.
As a result, yield stress was witnessed, and the value of cream was 10 times
greater than that of lotion. In addition, both of cream and lotion showed shear
thinning behaviour. In the oscillatory tests, oscillatory frequency sweep tests within
angular frequencies range from 0.025 to 100 rad s-1 was performed on the cream
and lotion, under a constant strain of 1% and 0.2% respectively. The result also
showed that, both for both of cream and lotion, the storage modulus was over loss
modulus through the whole measuring range, indicating elastic behaviour was
predominant within small amplitude (Kwak et al., 2015).
80
Chapter 3. Materials and Methodology
This chapter summarised experimental work involved in this project, where
theories and experimental procedures will be introduced. It is classified into three
sections: bio-surfactant production, cream formulation and characterisation
methods.
3.1 Sophorolipids (SLs) Production

The production of SLs in this work is referenced from the study of Ben et al.
(Dolman et al., 2017) in our group, including selection of producing microorganisms,
media preparation and strain cultivation strategy.
3.1.1 Producing Microorganisms

The yeast, Candida Bombicola ATCC-22214, was selected as the producer strain
for SLs production in this project, and the working stock was stored in cell vials at
-80 °C.
3.1.2 Chemicals
Chemicals and organic solvents that used for the media broth preparation and
product purification including yeast extract, peptone and monohydrate glucose
were obtained from Sigma Aldrich (UK), and Crisp ~N Dry oil providing rapeseed
oil that was obtained from Tesco. For purification of bio-surfactant product, ethyl
acetate and n-Hexane (Sigma Aldrich, UK) were applied.
3.1.3 Production Strategies

3.1.3.1 Fermentation Technology
In order to obtain single colony of cell, Candida bombicola from working stock was
firstly inoculated to the agar plate from cell vial, followed by cultivation for 48 h at
25 °C. Shake flask fermentation was used for SLs production. In order to produce
a high cell concentration and keep cell viability and peak cells at the same growth
stage, a pre-cultivation was carried out before the shake flask fermentation. 10%
(v/v) inoculum from pre-culture was added into fermentation media (Dolman et al.,
2019).
The composition of pre-culture media is the same as that of fermentation culture,

which contained yeast extract of 6 g L-1, peptone of 5 g L-1, glucose of 100 g L-1
and Crisp ~N Dry oil of 100 g L-1. 250 mL Erlenmeyer shake flask containing 25
81
mL media and 500 mL Erlenmeyer shake flask containing 50 mL media were
respectively prepared for pre-cultivation and shake flaks fermentation (Dolman et
al., 2019).
Except oil and glucose, the other ingredients were firstly added into the shake flask
and prepared according to the composition as mentioned above. Then they were
sterilized via autoclave, along with oil separately and other auxiliary glassware.
The glucose was filtered with 0.2 nm membrane to get sterilization.
After 48 hours of cultivation in agar plate, single colonies were inoculated to the
pre-culture shake flask, followed by incubation for 30 h at 25 °C with a rotating
speed of 200 rpm. Then the optical density (OD) of cells was measured using
spectrophotometer with the wavelength of 600 nm. The value of that could be taken
as a representative to immediately measure cell concentration, thereby
determining the percentage of pre-culture that used for further inoculation. As the
OD value of 20 was needed in this experiment, the pre-culture media was mixed
with supplementary culture media containing only 6 g L-1 peptone and 5 g L-1 yeast
extract. Subsequently, 10% (v/v) of the mixture with OD value of 20 was inoculated
into fermentation culture in 500 mL Erlenmeyer shake flask, stored in the incubator
for 8 days at 25 °C with the same shaking speed as pre-culture incubation. All
inoculation procedures were carried out under aseptic condition (Dolman et al.,
2019).
3.1.3.2 Isolation and Purification
3.1.3.2.1 Chemicals and Solvents
Solvent extraction was carried out for SLs isolation and purification, where ethyl
acetate (VWR, UK) and n-hexane (Fisher Scientific, UK) were used.
3.1.3.2.2 Experimental Procedure
Equal volume of n-hexane to culture media was firstly added into broth in order to
remove residual oil, thus the oil was extracted with the solvent in the supernatant.
After washing the broth with n-hexane twice and pipetting out the supernatant, SLs
was isolated by adding equal volume of ethyl acetate to the rest media broth
(Dolman et al., 2017). The solvent phase consisting of ethyl acetate and SLs was
separated from the broth by gravimetric method with the help of separating funnel.
In order to get rid of ethyl acetate and achieve purified SLs product, this solvent
phase was evaporated using rotary evaporator. Extracted SLs was stored in a
bottle and kept in the fridge at around 4 °C for further analysis.
82
3.1.3.3 SLs Concentration Determination
3.1.3.3.1 Gravimetric Method
Ethyl acetate (VWR, UK) and n-hexane (Fisher Scientific, UK) were applied in the
concentration determination on SLs using gravimetric method.
Gravimetric method for SLs concentration determination was carried out right after
the fermentation. 3 mL media broth was pipetted into centrifuge tubes. Equal
volume of n-Hexane (3mL) was twice added into the broth to extract the residual
oil, presenting in the upper layer. After removing this supernatant, media broth that
left in the tube was mixed with equal volume of ethyl acetate. With the help of
vortex to achieve a well mixing and complete extraction, glycolipids were fully
dissolved in ethyl acetate in the supernatant. Then this supernatant was poured
into pre-weighed drying dishes, denoted as W 1,0. After being left in the fume
cupboard for 24 h, the solvent was fully evaporated and the dish was weighed and
denoted as W 1. Thus the concentration of glycolipids can be estimated using
Equation 3.1,
𝑊1 − 𝑊1,0
× 100% 3.1
𝑉
Where, W1 is the dish and dried SLs, W1,0 is pre-weighed dish, V is the media broth
3.1.3.3.2 Exploratory Measurement with high performance

liquid chromatography (HPLC)
Acetonitrile in HPLC grade for gradient analysis (Fisher Scientific, UK), and water
in HPLC grade (Fisher Scientific, UK) were used in the measurement.
High performance liquid chromatography (HPLC) for SLs concentration analysis

was preliminary carried out, with the help of UltiMate 3000 instrument equipped
with a UV detector. C18 column was selected as the analytical column.
Sample for the measurement was prepared by scooping a quarter spoon amount
of extracted SLs (nearly 50 mg) using a Nickel Dual Spoon/Spatula utensil (Fisher
Scientific, UK) and fully dissolving in 20% (v/v) acetonitrile solvent. The mixture
was then filtered through a 0.22 µm membrane and stored in HPLC sample vials
(Dolman et al., 2017). Five bottles were prepared of the measurement.
The parameters for the measurement were pre-set and displayed in Table 3.1
(Dolman et al., 2017). 20 µl sample solution was injected into HPLC, and then
being measured according to the settings.
83
Table 3.1 Default settings for HPLC measurement for analysing SLs concentration
(Dolman et al., 2017)
Parameters Input
Elution Method Gradient
Mobile Phase Acetonitrile-Water
Elution Procedure Concentration of acetonitrile was increased from

20% to 70%
Elution Duration (min) 75
Flow Rate (mL min-1) 1
Wavelength of UV 207
Detector
3.2 Mannosylerythritol Lipids (MELs) Production

3.2.1 Producing Microorganisms
Pseudozyma aphidis DSM 70725 was selected as the producing strain for MELs
production. As train was freshly purchased, working stock was prepared prior to
the experiment. The purchased strain was streaked onto an agar plate containing
30 g L-1 glucose, 1 g L-1 NH4NO3, 0.3 g L-1 KH2PO4 and 1 g L-1 yeast extract, then
grown for 2 days at 30 °C (Dolman et al., 2019). Single colonies were inoculated
from agar plate to 50 mL cultivation media, followed by incubation for 30 h at 30 °C
with the rotating speed of 200 rpm. The media broth was centrifuged, after which
sterile media was added to replace the supernatant. After few times of this
refreshment, 15 mL of 30% glycerol, 3 mL of 30 g L-1 glucose and media was mixed
together and added up to 50 mL. 1 mL of the mixture was aseptically transferred
into each cryovial using sterile pipette tips and stored at -80 °C, as working stock
for further use.
3.2.2 Chemicals
Chemicals that used for MELs production included monohydrate glucose,
Ammonium Nitrate (NH4NO3), Monopotassium Phosphate (KH2PO4), yeast extract,
Sodium Nitrate (NaNO3), Magnesium Sulfate Heptahydrate (MgSO4·7H2O) and
Crisp ~N Dry oil. Except Crisp ~N Dry oil as the rapeseed oil source which was
purchased form supermarket, other chemicals were obtained from Sigma Aldrich.
The purification of MELs was also performed using solvent ethyl acetate and n-
Hexane.
84
3.2.3 Production Strategies
3.2.3.1 Fermentation Technology
Shake flask fermentation was initially carried out for the production of MELs, which
was partially adapted from the strategy applied for SLs production (Dolman et al.,
2019). The strain was inoculated from stock culture to agar plate for cultivation of
2 days at 30 °C. Single colonies were transferred and incubated in 250 mL
containing 25 mL pre-culture media (seed culture) [30 g L-1 glucose, 1 g L-1 NH4NO3,
0.3 g L-1 KH2PO4, 1 g L-1 yeast extract] at 30 °C under rotating of 200 rpm. After 2
days of incubation in pre-culture, the optical density of cells was measured to get
a preliminary understanding of the growth condition. After diluting the cell
concentration to OD value of 20, 10 % (v/v) of seed culture was sterilely added into
500 mL Erlenmeyer flask containing 50mL culture media [30 g L-1 glucose, 72 g L-
1
rapeseed oil, 2 g L-1 NaNO3, 0.2 g L-1 KH2PO4, 0.2 g L-1 MgSO4·7 H2O, 1 g L-1
yeast extract], followed by the cultivation of 10 days at 30 °C in the incubator with
a shaker rotating at 200 rpm.
Fed-batch fermentation was performed afterwards, aiming to achieve higher

production of MELs. In Fed-batch culture, concentrated media containing 500 g L-
1
glucose, 28 g L-1 NaNO3, 24 g L-1 yeast extract was added into each experimental
Erlenmeyer flask, as well as the Crisp ~N Dry oil offered rapeseed oil. According
to the analysis of pre-culture maximum consumption rate of glucose, NaNO3 and
yeast extract by Rau L et al (Rau et al., 2005b), the feeding rate of concentrated
medium was set as 0.1 mL h-1, and that of oil was set as 0.02 mL h-1. They were
added into the culture media after 4 days of cultivation.
3.2.3.2 Isolation and Purification
3.2.3.2.1 Chemicals and Solvents
Ethyl acetate (VWR, UK), n-hexane (Fisher Scientific, UK) and methanol in
analytical grade (Fisher Scientific, UK) were used during this procedure.
3.2.3.2.2 Experimental Procedure
Solvent extraction was also applied for MELs purification. After 10 days of batch
cultivation and 20 days of fed-batch cultivation, the culture broth was mixed with
an equal volume of ethyl acetate to extract MELs, where the upper organic phase
was separated. Vacuum rotary evaporator was then applied to get rid of solvent
and then the sticky crude MELs product was obtained. Three-time wash of the
85
crude MELs was carried out using the solvent of Hexane-methanol-water (1:6:3)
mixture, where two separated phases were obtained: one is the upper organic
phase containing oil and fatty acid, the other is the aqueous phase containing
MELs. After that, the aqueous layer was washed with hexane twice and the solvent
was then evaporated, followed by a freeze drying to get rid of water.
3.3 Preliminary Trials on Cream Formulation

At very first beginning, creams were formulated to investigate a feasible recipe and
proper mixing apparatus, thus this chapter conclude the exploratory experiments
for cream formulation. The recipe was preliminary created based on E45 cream,
where only active ingredients and some specified surfactants were applied. And
the weight concentration for each component was determined based on a nigh
cream formula from a formulation book (Flick, 2001).
3.3.1 First Trial for Formulation of Cream without Sodium

Lauryl Ether Sulfate (SLES), Using a Homogenizer
3.3.1.1 Chemicals
A trial cream was preliminary prepared, where light liquid paraffin (Scientific
Laboratory Supplies), white soft paraffin (Fisher Scientific), 1-Hexadecanol (Cetyl
Alcohol/CA) (95%, Sigma-Aldrich) and deionized water were applied in the
formulation.
3.3.1.2 Recipes
400 g of mimic cream was formulated, where only cetyl alcohol was applied as the
emulsifying agent in the formulation. Details of the composition is introduced in
Table 3.2.
Table 3.2 Formulation of first trial cream, with cetyl alcohol as sole emulsifying agent
Ingredients Weight Concentration Mass

(wt%) (g)
Oil Phase (A)

White soft paraffin 14.5 58
Light liquid paraffin 12.6 50.4
Cetyl alcohol 12 48
Aqueous Phase (B)

Deionized water 60.9 234.6
Fragrance and preservatives N/A
86
3.3.1.3 Apparatus and Configurations
A homogenizer (IKA T25 Ultra Turrax Homogenizer, IKA England LTD) was
applied for preparing cream at the first trial, equipped with a PYREX beaker of 500
mL as the mixing vessel. A stir and heater was used as the heating source for the
mixing.
3.3.1.4 Formulation procedure
Cream was prepared following the procedure introduced below:
1. White soft paraffin, liquid paraffin and CA were weighed separately using
an electronic scale, followed by mixing together in a laboratory beaker and
heating up to 70 °C with the help of a stir and heater. Then the beaker
containing oil phase mixture was kept in a water bath for keeping
temperature constant.
2. Specific amount of deionized water was measured using a cylinder and
then added into the mixing beaker. While being heated to reach 70 °C by
the heater, water was also being stirred using homogenizer at lower speed.
3. Oil phase was slowly poured into aqueous phase while mixing at 8000 rpm
using the homogenizer, and temperature was controlled at 70 °C.
4. Leave the mixture of oil phase and aqueous phase to be mixed for 10
minutes. Regularly check the temperature to maintain it at 70 °C.
5. After 10 min of mixing, the heater and stirrer was powered off. The cream
was cooled down for 10 min and reached room temperature (25±2 °C), by
immersing the mixing beaker in another plastic container filled with tap
water.
3.3.2 Second Trial for Formulation of Cream with Sodium

Lauryl Ether Sulfate (SLES), Using an overhead stirrer
For the second trial, sodium lauryl ether sulfate (SLES) was added into the formula
and an overhead stirrer was applied instead of the homogenizer for mixing.
3.3.2.1 Chemicals
Light liquid paraffin (Scientific Laboratory Supplies), white soft paraffin (Fisher
Scientific), 1-Hexadecanol (Cetyl Alcohol/CA) (95%, Sigma-Aldrich), Sodium
Laureth Sulfate (SLES) (Scientific Laboratory Supplies) and deionized water were
applied in the formulation.
87
3.3.2.2 Recipes
400g of mimic cream was formulated, where CA and SLES were applied as mixed
emulsifying agents in the formulation. Details of the composition is introduced in
Table 3.3.
Table 3.3 Formulation of second trial cream, with cetyl alcohol and SLES as mixed
emulsifying system
Ingredients Weight Concentration Mass
(wt%) (g)
Oil Phase (A)

White soft paraffin 14.5 58
Cetyl alcohol 6 24
Aqueous Phase (B)
Deionized water 60.9 234.6
SLES 6 24
Fragrance and N/A
preservatives
3.3.2.3 Apparatus and Configurations
A modification in the configuration of formulation was made in the second trial of

cream preparation. An overhead stirrer (IKA Overhead Stirrer, RW 20 digital, IKA
England LTD) equipped with a pitched 6-blade impeller, which was an agitator
providing axial flow, was introduced to replace the homogenizer.
As sketched in Figure 3.1, along with the photo of overhead stirrer, this simplified
configuration consisted of a 500 mL beaker (PYREX, USA) that used as the mixing
vessel, an overhead stirrer and a heater (IKA Magnetic Stirrer C-MAG HS 7, IKA
England LTD).
88
Oil
phase
Aqueous
phase
Figure 3.1 Schematic diagram of simplified configuration and photo of overhead stirrer
3.3.2.4 Formulation procedure
Cream was prepared following the procedure introduced below:
1. Oil phase components, including white soft paraffin, liquid paraffin and CA,
were weighed separately using an electronic scale, followed by mixing
together in a laboratory beaker and heating up to 70 °C with the help of a
stir and heater. Then the beaker containing oil phase mixture was kept in a
water bath for keeping temperature constant.
2. Aqueous phase consisted of SLES and water. SLES was weighed using
electronic scale. Specific amount of deionized water was then measured
using a cylinder and added into the mixing beaker. The mixture was heated
up to 70 °C, while mixing using the agitator at lower mixing speed (200 rpm).
3. Oil phase was slowly poured into aqueous phase, followed by being mixed
at 500 rpm for 10 min, and temperature was controlled at 70 °C. Regularly
check the temperature to maintain it at 70±2 °C.
4. After 10 min of mixing, the heater and stirrer was powered off. The cream
was cooled down for 10 min and reached room temperature (25±2 °C), by
immersing the mixing beaker in another plastic container filled with tap
water.
89
3.4 Modified and Standard Experimental Procedure for
Cream Formulation
Based on the previous trials for cream formulation, the standard formulation
system was established, where the selection of emulsifying system, the
composition and preparation process were determined. This chapter will introduce
the modified cream formulation process, where creams were formulated in lab
scale, with different emulsifying systems consisting of various concentration of
surfactant components. In this thesis, those formulated using chemically
synthesized surfactants are named mimic creams, and those involved bio-
surfactant are bio-creams.
3.4.1 Chemicals
Ingredients applied in the formulation included: light liquid paraffin (Scientific
Laboratory Supplies), white soft paraffin (Fisher Scientific), Groovy Food Organic
Extra Virgin Coconut Oil, Stork Original Baking Block (containing 75% vegetable
oils), 1-Hexadecanol (Cetyl Alcohol/CA) (95%, Sigma-Aldrich), Glycerol
Monostearate (GM) (purified, Alfa Aesar), Sodium Laureth Sulfate (SLES)
(Scientific Laboratory Supplies), biosurfactants (SLs and MELs that produced in
lab), deionized water. As summarised in Table 3.4, these ingredients are classified
into different groups according to roles that they played in the formulation.
Table 3.4 Classification of ingredients in the cream formulation
Phases Components
Oils Mixed paraffin oils Light liquid paraffin mixed with white
soft paraffin
Bio-oils Groovy Food Organic Extra Virgin
Coconut Oil, Stork Original Baking
Block
Emulsifying Chemical Sodium laureth sulfate, 1-
system surfactants Hexadecanol (cetyl alcohol), glycerol
monostearate
Biosurfactants Sophorolipids, mannosylerythritol
lipids
Water Deionized water
3.4.2 Recipes
3.4.2.1 Formulation_Ⅰ
The selection of oil and surfactants, and the determination of oil concentration was
referenced from the recipe of E45 cream. In order to formulate a mimic cream
90
exhibiting similar performance to the E45, recipes were created with different
surfactant compositions in the emulsifying system. This began with the formulation
of a night cream in Flick’s book (Flick, 2001), after which a few groups of
emulsifying systems were applied in the formulation. These mimic creams were
prepared in Formulation_Ⅰ, details of which is presented in Table 3.5.
Based on different compositions of fatty alcohols (cetyl alcohol and glycerol

monostearate), 16 creams, 50 g of each, were prepared and classified into four
groups, denoted as F1, F2, F3 and F4, where different concentrations of sodium
laureth sulfate (SLES) were involved. An assumption was made that 5 wt% of
residuals were not applied in the Formulation_ Ⅰ , such as fragrances and
preservatives.
Table 3.5 Formulation_Ⅰ of mimic creams prepared with varied proportion of

surfactant system
Mimic Creams
F1 F2 F3 F4
Ingredients
Component (wt%)
White soft
14.5 14.5 14.5 14.5
paraffin
Light liquid
12.6 12.6 12.6 12.6
paraffin
SLES 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6
Cetyl Alcohol
6 6 2 2
(CA)
Glycerol
Monostearate 6 2 6 2
(GM)
Deionized water added up to 95
Residuals 5
3.4.2.1 Formulation_Ⅱ
In order to further investigate the effect varied concentrations of fatty alcohols on

the performance of creams, Formulation_Ⅱ was prepared, where two groups of
creams were formulated with different concentrations of CA in two emulsifying
systems containing different concentration of SLES, denoted as F5 and F6
91
separately. The composition of Formulation_Ⅱ was introduced in Table 3.6. 50 g
of each cream was prepared.
Table 3.6 Formulation_Ⅱ of mimic creams prepared with varied concentrations of

fatty alcohols
Mimic Creams
F5 F6
Ingredients
Component (wt%)
White soft paraffin 14.5 14.5
SLES 2 4
CA 5 6 7 5 6 7
GM 2 2
Deionized water added up to 95
residuals 5
3.4.2.2 Formulation_Ⅲ
After preliminary analysis of mimic creams formulated with different concentrations

of chemically synthesized surfactants in Formulation_Ⅰ and Formulation_Ⅱ, the
recipe was optimized and determined for bio-creams preparation. In order to
compare the different performance between mimic creams and bio-creams, those
mimic creams containing specific concentration of surfactants were freshly
prepared in Formulation_Ⅲ. Details of the formulation were displayed in Table 3.7.
other components such as preservatives, fragrances and viscosity enhancers were
also not considered in this formulation, with an assumption of 5 wt% as residuals.
In addition, in replacement of paraffin mixed oils consisting of white soft paraffin

and light liquid paraffin, plant oils including coconut oil and vegetable shortening
were introduced as bio-oils in the Formulation_Ⅲ for the preparation of eco-friendly
products. Vegetable shortening is a fat made from vegetable oil which is in solid
state at room temperature.
As a summarise, in Formulation_Ⅲ, nine big groups of creams were formulated,

namely, group P1, P2 and P3, referring to creams that formulated using paraffin
mix oils (white soft paraffin and light liquid paraffin) with SLES, SLs and MELs as
surfactants respectively; group C1, C2 and C3, referring to creams that formulated
using coconut oil instead; group V1, V2 and V3, referring to creams that formulated
92
using vegetable shortening with SLES, SLs and MELs as surfactants respectively.
Prepared creams were stored in wide-opened plastic bottles for further analysis.
93
Table 3.7 Formulation_Ⅲ of mimic creams and bio creams with optimized surfactant system
Mimic creams (P1) Bio-SLs-creams (P2) Bio-MELs-creams (P3)
Ingredients Component Ingredients Component Ingredients Component
(wt%) (wt%) (wt%)
Paraffin mix 27.1 Paraffin mix 27.1 Paraffin mix 27.1
SLES 2 4 6 Sophrolipids 2 4 6 MELs 2 4 6
CA 6 6 6 CA 6 6 6 CA 6 6 6
GM 2 2 2 GM 2 2 2 GM 2 2 2
Deionized water Up to 95 Deionized water Up to 95 Deionized water Up to 95
Mimic creams (C1) Bio-SLs-creams (C2) Bio-MELs-creams (C3)

(wt%) (wt%) (wt%)
Coconut oil 27.1 Coconut oil 27.1 Coconut oil 27.1
CA 6 6 6 CA 6 6 6 CA 6 6 6
GM 2 2 2 GM 2 2 2 GM 2 2 2
Mimic creams (V1) Bio-SLs-creams (V2) Bio-MELs-creams (V3)

(wt%) (wt%) (wt%)
Vegetable shortening 27.1 Vegetable shortening 27.1 Vegetable shortening 27.1
CA 6 6 6 CA 6 6 6 CA 6 6 6
GM 2 2 2 GM 2 2 2 GM 2 2 2
94
3.4.3 Apparatus and Configurations
3.4.3.1 Simplified Configuration
The simplified configuration applied for Formulation_Ⅰ and Formulation_ Ⅱ of

cream formulation, was similar to the one introduced in chapter 3.3.2.3 (see Figure
3.1), including a 300 mL Tall-form beaker (PYREX, USA), an overhead stirrer (IKA
Overhead Stirrer, RW 20 digital, IKA England LTD) with a pitched blade impeller
and a heater (IKA Magnetic Stirrer C-MAG HS 7, IKA England LTD). The cooling
procedure was independent from this, which is realised by removing the beaker
from the configuration followed by immersing in a big plastic container filled with
cold tap water.
3.4.3.2 Continuous Configuration
By upgrading the simplified apparatus, a lab-scaled stainless jacket container,

used as the mixing vessel, was designed to replace the previous Tall-form beaker,
which realized the continuous heating and cooling procedure. This continuous
apparatus and its corresponding parameters are presented in Figure 3.2.
For assembling this refined configuration, a Thermos/HAAKE DC1-L Heating

Circulator Bath (Thermo Scientific HAAKE, Germany) was used for maintaining the
temperature while mixing, connecting to the mixing vessel using heat resistant
silicon rubber tubes. Rubber tube (a) was connected water bath out let with vessel
inlet, and tube (b) was between vessel outlet and water bath inlet.
For cooling, rubber tube (c) controlled the transportation of cold water from the
water tap, and circulated cooling was realized by simultaneously piping out water
to the storage sink with tube (d) opened. Each rubber tube was equipped with a
stainless-steel clamp for flow control as required.
95
Clam Clam
Storage p p
Sink (d) (b)
H Water
Bath
Clamp Clamp
Water Tap D
(c) T (a)
Parameters Values
D (mm) 60
T (mm) 70
H (mm) 137
Figure 3.2 Schematic diagram of continuous configuration and corresponding container

parameters that applied in Formulation_Ⅲ
3.4.4 Preparation Procedure for Standard Formulation
3.4.4.1 Formulation_Ⅰand Formulation Ⅱ
The preparation procedure could be referred to that described in chapter 3.3.2.4.

Tiny change was made according to the composition of oil phase and aqueous
phase, which is specified in Table 3.8.
Table 3.8 Ingredients for cream preparation in Formulation_Ⅰand Formulation_Ⅱ
Oil phase Aqueous Phase
Ingredients
White Soft Paraffin Deionized Water
Light Liquid Paraffin
Cetyl Alcohol (CA) Sodium lauryl ether sulfate
Glycerol Monostearate (GM) (SLES)
3.4.4.2 Formulation_Ⅲ
Creams (50g of each) in Formulation_ Ⅲ were prepared using continuous

configuration. The procedure for the cream preparation was introduced as below:
96
1. Oil phase consisting of different oils, CA and GM, was prepared, where
those components were weighed separately and mixed together in a
beaker, followed by melting at 70 °C using a stir and heater.
2. Liquid phase was then prepared while oil phase was kept homothermal by
the heater. Surfactant in aqueous phase including SLES, SLs and MELs
was weighed in the jacket container (mixing vessel), as required based on
the recipe. Then specific amount of deionized water measured using
cylinder was added.
3. The configuration was set up, where rubber tubes were applied to connect
water bath, jacked vessel and water tap. As specified before, tubes were
numbered (a) water bath outlet and vessel inlet, (b) water bath inlet and
vessel outlet, (c) vessel inlet and water tap and (d) vessel outlet and sink.
4. Lower down the stainless-steel impeller in order to make sure that the
pitched blade was fully submerged in the water phase mixture. Throttle the
connection between mixing vessel and water tap (c and d) and turn on the
water bath to fill the jacked of container. Adjust the temperature of water
bath and set to 72±2 °C. Meanwhile, power on the stirrer in order to mix
aqueous phase at 200 rpm.
5. Monitoring the temperature in mixing vessel using a thermometer. When it
reached to 70±2 °C, oil phase was added into the aqueous phase and the
mixing speed was increased to 500 rpm.
6. After 10 minutes mixing, water bath was turned off immediately and the
speed of agitator was turned down to 200 rpm. Then the clamp on tube (c)
and (d) was removed, while flow between water bath and mixing vessel
was chocked by clamping tube (a) and (b). Turn on the water tap in order
to cool the cream down for another 10 minutes to reach the room
temperature.
7. When the preparation finished, tubes were unplugged from the nozzles of
water bath and the tap, and the rest circulated water in the jacket of the
container was poured out into storage sink for the reuse in the water bath.
Creams were transferred into 100 mL wide-open plastic pots.
3.5 Modification of Preparation Process

Effects of different mixing time, mixing speed and different cooling procedure on
cream formulation was studied separately, where a model cream was prepared
using different procedures and cream performances were analysed with the help
of droplet size distribution analysis and rheological measurements.
97
3.5.1 Formulation of Model Creams
50 g of each model cream was prepared according to the recipe presented in Table
3.9.
Table 3.9 Formulation of model creams used for studying the effect of different
manufacturing strategies on cream performance
Weight Weight Weight

Component concentration concentration concentration
(wt%) (wt%) (wt%)
White soft paraffin 14.5 14.5 14.5
Light liquid
12.6 12.6 12.6
paraffin
SLES 2 4 6
CA 6 6 6
GM 2 2 2
Residules
(not in the 5 5 5
formulation)
Deionized water added up to 100 added up to 100 added up to 100
3.5.2 Preparation Procedure with Different Mixing Time During

Heating Procedure
Effect of different mixing time on the cream performance was studied with the help
of droplet size distribution measurement. Model cream was prepared following
recipe mentioned above, in the simplified configuration (see Figure 3.1). The
measurement was carried out following the procedure:
1. Oil phase consisting of white soft paraffin, liquid paraffin, CA and GM, was
prepared, where those components were weighed separately and mixed
together in a beaker, followed by melting at 70 °C using a stir and heater.
Then the beaker containing oil phase mixture was kept in a water bath for
keeping temperature constant.
2. Liquid phase was then prepared while oil phase was kept homothermal in
the water bath. SLES was weighed in another beaker using as the mixing
vessel, then specific amount of water was added. Then the configuration
was set up, where the heater and overhead stirrer was assembled properly.
3. Put the mixing beaker containing liquid phase mixture on the heater, then
lower the stainless steel impeller in order to make sure the pitched blade
fully submerged in the mixture. Turn the heater on. The temperature was
set at 90 °C at the beginning, and controlled by a thermometer at around
98
70°C while mixing. Meanwhile, stirrer was powered on and mixing speed
was set at 200 rpm.
4. When the temperature of liquid phase reached and maintained at 70 °C, oil
phased was poured into aqueous phase, and the mixing speed was
increased to 500 rpm.
5. 3 mL sample was then sequentially pipetted out from the mixing vessel at
different mixing times of 3 min, 5 min, 10 min, 15 min and 20 min, marking
as cream sample A, B, C, D and E, which is summarised in Table 3.10.
Table 3.10 Parameters of different mixing durations applied for study the effect of
different mixing procedure on product performance
Mixing Procedure
Cream Sample Mixing Speed Mixing Duration
(rpm) (min)
A 3
B 5
C 500 10
D 15
E 20
6. Each cream sample was directed to Mastersizer 3000 for droplet size
distribution analysis.
3.5.3 Preparation Procedure with Different Mixing Speed

During Heating Procedure
Effect of different mixing speed during heating procedure on the performance of
cream was studied, and droplet size distribution measurement was carried out for
the analysis. Model cream was prepared using the recipe specified in Table 3.9,
using simplified configuration. The measurement was carried out following the
procedure:
1. Preparation of oil phase and liquid phase, also the setting up of

configuration could be refer to the procedure introduced in chapter 3.5.2.
2. Creams A, B and C were then separately prepared at three different mixing
speed of 500 rpm, 700 rpm and 900 rpm (Boxall et al., 2010). For each
cream, mixing time of 10 min was pre-set. Then each of 1 mL hot cream
was pipetted out from the mixing vessel and transferred into different 20 mL
glass vials. These 1 mL sample was prepared for the following droplet size
distribution analysis. Mixing parameters are summarised in Table 3.11.
99
Table 3.11 Specification of different mixing speeds during heating procedure, applied
for study the effect of different mixing procedure on product performance, modified
from Boxall et al., 2010
Mixing Procedure
Cream Sample Mixing Speed Mixing Duration
(rpm) (min)
A 500
B 700 10
C 900
3.5.4 Preparation Procedure with Different Cooling Procedure

Effect of different cooling procedure on the performance of cream production was
studied, creams named A, B, C, D and E were respectively prepared with different
cooling procedure (Rønholt et al., 2014), and then the cream products were
analysed by rheological measurement. The mixing procedure was kept constant
for each cream, and the continuous configuration was applied. Parameters for
different cooling procedures were introduced in Table 3.12.
Table 3.12 Specification of different cooling procedures applied for study the effect of
different cooling procedures on product performance, adapted from Rønholt et al.,
2014
Mixing Procedure Cooling Procedure
Cream Mixing Mixing Cooling

Stirring speed
Speed Duration Duration
(rpm)
(rpm) (min) (min)
A 200 10
B 0 10
C 500 10 300 10
D 200 5
E 200 20
The procedure for the cream preparation could be referenced from that of
Formulation_Ⅲ in chapter 3.4.4.2. After resting for 20 min, prepared creams were
analysed with the help of rheometer.
3.6 Characterisation Methods

Creams were characterised using rheological measurements, for analysing their
flow properties, and differential scanning calorimetry, for analysing their
thermodynamic properties. Microscopy and droplet size distribution were also
100
conducted on some desired creams for providing information for microstructure
analysis.
3.6.1 Rheology
Rheological test is a useful method for rapidly predicting the performance of a
material such as spreadability, rigidity and thixthotropy, where non-linear steady
state rotational test and linear oscillatory test are two main rheological
characterisation methods. Basic principles and background knowledge of rheology
applied in this study will be preliminary introduced, mainly including viscosity with
corresponding flow models and viscoelasticity with corresponding models.
3.6.1.1 Theory of Flow Behaviour
The two-plate model generally used to express the rotational tests and define
rheological parameters, where flow goes through two parallel plates (Barnes et al.,
1989). An external force is applied constantly to the upper plate along positive
direction of axis resulting a velocity, while the lower plate is stationary. With the
assumption that no wall-slip effects and laminar flow is involved, the adherence of
flow to surfaces of both plates and the flow is imagined in the form of numerous
layers that clinging to each other. The flow rate of one flow layer is different from
another, leading to relative movement and velocity gradient between flow layers
and the velocity. Therefore, a shear force F which is parallel to the flow layer arises
between two layers. If the shear area is A, the shear stress τ can be expressed in
Equation 3.2,
𝐹
𝜏= 3.2
𝐴
Where, τis shear stress, F is shear force, A is shear area
Shear strain 𝛄 is defined as the displacement (deformation) of the plate ( Δx)

divided by the distance between two plates (Δy), shown in Equation 3.3,
∆𝑥
𝛾= 3.3
∆𝑦
Where, γis shear strain, Δx is displacement of the plate, Δy is distance between

two plates
Shear rate 𝛄̇ is defined as the time rate of shear strain, which is notated using 𝛾̇
with a unit of s-1, shown in Equation 3.4. This value is applied to indicate the flow
velocity u.
101
𝑑𝛾 𝑑 𝑑𝑥 𝑑 𝑑𝑥 𝑑𝑢
𝛾̇ = = ( )= ( )= 3.4
𝑑𝑡 𝑑𝑡 𝑑𝑦 𝑑𝑦 𝑑𝑡 𝑑𝑦
Where, 𝛾̇ is shear rate, u is flow velocity
For Newtonian fluids, shear stress is proportional to the velocity gradient, and the
coefficient is known as viscosity μ, with a unit of Pa∙s, which is shown in Equation
3.5 and 3.6.
𝑑𝑢
𝜏 = −𝜇 ( ) 3.5
𝑑𝑦
𝜏
𝜇= 3.6
𝛾̇
Where, μis the viscosity for Newtonian fluids
Viscosity μ is constant for Newtonian fluids, indicating an independent of internal

flow resistance is independent of external forces. Whereas for non-Newtonian
fluids, known as structured or complex fluids, the viscosity η is inconstant that
alters with the external stress (see Equation 3.7). The classification of non-
Newtonian fluids is shown in Table 3.13, and their flow behaviours are plotted in
Figure 3.3, displaying shear stress (τ) and viscosity (η) dependent on shear rate
(𝛾̇ ) (Mezger, 2020).
𝑑𝑢
𝜏 = −𝜂 ( ) 3.7
𝑑𝑦
Where, η is the viscosity for non-Newtonian fluids
102
Table 3.13 Classification of Non-newtonian fluids, according to Mezger, 2020
Categories Classification
Time Newtonian fluid
independent Pseudoplastic fluid
Dilatant fluid
Pure
viscous
fluid Bingham’s fluid Non-
Yield- Pseudoplastic fluid Plastic Newtonian
Yield- dilatant fluid fluid fluid
Time Thixotropic fluid

dependent Rheopectic fluid
Viscoelastic fluid More types of fluid
Figure 3.3 Flow behaviour of fluids plotted in shear stress-shear rate (left) and
viscosity-shear rate (right) diagram, according to Mezger, 2020
3.6.1.2 Theory of Rheological Measurements
Various rheological measurements were carried out experimentally to study the

flow properties of materials, such as steady state shear test, dynamic oscillatory
sweep test, creep-recovery test and stress relaxation test. Generally, these
experiments are carried out by exerting an external force (shear or sweep) on the
product sample, simulating conditions that encountered during product life, and the
obtained rheological profiles will be introduced in this part.
103
3.6.1.2.1 Steady state rotational shear test (non-linear)
Steady state rotational test involves forcing sample being sheared under increased
stress or rate within pre-set range. Through simulating processes that the sample
will experience in real practice such as spreading, the rheological properties
including shear thinning or thickening behaviour and apparent viscosity could be
predicted (Mezger, 2020). Figure 3.4 schematically illustrates the sample laded
between bob (cone in the fig) and plate geometry, provided with the generate shear
profile. The profile could be interpreted with two-parallel plate model where flows
are depicted as layers sliding over each other.
Figure 3.4 Schematic diagram of steady state shear and generated shear profile,
according to Mezger, 2020
Rheological profile of time-independent shear thinning fluids
Rotationally shearing sample within a wide range of shear stress from low to high,
the change of apparent viscosity of a sample with increased shear stress is
obtained, and the rheological profile is usually logarithmic presented. Take shear
thinning fluid as an example, a typical S-shape flow curve is generally achieved
and plotted in a log (viscosity)-log (shear rate 𝛾̇ or shear stress 𝜏) graph, shown in
Figure 3.5 (Tatar et al., 2017). During 1st Newtonian plateau, zero shear viscosity
(η0 ) indicates the strength of system microstructure to resistant external forces,
after exceeding the yield stress, it starts to flow, and another plateau will be
achieved when molecules already realigned in a same direction and no further
decrease in viscosity witnessed, showing infinite shear viscosity (η∞ ). In addition,
the orange curved line in the figure between 1st Newtonian Plateau and shear
104
thinning is defined as the transition region where the microstructure of system
starts to alter.
𝑙𝑜𝑔 𝜂
Cross/ Bird-Carreau-Yasuda model
st
1 Newtonian Shear Thinning 2nd Newtonian Plateau
Plateau
𝜂0
Ellis model
𝜂∞
Sisko model
𝑙𝑜𝑔 𝛾̇ or 𝑙𝑜𝑔𝜏
Figure 3.5 Qualitative S-shape rheological curve for typical shear thinning fluids and
corresponding model fitting range, according to Tatar et al., 2017
Various mathematical models were developed and applied to interpret time-

independent non-Newtonian flow behaviours. The constitutive equations of non-
Newtonian models are summarised in Table 3.14 (Mezger, 2020), where τ is the
shear stress, 𝛾̇ is the shear rate, and the apparent viscosity (effective viscosity) is
notated as 𝜂𝑒𝑓𝑓 . The application of models fitting in the S-shape curve is presented
in Figure 3.5.
Table 3.14 Non-Newtonian models with constitutive equations, according to Mezger,

2020
Models Constitutive equations
Bingham Model 𝝉𝒚
𝜼𝒆𝒇𝒇 = 𝜼𝒆𝒇𝒇,∞ +
𝜸̇
 Describe Bingham plastic
Where,
fluids which exhibit a  𝜏𝑦 is the yield shear stress
Newtonian behaviour (linear  𝜂𝑒𝑓𝑓,∞ is the limiting viscosity of
relationship between shear plastic fluids above the yield
stress
stress and shear rate) when
above yield point
Ostwald-de Waele (power law) 𝜼𝒆𝒇𝒇 = 𝒌(𝑻) ∙ 𝜸̇ 𝒏−𝟏

Model Where,
105
 Represent shear thinning  k is the flow consistency index
(dependent on temperature)
region in 𝒍𝒐𝒈𝜼 − 𝒍𝒐𝒈𝜸̇ or
 n is the flow behaviour index
𝒍𝒐𝒈𝜼 − 𝒍𝒐𝒈𝝉 curve
 Cannot fit in 1st Newtonian
plateau
Herschel-Bulkley Model
 Combination of Bingham and 𝝉 = 𝝉𝒚 + 𝒌(𝑻) ∙ 𝜸̇ 𝒏
power law model Where,

 k is the flow consistency index
 Describe the fluids which (dependent on temperature)
exhibit shear thinning  n is the flow behaviour index
behaviour (non-linear
relationship between shear
stress and shear rate) when
above yield point
Bird-Carreau-Yasuda Model 𝜂𝑒𝑓𝑓 (𝛾̇ ) − 𝜂∞ 𝑛−1

= (1 + |𝜆 ∙ 𝛾̇ |𝑎 ) 𝑎
𝜂0 − 𝜂∞
 Interpret 1st Newtonian
𝜂𝑒𝑓𝑓 (𝛾̇ ) = 𝜂∞ + (𝜂0 − 𝜂∞ )
plateau and shear thinning 𝑛−1
∙ (1 + |𝜆 ∙ 𝛾̇ |𝑎 )𝑎
region in 𝒍𝒐𝒈𝜼 − 𝒍𝒐𝒈𝜸̇ curve
Where,
 Describe pseudoplastic flow or  𝜆 is the relaxation time constant,
thermoplastic materials for 1⁄ is the critical shear rate at
𝜆
which there is a typical which viscosity begins to
decrease
curvature of the viscosity in  𝑛 is the power law index, giving
the transient area. the degree of shear thinning
 𝑎 describe the width of the
 Involving two fitting transition region between low
parameters, 𝐧 and 𝛌 shear rate and when the power
law region starts, equals 2 in
original model
When the viscosity (𝜂∞ ) at infinite shear
rate is negligible, the model is simplified
as follow:
𝜼𝟎
𝜼𝒆𝒇𝒇 (𝜸̇ ) = 𝒏−𝟏
(𝟏 + |𝝀 ∙ 𝜸̇ |𝒂 ) 𝒂
Cross Model 𝜂𝑒𝑓𝑓 (𝛾̇ ) − 𝜂∞ 1
=
𝜂0 − 𝜂∞ 1 + 𝐾 ∙ 𝛾̇ )1−𝑛
(
 Similar to the Bird-Carreau-
Where,
Yasuda model, describing
 𝐾 is the cross constant,
both Newtonian and shear indicating the onset of shear
thinning
106
thinning behaviour in 𝒍𝒐𝒈𝜼 − When 𝜂∞ is negligible, the model is
𝒍𝒐𝒈𝜸̇ curve simplified,
𝜼𝟎
 Involving two fitting 𝜼𝒆𝒇𝒇 (𝜸̇ ) =
𝜼 ∙ 𝜸̇ 𝟏−𝒏
𝟏 + ( 𝟎𝝉∗ )
parameters, 𝐧 and 𝐊
𝜂
Where, 𝜏 ∗ = 0⁄𝐾
Ellis Model 𝜼𝟎
𝜼𝒆𝒇𝒇 (𝜸̇ ) = 𝜶−𝟏
 Describe time-independent 𝝉
𝟏 + (𝝉 )
𝟏⁄
𝟐
shear-thinning non-Newtonian
Where,
fluids in 𝒍𝒐𝒈𝜼 − 𝒍𝒐𝒈𝝉 curve
 𝜏1⁄ represents the shear stress
2
focusing on the 1st Newtonian when the apparent viscosity
𝜂
plateau and shear thinning 𝜂𝑒𝑓𝑓 decreased to 𝑒𝑓𝑓 ⁄2
region When 𝜂∞ is negligible, the model is
simplified,
Sisko Model 𝜼𝒆𝒇𝒇 (𝜸̇ ) = 𝑲 ∙ 𝜸̇ 𝒏−𝟏 + 𝜼∞

 Describe time-independent Where,
shear-thinning non-Newtonian
 K is the cross constant,
fluids in 𝒍𝒐𝒈𝜼 − 𝒍𝒐𝒈𝝉 curve indicating the onset of shear
thinning
focusing on the shear thinning
 n is the power law index
and 2nd Newtonian plateau
region
Rheological profile of time-dependent fluids
The flow properties of time-dependent non-Newtonian fluid, such as thixotropic and

rheopectic fluids, depend on both of the amount and the duration of external forces.
The hysteresis loop analysis is an applicable method for their study. As shown in
Figure 3.6, where shear stress against shear rate, thixotropic fluids presents a
clockwise loop while rheopectic fluids shows an anticlockwise one. The larger the
loop area, greater extend is the dependent on time (Maazouz, 2020). Conversely,
if the loop area is zero, flow behaviour of the material is time independent. Also the
area between curves represents energy loss of the system, and maximum viscosity
is identified from the apex.
107
Shear stress 𝜏
Thixotropic fluid
Rheopectic fluid
Shear rate 𝛾̇
Figure 3.6 Typical hysteresis loop of shear stress-shear rate behaviour for thixotropic
and rheopectic material, according to Maazouz, 2020
.
3.6.1.2.2 Creep and recovery test
Creep test is applied for the analysis of viscoelasticity of complex fluids, where the
sample is under a constant shear stress in linear viscoelastic region over a period
of time and the resultant shear strain is measured. In the following recovery step,
the stress is removed, and the shear strain in the system is measured for a period
of time. Hook’s Law, representing by spring as elastic response (Equation 3.8) and
Newton’s Law, representing by dashpot as viscous element (Equation 3.9), are
basic theories for viscoelasticity interpretation, which is schematically presented in
Figure 3.7 (Mezger, 2020).
Δx
F/τ Δx
F/τ
Figure 3.7 Schematic diagram of spring represents elastic behaviour (left) and dashpot
represent for viscous behaviour (right)
108
𝜎𝐺 = E ∙ ε𝐺 3.8
Where, σG is tensile stress, E is the Young’s modulus, εG is the spring strain.
𝑑𝛾𝜂
𝜏𝜂 = 𝜂 ∙ = 𝜂 ∙ 𝛾̇𝜂 3.9
𝑑𝑡
Where, τy is the shear stress, 𝛾̇𝜂 is the shear rate, η is the viscosity
The responses of linearly elastic material (spring element model) and viscous liquid
(dashpot element model) subjecting to creep and recovery test is presented in
Figure 3.8. When given an external force at constant shear stress of 𝜏0 from
𝜏
time 𝑡0 = 0 to 𝑡1 , the linearly elastic material responses an instant strain 𝜀0 = 0⁄𝐸
at 𝑡0 = 0, lasting until t1 when the load is removed (Figure 3.8 (b)). However, as
Figure 3.8 (c) presented, the strain of dashpot increased gradually when the
𝜏0
external force applied, building up the strain to 𝛾0 = (𝑡1 − 𝑡0 ) until t1, and the strain
𝜂
that built up is permanent and irreversible after the force removed.
a b c
τ ε γ 𝜏0
𝜏 𝛾0 = (𝑡 − 𝑡0 )
𝜖0 = 0⁄𝐸 𝜂 1
τ 𝜖0 γ
0 0
t t t
t0=0 t1 t0=0 t1 t0=0 t1
Figure 3.8 Creep and recovery test (a) and expected response of different materials:
response of linearly elastic material (b), response of viscous liquid (c)
The Maxwell fluid model
Maxwell model consists of a spring representing for the instantaneous response of

the elastic solid in tandem with a dash pot presenting the react of the viscous fluid,
showed in Figure 3.9. In theory, when the force added to the Maxwell model, the
system is preliminary dominated by elastic E during very short time, followed by
the viscous behaviour emerging and η is gradually predominant. The equation for
Maxwell model can be deduced to Equation 3.10.
dγ𝑡𝑜𝑡𝑎𝑙 1 dτ𝑡𝑜𝑡𝑎𝑙 𝜏𝑡𝑜𝑡𝑎𝑙

= ∙ + 3.10
dt 𝐸 dt 𝜂
Where, E is the Young’s modulus, τtotal is the total shear stress, γtotal is the total
shear strain, η is the viscosity
109
t=T t=T t=T+ΔT
𝑑𝛾𝜂
𝜏𝜂 = 𝜂 ∙ η
𝑑𝑡 η η
∆𝑥𝜂
𝜏𝐺 = E ∙ ε𝐺 E E E
F
∆𝑥𝐸 F
F
Figure 3.10 Schematic diagram of Maxwell model
Maxwell model could be used to predict Newtonian behaviour, especially for
viscoelastic liquid. Figure 3.10 shows the stress applied to Maxwell model system
(a) and the strain response of creep and recovery test (b). The model gives an
𝝉
instant elastic response ( 𝜺𝟎 = 𝟎⁄𝑬 ) at t0, then the behaviour during most of creep
loading duration presents strain linearly increasing with time and the model
showing viscous dominant governing by the dashpot. When the external force is
𝝉
removed, the elastic strain which is valued 𝟎⁄𝑬 is recovered right away, a
permanent strain (𝜺1) caused by the dashpot remains (Mezger, 2020).
a b
τ ε
𝜀0
τ0 𝜀1
𝜀1
𝜀0
t t
t0=0 t1 t0=0 t1
Figure 3.9 Creep and recovery test (a) and expected response of Maxwell model (d)
The Kelvin-Voigt solid Model
Kelvin-Voigt is made up of a spring and a dashpot connected in parallel, shown in

Figure 3.11. The spring and the dashpot will undergo the same strain when
external force applied, and the total stress is the sum of individually experienced
stress of spring and dashpot. Equation 3.11 expressed the responded strain and
time in Kelvin-Voigt model
110
𝛾𝜂 γ𝐺
𝑑𝛾𝜂 𝜏𝐺 = G ∙ γ𝐺
𝜏𝜂 = 𝜂 ∙
𝑑𝑡
F, 𝜏𝑡𝑜𝑡𝑎𝑙
Figure 3.11 Schematic diagram of Kelvin-Voigt model
𝑑γ𝑡𝑜𝑡𝑎𝑙 𝜏𝑡𝑜𝑡𝑎𝑙 E
= − ∙ γ𝑡𝑜𝑡𝑎𝑙 3.11
𝑑𝑡 𝜂 𝜂
Where, E is the Young’s modulus, τtotal is the total shear stress, γtotal is the total
shear strain, η is the viscosity
From there, strain is exponentially decays with time. Thus Voigt model could be
used for predicting creep response for viscoelastic materials. Figure 3.12 presents
response of Kelvin-Voigt Model to a constantly external stress 𝝉𝟎 lasting from 𝑡0 =
0 to 𝑡1the dashpot hinders the stretching of spring and takes stress 𝝉𝟎 and
𝝉
response with an increasing of strain with a slope of 𝟎⁄𝜼. As strain increased, part
of the stress will transferred to the spring from the dashpot, and the slope of the
𝝉
increased strain changes to 𝜼⁄𝜼 (where 𝝉𝜼 is the residual stress in dashpot).
When all the stress is taken by the spring, the maximum strain is reached which
𝝉
is 𝟎⁄𝑬 . At t1 when the stress is removed, the strain decreased gradually. No
permanent strain remains eventually and the system will achieve full recovery,
because the spring will eventually contract to its original position and the parallel
arrangement allows same strain for spring and dashpot (Mezger, 2020).
111
a b
τ 𝛾
τ0
t t
t0=0 t1 t0 t1
Figure 3.12 Creep and recovery test (a) and expected response of Voigt model (b)
Burgers Model
Compared with creep-recovery response between Maxwell and Kelvin-Voigt

models, the ever-decreasing strain rate type creep and anelastic recovery could
be predicted with Kelvin-Voigt model, but not with Maxwell one; but the
instantaneous elastic response and permanent strain could be only witnessed with
Maxwell model. In real practice, some advanced models involved three or more
elements are proposed for the interpretation of more complex materials, such as
the Standard Linear Model and Burgers Model. Burgers model is applicable in the
rheological analysis for viscoelastic models, which is schematically as a Maxwell
model in series connection with a Kelvin-Voigt model (Figure 3.13).
𝜖
a) b)
η2
Creep E1_R
η η4
E1
strain 4
Ⅱ F, τtotal
E3,
E3, η2_C η2_R Ⅰ E3 Ⅳ
E1_C Permanent Ⅲ
strain t
t0 t1
Figure 3.13 Typical creep and recovery response (a) for Burgers model accompanied
with its schematic diagram (b)
As seen in Figure 3.13 (a) showing strain response of Burgers model to external
stress, spring element Ⅰ stretches immediately resulting in an instantaneous strain,
followed by a creep strain consisting of a delayed elastic response ( E3, η2_C) and a
linear viscous response (η4). As soon as the force is removed, an elastic response
caused by spring element Ⅰ (E1) is initially observed, after which the recovery of
Kelvin-Voigt element (paralleled system involving viscous element Ⅱ (η2) and
112
spring element Ⅲ (E3)) shows anelasticity. Permanent strain exists due to the
viscous deformation by viscous element Ⅳ (η4).
Instead of the strain, compliance J(t) is normally applied for the presentation of
creep and recovery response curve, which is expressed as the measured strain
divided by the applied stress, shown in Equation 3.12.
𝛾 (𝑡)
𝐽(𝑡) = 3.12
𝜏
Where, J is the compliance, τis the applied stress, γ is the measured strain
Figure 3.14 simply illustrates response of pure viscous and elastic materials
subjecting to creep test in terms of interpretation of creep compliance against time.
log creep compliance J
Viscous material
Elastic material
t0
log t
Figure 3.14 Response of viscous material and elastic material to creep test, expressed
with creep compliance with time in log-log plot
113
3.6.1.2.3 Dynamic oscillatory sweep test (linear)
Dynamic oscillatory sweep test is often carried out to obtain the similar information
as creep and recovery test for the viscoelasticity characterisation, where a shear
strain with a sinusoidal waveform is usually induced to the system expressing with
two-plate model shown in Figure 3.15.
27 90
0°/360 0° °0
° °
27 90 90
0° ° °
180° 180
°
270
°
360
°
Figure 3.15 Two-plate model for oscillatory shear test and the applied oscillatory shear
profile
In oscillatory shear test, one type is applying stress (torque) to the bob and
measuring the resultant strain γ (angular displacement), the other is controlling
the strain and then measuring the stress. When the frequency of sinusoidal wave
is 𝑓, the complex shear strain that applied to a material is expressed in Equation
3.13 (Mezger 2006),
𝛾 = 𝛾𝑚𝑎𝑥 sin 𝜔𝑡 = 𝛾𝑚𝑎𝑥 𝑒 𝑖𝜔𝑡 3.13
Where, 𝜔 is angular frequency (𝜔 = 2𝜋𝑓 ) with a unit of rad · s-1, 𝛾𝑚𝑎𝑥 is the
complex shear strain amplitude, t is time with unit of second, 𝑖 = √−1.
Generally, the corresponding linear response of material in terms of complex shear

stress is expressed in Equation 3.14,
𝜏 = 𝜏𝑚𝑎𝑥 sin(𝜔𝑡 + 𝛿 ) = 𝜏𝑚𝑎𝑥 𝑒 𝑖(𝜔𝑡+𝛿) 3.14
Where 𝛿 is defined as phase angle with a unit of degree (°), 𝜏𝑚𝑎𝑥 is complex stress
amplitude.
When 𝛿 = 0°, the stress in material is proportional to the strain which is known to
be in phase, and the material is purely elastic. If the phase angle 𝛿 equals to 90°,
114
the stress is proportional to the rate of strain where the stress and strain is said to
be out of phase, the material is purely viscous. For a material showing both of
elastic and viscous properties, the response of which contains both in phase and
out of phase contributions, so phase angle will lie between of two extremes (0° <
𝛿 < 90°) (Lade et al., 2019).
Complex shear modulus (𝐺 ∗ ) is introduced for quantifying the resistance of a

material to deformation, which is the combination of viscous component and elastic
component. It could be expressed as the ratio of applied stress (strain) to the
response in terms of strain (stress), see Equation 3.15.
𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝑠𝑡𝑟𝑒𝑠𝑠 𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒 𝜏𝑚𝑎𝑥

𝐺∗ = = ⁄𝛾𝑚𝑎𝑥 3.15
𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝑠𝑡𝑟𝑎𝑖𝑛 𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒
Where, G* is complex shear modulus, τmax is complex stress amplitude, γmax is

the shear strain
The viscous component contributing to complex modulus is defined as loss

modulus (𝐺 ′′), representing for energy loss; the elastic component contributing to
complex modulus is defined as storage modulus (𝐺 ′ ), representing for energy
storage. Equation 3.16~3.19 mathematically expressed of relationships between
these terms.
𝜏𝑚𝑎𝑥
𝐺 ′ = 𝐺 ∗ cos 𝛿 = cos 𝛿 3.16
𝛾𝑚𝑎𝑥
𝜏𝑚𝑎𝑥
𝐺 ′′ = 𝐺 ∗ sin 𝛿 = sin 𝛿 3.17
𝛾𝑚𝑎𝑥
𝐺 ∗ = √𝐺 ′ 2 + 𝐺 ′′ 2 = 𝐺 ′ + 𝑖𝐺 ′′ 3.18
𝐺 ′′
tan 𝛿 = 3.19
𝐺′
Where, G’ is storage modulus, G’’ is loss modulus, G* is complex shear modulus,
τmax is complex stress amplitude, γmax is the shear strain, δis phase angle
Complex viscosity is determined during oscillatory shear test which is the

frequency dependent viscosity, indicating the total resistance of material to flow or
deformation, defined with Equation 3.20.
∗
𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝑠𝑡𝑟𝑒𝑠𝑠 𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒 𝜏𝑚𝑎𝑥 𝜏𝑚𝑎𝑥 𝐺∗
𝜂 = = = = 3.20
𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝑠𝑡𝑟𝑎𝑖𝑛 𝑟𝑎𝑡𝑒 𝑎𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒 𝛾𝑚𝑎𝑥
̇ 𝛾𝑚𝑎𝑥 𝑖𝜔 𝑖𝜔
115
Similar to the definition of 𝐺 ∗ , 𝜂∗ could be regarded as the combination of real part
and imaginary part as well, see Equation 3.21 and 3.22 (Mezger, 2020).
𝜂∗ = 𝜂′ + 𝑖𝜂′′ 3.21
𝐺 ′′ ′′ 𝐺 ′
𝜂′ = ,𝜂 = 3.22
𝜔 𝜔
Where, 𝜂′ represents viscosity for real portion; 𝜂′′ represents viscosity for the
imaginary portion.
Oscillatory amplitude sweep
Oscillatory amplitude sweep refers to the test where a material is being oscillated
sheared by varying the amplitude of the deformation or shear stress (generally
with %strain) while keeping the frequency (generally with angular frequency) as
constant. The typical response of a complex fluids to an oscillatory amplitude
sweep is shown as the storage modulus 𝐺 ′ and loss modulus 𝐺 ′′ changing with the
increased strain or stress (Mezger, 2020). Linear viscoelastic region (LVER) is a
key achievement by carrying out oscillatory amplitude tests, where moduli are
independent with applied strain or stress and remaining constant at a plateau value.
The value of storage modulus 𝐺 ′ in LVER gives the information of rigidity of
material at rest, while that of loss modulus 𝐺 ′′ reveals the information of viscosity
of undisturbed material. Another point of oscillatory amplitude is the determination
of crossover point of curves of 𝐺 ′ and 𝐺 ′′, which is known as the flow point after
which the dominate contribution to the material system will change.
Oscillatory Frequency sweep
Oscillatory frequency sweep refers to the test where a material is being oscillatory
sheared varying the frequency at a constant strain or stress amplitude. The storage
modulus, 𝐺 ′ , and loss modulus, 𝐺 ′′, is quantified against angular frequency which
is measured in rad s-1. Lower frequencies indicating longer time scale and high
ones for short time scale. Due to time-dependent property of viscoelastic materials,
moduli are expected to change with varied frequency. Small amplitude oscillatory
frequency sweep that applied in this study refers to the test carried out during LVER,
whereas large amplitude oscillatory frequency sweep refers to nonlinear response
of materials due to large deformations or structural disruptions and material
functions are not only dependent on frequency, which will not be discussed in
details here.
116
Small Amplitude Oscillatory Shear (SAOS)
As previous introduction, at low amplitudes of strain range (LVER), material is

expected to give linear response in terms of shear stress when subjecting to
𝜂
applied strain. Introducing relaxation time, 𝜆 (𝜆 = 𝐺 ), response of Maxwell model in
terms of 𝐺 ′ and 𝐺 ′′ is expressed with Equation 3.23 is obtained (Mezger, 2020)
𝜆𝜂𝜔2 𝛾0 𝜂𝜔𝛾0
𝐺′ = 2 2
, 𝐺 ′′ = 3.23
1+𝜆 𝜔 1 + 𝜆2 𝜔 2
It can be conclude from equations above, at low frequencies, 𝐺 ′ ⋉ 𝜔2 and 𝐺 ′′ ⋉ 𝜔,

indicating that 𝐺 ′′ is larger than 𝐺 ′ , so the response of Maxwell model-material is
viscous dominant, while at very high frequencies, the situation is reversed (Figure
3.16 (a)). As for Voigt model which describes viscoelastic solids, storage
modulus 𝐺 ′ is a constant value and independent with time, and loss modulus 𝐺 ′′ is
linearly increase with frequency. At very low frequencies, solid behaviour
dominates. With the increase of frequency, storage modulus remains constant and
loss modulus increases linearly, therefore 𝐺 ′′ will be larger than 𝐺 ′ at high
frequencies and material behaves more liquid-like (Figure 3.11 (b)) (Mezger, 2020).
(a) (b)
Log modulus
Log modulus
𝐺′
𝐺 ′′
𝐺 ′′
𝐺′
Log angular frequency Log angular frequency
Maxwell model (For viscoelastic liquid) Voigt model (For viscoelastic solid)
Figure 3.16 Typical frequency response of Maxwell model for a viscoelastic liquid (a)
and Voigt model for a viscoelastic solid (b)
117
3.6.1.3 Experimental Section
3.6.1.3.1 Measuring System and Geometries
In this project, the flow properties of manufactured creams were examined after 20
minutes from preparation, using a controlled stress AR 2000 rheometer (TA
instrument) equipped with a cone and plate geometry (cone angle of 1°59 and
radius of 40 mm). Samples were loaded on the plate, and the cone was lowered
to reach a gap of 57 mm with the plate. The physical model of rheometer system
is presented in Figure 3.17. As the flow resistance exist in the flow behaviour, and
the internal friction process occurring between particles will result in viscous
heating of the sample, the water bath is used for controlling the temperature at a
required value for the experiment.
Water bath Computer Rheomete
r
Figure 3.17 Physical model of rheological measuring system
In the schematic diagram Figure 3.18, Ω represents for angular velocity of the cone
(Ω = 2𝜋𝑛⁄60, where n is the rotor speed with the unit of 𝑟 ∙ 𝑚𝑖𝑛−1 ); T represents for
the resulting torque (with the unit of 𝑁 ∙ 𝑚) which is needed to rotate the cone. Ω,
T, and the total force F normal to the fixed plate are quantities that were measured
in the experiment. Rc is the radius with a unit of m, and α is the gap angle with a
unit of rad.
According to the research of Khan and Mahmood, in the measuring system with
cone and plate geometry, the shear rate 𝛾̇𝑐 could be expressed with the Equation
3.24 (Hellström et al., 2014).
118
Torque T
Transducer for torque measurement
Rc
Tested sample
Cone
α
Plate
Figure 3.18 Schematic diagram of cone and plate geometry
1
𝛾̇𝑐 = ∙Ω = 𝑀∙Ω 3.24
𝑡𝑎𝑛𝛼
Where, M represents for shear rate factor with the unit of rad-1. This value is
constant for a specific cone and plate measuring system, 𝛾̇𝑐 represents for shear
rate with the unit of s-1.
The shear stress can be related to the measured torque, see Equation 3.25,
assuming that the torque working on the cone equals to that working on the plate
(Mezger, 2020).
3
𝜏𝑐 = ( )∙𝑇 3.25
2𝜋 ∙ 𝑅𝐶 3
Where, 𝜏𝑐 represents for shear stress on cone and plate with the unit of Pa.
Then Equation 3.26 for viscosity function is obtained,
𝜏𝑐 3∙𝑇 𝛼
𝜂 (𝛾̇𝑐 ) = =( 3) ∙ 3.26
𝛾̇𝑐 2𝜋 ∙ 𝑅𝐶 Ω
Where, 𝛾̇𝑐 is the shear rate, η is the viscosity, τc is the shear strain, T is the torque,
αis the gap angle, Ω is the angular velocity, Rc is the radius
3.6.1.3.2 Measuring Procedure

After 20 min of preparation, rheological tests were at least duplicated carried out
for every sample, where samples were freshly loaded following consistent routine
in order to achieve the reproducible results. The procedure of characterisation is
summarised as below, where parameters that selected are according to the results
of characterisation of E45 cream (see Chapter 4).
119
1. Steady state shear test (SSS) was firstly performed on creams. The
Sample was rotational sheared under varied shear stress, thus viscosity
change with shear stress was obtained. Details of test including conditions
and setting parameters are displayed in Table 3.15.
Table 3.15 Parameters for steady state shear test (SSS)
Conditioning Step for SSS
Geometry gap 57 mm Temperature 25 ºC
Equilibrium time 10 minutes Pre-shear No pre-shear

procedure
Steady State Flow Step
Variables Shear stress ranging from 1 Pa to 300 Pa
Number of points 10 points per decade in log mode
2. Oscillatory sweep test was then performed. Oscillatory amplitude (strain)

sweep (OSS) was performed in order to determine linear viscoelastic region
(LVER). Then an oscillatory frequency test (OFS) was carried out at a
constant strain selected within LVER. Details of tests are displayed in
Table 3.16 and 3.17.
Table 3.16 Parameters for oscillatory strain sweep test (OSS)
Conditioning Step for OSS
Equilibrium time 10 Pre-shear procedure No pre-shear

minutes
Oscillatory Strain Sweep Step
Variables %strain ranging from 0.001 to 1000
Controlled variable Frequency controls at 1 Hz
Table 3.17 Parameters for oscillatory frequency sweep test (OFS)
Conditions for OFS
120
Equilibrium time 10 minutes Pre-shear procedure No pre-shear
Oscillatory Frequency Sweep Step
Variables Frequency ranging from 0.01Hz to 100 Hz
Controlled variable strain within LEV range selected from oscillatory amplitude
test (0.1% for mimic creams, at 0.01% for bio creams)
3. Creep and Recovery test for creams was carried out for further
analysis of their viscoelastic properties. Constant stress was applied on
the sample for a period of time, followed by a strain relaxation process
where external stress was removed. Details of the test are introduced in
Table 3.18.
Table 3.18 Parameters for creep and recovery test
Conditions for creep and recovery test
Geometry 57 mm Temperature 25 ºC
gap
Equilibrium 10 minutes Pre-shear procedure No pre-shear

time
Creep Step
Controlled Shear stress of 10 Pa for Duration 30 minutes

variable mimic creams, shear
stress of 1 Pa for bio
creams
Recovery Step
Controlled Shear stress of 0 Pa Duration 30 minutes

variable
Number of 10 points per decade in log mode

points
3.6.2 Differential Scanning Calorimetry (DSC)
3.6.2.1 Theory
Thermal analysis refers to the measurement that monitors the properties of a

material changing as a function of temperature or time. The sample is prone to be
heated, melted, oxidized and decomposed while increasing temperature, as a
121
result, melting point, crystallization behaviour, glass transition temperature and
stability are acknowledged. Differential scanning calorimetry (DSC) is a type of
thermos analysis method, where the difference in the heat to or from the sample
and the reference (air) was measured against temperature while the sample is
heated or cooled. In practice, two types of DSC measurement theory are widely
applied, which are known as heat-flux DSC and power compensation DSC (Höhne
et al., 2013).
3.6.2.1.1 Power compensation DSC

For power compensation DSC, the input energy that applied to the sample and
reference (air), for maintaining their temperature difference close to zero, is
measured while the sample is scanned. This resulting energy difference is
proportional to heat flow, and recorded as a function of sample temperature. The
schematic configuration of power compensation DSC is depicted in Figure 3.19
(Danley, 2002).
pans (with lids)
Sample Reference
Platinum
Platinum
resistance
resistance
thermomete
thermomete
rs (TS)
rs (TR)
Insulating heat sink Individual heaters

Temperature
programmer
(∆T=0) ∆ Controller
P
Figure 3.19 Schematic diagram of power compensation DSC, adapted from Danley,
2002
The sample and reference are enclosed in two separate aluminium or platinum
pans (with lids) placing in two platforms, where they are heated up by two individual
heating sources. The temperature of sample (TS) and reference (TR) are controlled
to be equal (∆T= TS-TR=0) through supplying differential power input ∆P when the
sample undergoing endothermal or exothermal process, which is monitored by
separate two sensors (platinum resistance thermocouples or thermometers). The
power signal ∆P is proportional to the endothermic and exothermic heat.
122
3.6.2.1.2 Heat flux DSC
For heat flux DSC, the sample and reference (air) are heated by a single heating
source, resulting in same heat flowing into them, and the temperature difference
between them, due to variation of thermal properties (enthalpy or hear capacity) of
the sample while scanning, is measured (Drzeżdżon et al., 2019).
Pans (with lids)
Insulating
heat sink Sample Reference
Heat flux
plate
Thermocouples
Material 2 Material 1
(Chromel wire) T T (Alumel wire)
S R
∆
T
Temperature programmer
Figure 3.20 Schematic diagram of heat flux DSC
In terms of the configuration of heat flux DSC, seen from Figure 3.20, sample and
reference (usually air) encapsulated in pans, are placed together in an insulating
heat sink. A heat flux plate (usually a constantan disc) is connected to the heater
(not shown in figure) and provide heat flow to the sample and reference platforms
through heat resistor (not shown in figure). Thermocouples, junctions that
produces voltage due to temperature difference, are used as sensors in the
configuration. A Chromel wafer (grey block underneath the pan) is equipped at the
bottom of pans, with which chromel-constantan thermocouples are formed for
detecting the differential temperature ∆T between sample and reference. This is
measured as the voltage difference ∆U. Alumel wires are connected to the chromel
wafer, resulting chromel-alumel thermocouple junctions, by which the
temperatures of sample (TS) and reference (TR) are measured individually.
Temperature programmer helps control temperature to satisfy the experimental
demand, with the help of another thermocouple set in the heater. As the
temperature difference between sample and reference is directly related to the
123
differential heat flow, for an accurate detection of the differences of temperature, a
vacuum working environment with purge gas flow through the sink is practically
applied.
In heat flux DSC, the response of sample could be expressed with Equation 3.27
(Höhne et al., 2013),
𝑑𝐻 𝑑𝑇
𝑞= = 𝐶𝑝 + 𝑓 (𝑇, 𝑡) 3.27
𝑑𝑡 𝑑𝑡
Where, 𝑞 represents for heat flow, with a unit of J min-1, which is the DSC heat flow
𝑑𝑇
signal; 𝐶𝑝 is the specific heat, with a unit of J g-1 ºC-1; 𝑑𝑡
is the heating rate, with a
unit of ºC min-1; 𝑓(𝑇, 𝑡) is the kinetic response of sample in terms of heat flow as a
function of time at an absolute temperature.
3.6.2.2.1 Measuring System

TzeroTM DSC 2500 system (TA Instrument) was applied for measuring
thermodynamic properties of creams in this project, equipped with TRIOS software.
As the sample and reference calorimeters are rarely designed to be symmetrical
in real practice, the conventional calculation of heat flow based on those
assumptions involves unavoidable error. Tzero DSC 2500 system equips with
another Tzero thermocouple as a control sensor in the middle position of sample
and reference platforms, which allows measuring the asymmetry in terms of
imbalanced heat flow at sample and reference calorimeters. The schematic of
Tzero heat flow model is shown in Figure 3.21.
Sample Reference
T0
RS Tzero Rr
thermocouple
CS CR
TS TR
qS qR
Figure 3.21 Schematic diagram of Tzero measurement model for DSC
124
Thus, the heat balance equation for sample and reference are written as Equation
3.28 and 3.29 (Arias et al., 2018).
𝑇0 − 𝑇𝑆 𝑑𝑇𝑆
𝑞𝑆 = − 𝐶𝑆 3.28
𝑅𝑆 𝑑𝑡
𝑇0 − 𝑇𝑅 𝑑𝑇𝑅
𝑞𝑅 = − 𝐶𝑅 3.29
𝑅𝑅 𝑑𝑡
Where, 𝑇0 represents the temperature for control; 𝐶𝑆 and 𝐶𝑅 represent for heat
capacity of sample sensor and reference sensor separately. Then the resultant
Tzero heat flow equations are obtained (see Equation 3.30~3.32).
∆𝑇 1 1 𝑑𝑇𝑆 𝑑∆𝑇
𝑞 = 𝑞𝑆 − 𝑞𝑅 = − + ∆𝑇0 ( − ) + (𝐶𝑅 − 𝐶𝑆 ) − 𝐶𝑅 3.30
𝑅 𝑅𝑆 𝑅𝑅 𝑑𝑡 𝑑𝑡
∆𝑇 = 𝑇𝑆 − 𝑇𝑅 3.31
∆𝑇0 = 𝑇0 − 𝑇𝑆 3.32
Where, ∆𝑇 is the measured temperature difference between sample and reference

and ∆𝑇0 is the measured base temperature difference between sensor sample.
a) Sample cells preparation
Proper sample preparation was carried out for the following measurement. 5~10
mg of samples, including creams and raw materials (mixed paraffin oils, Sodium
Laureth Sulphate, Cetyl Alcohol Glycerol Monostearate, SLs and
Mannosylerythritol lipids), were weighed into the alumina pan respectively,
followed by being hermetically sealed using Tzero sample encapsulation press kit.
Another empty reference pan was also enclosed with the same procedure.
b) Method setting for DSC measurement
Test was edited using TRIOS software. Details of sample information was entered,
including sample and reference names with assigned pan location number,
measured weight of samples and pans (including lid). Autosampler was applied for
precisely picking up sample and reference pans from their location and releasing
them at their position in the cell, thereby realising consistent cell closure and
improving the reproducibility of the test.
125
A method for analysing mimic cream in terms of thermodynamic properties was
created in the software for the analysis according to cream system. The sample
was heated from 25 ºC to 90 ºC at a constant rate of 3 ºC min-1. An equilibration
step was taken at 90 ºC for three minutes, followed by a backward cooling process
to -20 ºC at the same scan speed of 3 ºC min-1. After being maintained equilibrium
at -20 ºC for three minutes, the sample was undergoing a heating process to 25
ºC. As a result, thermal properties of samples during heating and cooling cycles
were measured, presenting as a thermo-diagram.
3.6.3 Droplet Size Distribution Analysis
3.6.3.1 Theory
Droplet size distribution (DSD) of the cream was characterised using the technique
of laser diffraction. When light from laser beam passing through different sizes of
particles or droplets, different angle of light diffraction will be generated. As
schematic diagram illustrates (Figure 3.22), large droplets scatter light at narrow
angles while small droplets scatter light at wide angles (Perlekar et al., 2012).
Incident Light Incident Light

Small angle scattering Large angle scattering
Figure 3.22 Schematic diagram of Laser diffraction when encountering different size of
particles
A simplified schematic diagram of optical part of laser diffraction droplet size

analyser is shown in Figure 3.23. When a sample containing droplets subjects to
the laser beams, a light intensity diffraction pattern is generated from the forward
scattered light and displayed on a detecting plane. Light being diffracted from side
and backward will be detected by side scatter light sensor and backward scatter
light sensor separately.
126
Sample with droplets
Laser Light source

Incident Light
Diffracted image
Sideofscatter
Figure 3.23 Schematic diagram laser light sensor particle size analyser
diffraction
Simply consider a sample containing spherical particles or droplets of same sizes.
Airy Disk could be used as an example in order to interpret diffraction pattern. As
can be seen in Figure 3.24, it consists of an innermost circle surrounding with a
series of concentric rings of decreasing intensity. Also the profile of irradiance is
displayed with red wave patterns (Pan et al., 2016).
The angular radius of the Airy disk pattern where from the peak of irradiance to the
I/I I/I
a (θ) b (θ)
Sin Sin
θ θ
Figure 3.24 Diffraction patterns and the corresponding radial intensity for two
spherical particles 1 (a) and 2 (b) in different sizes
first minimum is expressed with Equation 3.33, in the situation when using small
angle (sin 𝜃 ≅ 𝜃) (Pan et al., 2016)
1.22𝜆
∆θ = 3.33
𝑑
Where, ∆θ is the angular resolution, 𝜆 is the wavelength, 𝑑 is the diameter of

particles or droplets.
127
Thus it is clearly to find that the size of Airy disk is directly proportional to the
wavelength λ and inversely proportional to the size of particle d. In addition to that,
Δθa which equals to 1.22 λ/d1 is smaller than Δθb which equals to 1.22 λ/d2,
therefore 𝑑1 is larger than 𝑑2 , indicating that the diffraction pattern of larger
particles is denser than that of smaller ones.
A real sample contains droplets or particles of different sizes and may also in
different shapes, thus the resulted diffraction pattern is overlapped by each specific
diffraction pattern, and the generated intensity profile will be the sum of intensity
plot of each particle. The particle analyser records this intensity plot as raw intensity
data, and the distinguish individual diffraction patterns from the summed intensity
profile, where this profile will be divided into different individual intensity plots
representing for groups of particles in similar size. These groups are known as size
classes. Theoretically calculated intensity profiles of every size classes, using Mie
theory, are compared to the experimental ones measured by instrument. From
there, the percentage of particles in specific size class, namely particle or droplet
size distribution, is obtained (Wriedt, 2012).
Volume density (%)
Droplet size
Figure 3.25 Droplet size distribution of a sample, and the corresponding illustration of
size classes
As can be seen from Figure 3.25, droplet size distribution is plotted as the amount
of each size by volume (volume fraction) as the function of diameters, also the
illustration of size classes consisting of representative droplets is presented.
3.6.3.2 Interpretation of particle size distribution
The interpretation of the result of droplet size distribution depends on the type of
measurement applied and the corresponding basis of calculation. There are three
common distribution-based systems: number distribution, surface distribution and
128
volume distribution, where a few of statistical parameters are calculated in order to
interpret droplet size distribution data (McClements and Coupland, 1996).
Central values, including mean, median and mode, are calculated for interpreting
the commonest droplet size in a sample. Noticeability, if the droplets size
distribution is a symmetric plot, those central values are equivalent, namely,
mean=median=mode. “Mean” refers to a calculated value of the average of droplet
sizes. Depending on different distribution based systems including number
distribution, surface distribution and volume distribution, different definition and
corresponding calculation for mean value is generated, such as number means
(e.g. D [1,0]) and moment means including surface area moment mean (D [3,2])
and volume or mass moment mean (e.g. D [4,3]).
Surface area moment mean is called Sauter Mean Diameter (SMD), termed D [3,2].
It is calculated by involving both volume and surface area. The definition of SMD
refers to the diameter of a sphere that has the same volume-to-surface ratio as a
target droplet or particle in particulate material, thus it is also known as surface-
volume mean. Equation 3.34 is applied for SMD calculation, when the size
distribution is applied to characterize the material (Canu et al., 2018).
∑𝑛𝑖=1 𝑛𝑖 𝑑𝑖3
D[3,2] = 3.34
∑𝑛𝑖=1 𝑛𝑖 𝑑𝑖2
Where, 𝑛𝑖 is the number of droplets in a size fraction and 𝑑𝑖 is the diameter of
droplets in this size fraction.
In terms of the physical meaning, SMD for a given droplet is formulated according
to Equations 3.35~3.37,
𝑑𝑣3
D[3,2] = 𝑑32 = 3.35
𝑑𝑠2
1
6𝑉𝑝 3
𝑑𝑣 = ( ) 3.36
𝜋
𝐴𝑝
𝑑𝑠 = √ 3.37
𝜋
Where, 𝑑𝑣 is the volume diameter of droplet, 𝑑𝑠 is the surface diameter of

droplet, 𝑉𝑝 and 𝐴𝑝 represents for volume and surface area of droplet respectively.
129
A particle size analyser, Mastersizer 3000 (Malvern Instruments Ltd, UK), was
applied, equipping with Hydro EV which is a dip-in and semi-automated wet sample
dispersion unit, which is illustrated in Figure 3.26. In this study, a 500 mL laboratory
beaker was applied. Physical diagram of the instrument is shown in Figure below.
With an accuracy of ±0.6%, this instrument is capable of measuring particle size
ranging from 10 nm to 3.5 mm.
The dispersion unit is applied to circulate the sample through the cell where the
sample flow passes through the instrument’s laser path. Then the sample is
measured by optical unit using red and blue light wavelengths. The optical unit is
the key component of the system, which directs light through the sample and then
collect the diffracted light by the droplets. Cell window is a key art of wet cell, which
is the direct path of sample passing through. Thus it has to be kept clean for a
desired result.
1. Optical unit 3. Wet cell

2. Wet dispersion 4. Computer running the master sizer application
unit software
Figure 3.26 Illustration of instrument Mastersizer 3000 connecting to the wet

dispersion unit
130
a) Preparation for the test
For the measurement of sample taken from the hot mixture during preparation, 3
mL of sample was pipetted out and transferred to 8 mL snap-cap specimen vials
filled with 2 mL hot water at 50°C. After being well mixed, 3 mL of mixture was
pipetted into the dispersion unit containing 500 mL pure degassed water which is
used as dispersant. Slightly change of the amount of added sample in order to
ensure that the obscuration bar indicated in the system was in the right range
around 5% to 15%.
For the measurement of sample taken originally from prepared solid-like cream, in
order to allow cream sample being homogenized stirring in dispersant unit and also
avoid lump of cream sample blocking wet cell and the flowing path, treatment was
carried out before adding it into the dispersant beaker. Half teaspoon amount of
cream which is nearly 2 g was added into a beaker. Then some hot water heated
at around 50 °C was poured inside. The mixture was homogenized using a stir and
heater, where the temperature was set as 70 °C. After the mixture was visually
observed to be homogenized, 3 mL diluted sample was pipetted into the dispersion
unit containing 500 mL pure degassed water which is used as dispersant.
Obscuration bar was monitored within 5% to 15% by changing the amount of
injected sample.
Refractive index of the dispersant was quickly measured, where a refractometer

was applied. The refractive indexes of water and paraffin oils were determined
respectively. The particle density of mixed paraffin oils was approximately
measured, by weighing a specific volume, v, of mixed paraffin oils. If the weight is
denoted as m, the average particle density was estimated, see Equation 3.38
(Singh, 2002).
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑚
Particle density = = 3.38
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑣
Where, v is the volume, m is the mass
b) Experimental set-up
Before carrying out the measurement, a standard operating procedure (SOP) was
preliminarily set up using software of the instrument, and details of parameters are
listed in Table 3.19. The measurement was carried out following the induction from
the instrument.
131
Table 3.19 Details for SOP applied in droplet size analysis for mimic cream
Accessory (Hydro EV) Material (Mixed Paraffin Dispersant (Water)

Oils)
Stirrer Speed 1500 Refractive Index 1.466 Refractive index 1.33

(rpm)
Ultrasound None Particle Density 0.89

Mode
Analysis
Model General purpose Sensitivity Normal Scattering model Mie
Measurement Sequence
Number of 5 Background measurement duration (red/blue) 10

measurement (seconds)
Obscuration 0.1-20 Sample measurement duration (red/blue) 10

limits (%) (seconds)
3.6.4 Microscopy
Sample of cream was examined under a polarized light microscope one day after
preparation, under a magnification of x64, where The Axioplan 2 imaging
microscope was applied (Zeiss, Germany). Samples were prepared by smearing
tiny amount of creams on to microscope slides, with glass cover slips on top.
3.6.5 Surface and Interfacial Tension Measurement

Surface activity was preliminary carried out on SLs using Du Noüy ring method,
where surface tension between SLs solution and air was analysed.
3.6.5.1 Theory
Liquid surface tension γ (N m-1) refers to a phenomenon caused by the unbalance

cohesive forces of molecules on the surface (between liquid and gas) or interface
(between two immiscible liquids), which is reflected in the tendency of fluid surface
to contract to the minimum. Physically, surface tension is defined as a tensile force
F per unit length L. As illustrated in Figure 3.27, the dark blue bar has a tendency
132
to be pulled towards left due to the surface tension, and the force F is required to
balance it and increase the surface area (Hartland, 2004).
Surface
F
L
dx
Figure 3.27 Schematic diagram of force that applied to increase the surface area, and
the surface tension is proportional to this measured force
The measurements of surface and interfacial tension for liquid are generally
classified into equilibrium methods, such as du Nouy ring method, Wilhelmy plate
method and pendent drop method, and dynamic methods, such as bubble
pressure (Hartland, 2004). Besides, due to the different measuring principle, Nouy
ring and Wilhelmy plate methods are also known as force tensiometry where
pulling force is measured and related to the tension, while pendent drop belongs
to optical tensiometry where the shape of drop is optically determined and related
to the tension. In this project, force tensiometry was applied.
133
The Krüss K11 tensiometer (Krüss GmbH, Germany) instrument was applied for
surface and interfacial tension measurement of SLs. Figure 3.28 displayed photo
of physical model of the tensiometer.
Figure 3.28 Physical model of tensiometer
Du Noüy ring method was applied. The ring is made of platinum-iridium which has
high solid surface free energy, and a contact angle of 0ºis generally obtained,
thereby realising superb wettability when contacting with liquid. Based on Du Noüy
theory, the ring method measures the maximum pulling force Fmax on a ring by the
surface or interface. Referring to Figure 3.29, when exerting a force on the fully
submersed ring to pull it out of liquid bulk through the phase boundary, a lamellar
meniscus of liquid will be produced and lifted up to the maximum height then
eventually teared, reflecting on the force firstly increasing to a top value followed
by a decrease after the lamella tears from the ring. The measured maximum force
is related to the surface tension. With the wetted length of ring of L = 2πR, the
relationship between force 𝐹 and measured surface tension γ is expressed as
below, see Equation 3.39 and 3.40 (Lee et al., 2012).
𝐹 = 2γL cos 𝜃 = γ ∙ 4πR ∙ cos 𝜃 3.39
𝐹
γ= 3.40
𝐿 cos 𝜃
134
Where, L is the wetted length of ring, F is the force, γ is the surface tension, θ is
the contact angle, R is the inner radius of the ring
F F
ring
L rin Lamella
θ g
Liquid Liquid
Figure 3.29 Schematic illustration of Du Noüy ring method (left) and its cross-section
view (right)
3.6.5.2.2 Measuring Procedure for surface tension
a) Preparation for the test
0.8 mg, 1 mg, 1.84 mg, 2 mg, 2.8 mg, 3.84 mg, 5.4 mg, 9 mg and 12 mg of SLs
were respectively weighed, and certain amount of distilled water was used for
dissolution and added up to 40 ml for each of them. Then prepared SLs solutions,
with concentrations of 20 mg L-1, 25 mg L-1, 46 mg L-1, 50 mg L-1, 70 mg L-1, 96 mg
L-1, 135 mg L-1, 225 mg L-1 and 300 mg L-1 (theoretical concentration), were stored
in 50 mL centrifuged tubes separately and ready for the measurement.
The platinum-iridium ring has to be nearly perfect, as small blemish or scratch can
greatly affect the accuracy of the results. Thus the pre-treatment of ring was done
right before every single test. When no solvent attached to the ring, distilled water
was used for the cleaning, where the ring was fully sprayed using the wash bottle
filled with distilled water. If oily media was attached to the ring after the experiment,
methanol was applied instead. Then the wetted ring was dried with the help of
Bunsen burner. Proper and moderate operation is required, because no
overheated is allowed for maintaining the perfection of the ring.
b) Experimental procedure setting
The experiment was done following the procedure as inducted. Template of Du

Noüy Ring (SFT) was selected as the measuring method for the surface tension
measurement, where standard parameters are included and they are suitable for
most of common cases. Among those parameters, correction method was selected
135
as Harkins & Jordan and immersion depth was set as 3 mm. The measurement
was started by selecting “Run the measurement”. A measuring sequence guide
from the system was followed for the measurement.
c) Experimental accessories cleaning
After every test, glass sample vessels were filled with Decon 90 and rest for 2 h,
after which they were fully cleaned with distilled water. Only well cleaned vessels
could be used for the new sample. The ring was cleaned after testing one type of
material, which is submersed in a beaker filled with Decon90 and rest for 2 h. Then
the ring was washed with distilled water and dried with Bunsen burner flame.
3.6.6 Mass Spectrometry (MS) and Tandem Mass

Spectrometry (MS-MS)
Structural analysis was carried out for both of sophorolipids (SLs) and
Mannosylerythritol lipids (MELs) with the help of Mass Spectrometry technology.
And further confirmation was made by applying tandem mass spectrometry and
liquid chromatography-mass spectrometry.
3.6.6.1 Theory
3.6.6.1.1 Mass spectrometry (MS)
Mass spectrometry (MS) is a universally applied analytical technique for identifying
unknown compounds in a sample, through converting neutral molecules in the
sample to rapidly moving ionized fragments using different ionisation method and
then charged particles are separated in to different populations based on their
masses. Generally, mass spectrometry process consists of four main stages which
are ionisation, acceleration, deflection and detection (Ruhaak et al., 2018).
As Figure 3.30 illustrated, where high vacuum system of spectrometer, consisting

of ion source, mass analyser and detector, was displayed, neutral molecules in the
vaporised sample will be initially ionised with the present of an ionization source,
thereby converting to charged particles, either positive or negative through
removing or absorbing of electrons. After being accelerated when passing through
a set of charged parallel plates at different volts, ions enter into the magnetic field
where ions are subjected to a sideway force and deflected based on their masses
and the charge on it. Therefore, mass-to-charge ratio, denoted as m/z, is
introduced for combination of those two factors. Referring to the diagram, green
stream consisting of ions with greatest m/z value deflected least, while red stream
deflected the most which contains ions with the smallest m/z. Only those ions in
purple stream could eventually reach the detector and are quantified by ion counter.
136
Others will be neutralised and pumped out of the spectrometer (McLafferty, 2012).
After that, those detected ions will be converted to the form of current and analysed
by the recorder, presenting as a mass spectrum which is intensity or abundance
as a function of their m/z.
Ionisati Accelerati vacuum

on on
electromagnet
Vaporised sample
Detectio
n
Deflectio
n
Ion Mass analyser Detecto

source r record
er
Figure 3.30 Schematic diagram of the theory of a mass spectrometry
3.6.6.1.2 Tandem mass spectrometry (MS-MS)
Based on the principle of mass spectrometry, where sample molecules are ionized
to separate into charged fragments according to their mass-to-ratio value, tandem
mass spectrometry refers to that the a second or more mass spectrometers are
coupled to the previous one, thereby further breaking down selected ions into
smaller fragments. The work system of MS-MS could be interpreted schematically
in Figure 3.31, where sample molecules are firstly ionised followed by m/z
separation using mass spectrometer MS1. The red ion selected from MS1
represents for precursor ions which possess particular m/z value, which are
fragmented into smaller product ions. These particles are transferred to the second
mass spectrometer MS2 for m/z separation, followed by detection and analysis
with the help of detector (Hiraoka, 2013). As an outcome, a mass spectrum is
obtained presenting as intensities of molecules upon corresponding m/z values.
137
sample
+ -
- - detector
ioniser MS - fragment MS
- -
-
+ 1 2
Ionisation m/z fragmentatio m/z detection

separation n separation
Figure 3.31 Schematic diagram of the theory of mass spectrometry

The mass spectrometer (Waters UK) with electrospray ionisation (ESI) method
was used for MS and MS-MS measurements on SLs. Negative ionisation mode is
selected, and deprotonated molecules were expected to be observed in the mass
spectra. Time of flight (TOF) detection was equipped. Same mass spectrometer
was used for MS measurements on MELs, where ESI was applied as ionisation
technique and TOF analyser was applied for the determination of mass-to-ratio
values of ions. While, positive ionisation mode was selected for MS analysis on
MELs, thereby obtaining protonated or alkali adduct sample molecules. Acetonitrile
was the solvent in mobile phase for the measurements.
Samples of SLs and MELs were prepared for MS and MS-MS respectively. A small
amount of extracted product, which is nearly 50 mg, was transferred from sample
bottle to a drying dish using a laboratory micro spatula. Proper amount of ethyl
acetate was added into the drying dish for fully dissolve the product. Then this
mixture was diluted 30 times with ethyl acetate, followed by a filtration using 0.22
μm membrane. The 1 μL filtered sample solution was stored in 2 mL glass sample
chromatography vials. Five samples were prepared for each product.
138
Chapter 4. Preliminary Characterisation of E45
Cream
Performance of E45 cream, in terms of rheological properties, droplet size
distribution and thermodynamic properties, was preliminary studied. The
conclusion could be used as a standard for the following mimic and bio cream
preparation.
4.1 Rheological Characterisation of E45 cream

Dermatological E45 cream, 350 g, was purchased from The Boots Company PLC
(UK), which is packed in a jar on shelf. Different rheological characterisations were
carried out for studying the flow property of E45 cream, including steady state
shear and oscillatory sweep. A controlled stress AR 2000 rheometer (TA
instrument) was applied, equipped with cone and a 40 mm plate geometry with a
cone angle of 2°. All measurements were repeated at least twice at same
temperature condition. This enabled a coefficient of variation of 5% in all cases for
making sure that highly reproducible date was obtained. Before the measurement,
the instrument was checked for proper function by measuring the viscosity of
silicon oil (Newtonian flow).
4.1.1 Preliminary Testing: Conditioning Step Determination

In order to obtain a relatively accurate rheological behaviour and reproducible
results, samples should get rid of history structures.
4.1.1.1 Experimental Procedure
The test introduced in this chapter was applied for seeking a proper stress for pre-
shear and a minimum equilibrium time before staring the experiment.
4.1.1.1.1 Pre-shear Stress Determination
The measurement was carried out following the procedure for pre-shear stress
determination:
1. Check whether the air supply is sufficient for the rheological measurement,
where the pressure should be no less than 30 psi.
2. Turn on the water supply, which is a water bath.
3. Power on the Rheometer and access the rheology software on the
computer.
139
4. Inertia calibration and bearing friction correction. Instrument inertia was
firstly calibrated following the induction in the software, which is expected
in the range of 14-16 µNms2. Then the cone-plate geometry was attach to
the rheometer, followed by a geometry calibration. After that, go to the
Instrument>Miscellaneous page and carry out bearing friction calibration,
where a value between 0.5 and 1.1 µNm (rad s-1)-1 is accepted.
5. Perform rotational mapping.
6. Set the zero gap following the software induction, which is set to be 57mm
in the test. After that, raise the head up and load the sample with correct
filling.
7. Lower down the head to reach zero gap. Then select Peltier Plate system
for temperature control.
8. Create a new procedure as the test program, where steady state flow was
selected for the test. Input parameters in the procedure, which is specified
in Table 4.1. Then start the test.
Table 4.1 Parameters of pre-shear stress determination for E45 cream characterisation
Conditioning Step
Temperature (°C) 25
Equilibrium time (min) 0
Pre-Shear No
Steady State Shear Step
Variables Shear stress (Pa) 10-500
Number of Points 10 points per decade in log mode
9. After the measurement finish, raise the head up and remove the measuring
geometry. Clean the plate and cone.
10. Exit the software and export date. Then power off the rheometer and the
water bath.
4.1.1.1.2 Equilibrium Time Determination
Oscillatory time sweep (OTS) test was carried out to determine minimum time for
the structure of E45 cream to reach steady state after loading, where E45 cream
was swept under constant oscillatory stress and frequency during certain time slot.
Before this, oscillatory stress sweep (OSS) test was carried out, in order to obtain
a proper controlled variable (oscillatory stress) that could be used in OTS test to
140
make sure the test was carried out within linear viscoelastic region (LVER). The
procedure was introduced as follow.
1. Follow step 1 to 4 described in chapter 4.1.1.1.1 for pre-shear stress

determination test.
2. Perform oscillatory mapping.
3. Set the zero gap following of 57mm in the test. After that, raise the head up
and load the sample with correct filling.
4. Lower down the head to reach zero gap. Then select Peltier Plate system
for temperature control.
5. Create an oscillatory stress sweep procedure as the test program. Input
parameters in the procedure, which is specified in Table 4.2. Then start the
test.
Table 4.2 Parameters for preliminary linear viscoelastic range (LVER) determination for
E45 cream characterisation
Conditioning Step
Pre-Shear No
Oscillatory Stress Sweep Step
Variables Oscillatory stress (Pa) 0.01-1000
Controlled variable 1 Hz
From the result of OSS test, an oscillatory stress of 4 Pa was selected for the
following OTS test (Result will be introduced in chapter 4.1.1.2). Then the OTS test
program was create for E45 cream, following procedure steps described below. In
addition, pre-shear was performed in conditioning step, where stress was
determined as 50 Pa (Result will be introduced in 4.1.1.2)
1. Follow step 1 to 4 described in chapter 4.1.1.1.1 for LVER determination

test.
2. Create an oscillatory time sweep procedure. Input parameters in the
procedure, which is specified in Table 4.3. Then start the test.
3. After the measurement finish, raise the head up and remove the measuring
geometry. Clean the plate and cone.
141
4. Exit the software and export date. Then power off the rheometer and the
water bath.
Table 4.3 Parameters for equilibrium time determination for E45 cream
characterisation
Conditioning Step
Temperature (°C ) 25
Pre-Shear Yes Shear stress 60 Duration 5

(Pa) (min)
Oscillatory Time Sweep Step
Controlled variable Oscillatory stress (Pa) 4
Frequency (Hz) 1
Time Duration (min) 30, 70, 100 had been applied separately
Sampling Time (second) 5
4.1.1.2 Results and Analysis

Pre-shear stress determination
A representative result of steady state shear that carried out on E45 cream without
any pre-shear and equilibration was presented in Figure 4.1. E45 cream presented
shear thinning behaviour, where the apparent viscosity decrease with increasing
shear stress. In addition, 1st Newtonian plateau (purple dash line), shear thinning
(red dash line) and 2nd Newtonian plateau (orange dash line) presented in the flow
profile of E45. This preliminary shear test was carried out for determination of the
stress applied during pre-shear. Selection of the value should lie beyond the 1st
Newtonian plateau, but not way too large in order to ensure rebuilt structure.
Therefore, referring to the viscosity behaviour presented in rheogram, shear stress
could be a value selected from 30 to 60 Pa, which is determined to be 50 Pa.
The 1st Newtonian plateau could refer to the resistance of microstructure to the
external shear force due to the presence of yield stress, where the apparent
viscosity showed independent with shear stress and no obvious flow or
deformation was witnessed, when the wall depletion effect is eliminated or
neglected. However, for highly concentrated dispersions with large droplets that
confined in a gap contacting with smooth surface, wall slip usually occurred due to
the displacement of the disperse phase away from solid boundaries (Barnes, 1995),
142
where the overall deformation of the material is localized in a thin layer of thickness
adjacent to the confining walls, resulting in a large velocity gradient at the wall.
Thus the actual deformation experienced by material is highly different from the
effective shear rate that applied, resulting in an underestimation of the actual
viscosity (Mukherjee et al., 2017). As indicated that, wall depletion mostly affects
yield stress and sometimes apparent viscosity at 1 st Newtonian plateau, namely,
resulting in lower yield stress which is approximately 65% lower compared to the
actual value for a hand lotion (Saarinen et al., 2014). The reason for the
phenomena may be steric, hydrodynamic, viscoelastic and chemical forces and
constraints acting on the disperse phase immediately adjacent to the walls
(Hatzikiriakos, 2012).
1000000
100000
10000
1000
Viscosity /Pa S
100
10
0.1
10 100 1000
Shear Stress /Pa
Figure 4.1 Exploratory flow characterisation of E45 cream for pre shear stress
determination, where viscosity varied as a function of shear stress
However, in this study, rheological characterisations of all creams were conducted

using the same smooth cone and plate geometry and confined within the gap of
57 mm, plus their nature which are semisolid systems with large size droplets
dispersed, and no measures have been taken to inhibit wall depletion phenomenon,
thus without carrying out further investigations for detecting whether a wall
depletion existed or the effect degree of this phenomenon, it has to point out that
wall slip phenomenon may occur as it is a common phenomenon for most complex
materials. Even though, as all rheological measurements are consistently carried
out in terms of geometry, gap and other measuring parameters, also reduplicative
results were obtained for every single cream, thus the rheological data that
143
measured could be utilized as qualitative indices for comparing the relative
differences between creams.
Equilibrium time determination
Linear viscoelastic range (LVER), where storage modulus and loose modulus are
independent with applied stress, was determined by carrying out oscillatory
amplitude sweep for the following dynamic measurements. As a result, change of
storage modulus G’ and loss modulus G’’ of E45 cream as a function of oscillatory
stress was obtained in rheogram presented in Figure 4.2. G’ and G’’ kept constant
until the applied stress increased to around 10 Pa and G’ was always over G’’
during this range, where is known as LVER. Afterwards, both of G’ and G’’ started
to decrease. When applying oscillatory stress of over 50 Pa, G’’ was predominant
in the system, indicating a viscous behaviour dominated system. An oscillatory
stress of 4 Pa was selected for the following oscillatory time sweep
10000
1000
100
G',G'' /Pa
10 G'
G''
1
0.1 1 10 100
Osc. Stress /Pa

Figure 4.2 Oscillatory amplitude sweep of E45 cream for determination of oscillatory
stress within linear viscoelastic range
Oscillatory time sweep of E45 cream was carried out after pre shearing cream
sample at 50 Pa for 5 minutes. As an output of oscillatory time sweep, E45 cream
was swept under constant amplitude and frequency for a period of time, where
changes of storage modulus G’ and loss modulus G’ were recorded. As seen in
Figure 4.3, G’ and G’’ began to level off roughly after 50 min of sweep, and they
tend to reach plateau until 100 min.
144
However, equilibrating cream for completely rebuilding the structure also has
drawbacks. Too long time equilibration may cause water evaporation of E45 cream,
thereby bringing edge effect which happens on the boundary of sensory system
when the measurement is running. The large effect may cause extra shear strain
to be recorded by the measuring system, then inaccurate higher viscosity of cream
will be measured as a result. In another aspect, the edge cracking may lead to
discontinuity of shear rate happen in viscous emulsions and gel dispersions. Under
this circumstance, part of sample was edged out by the geometry (cone here).
Subsequently for the remaining cream sample, portion of which rotates with the
movement of boundary; portion of which may rotate at the same speed as the
boundary does. And those in the centre of geometry do not behave with a
consistent velocity gradient. Thus for a compromise, 55 min was selected as the
applicable equilibrium time for E45 structure built up.
100000
G'
G''
10000
G'/G'' Pa
1000
100
0 20 40 60 80 100 120 140
Time min
Figure 4.3 Oscillatory time sweep of E45 cream for determination of equilibrium time
4.1.1.3 Conclusions of Preliminary Testing

As a result, a pre shear step was set up, where E45 would be sheared at 50 Pa for
5 min followed by an equilibration for 55 min. Rheological measurements were
carried out in this chapter just for setting up conditioning step for the following
experiments, so they may not truly interpret the rheological behaviour of E45 cream.
145
4.1.2 Rheological Characterisation on E45 Cream
In this chapter, standard rheological tests, which were carried out after previously
determined conditioning step, were introduced.
4.1.2.1 Experimental Procedure

4.1.2.1.1 Steady State Shear
Steady state shear (SSS) test was performed to investigate shear dependent non-
Newtonian flow behaviour of E45 cream. By spinning the cone geometry to shear
the cream on a stationary lower plate with increased shear stress, the apparent
viscosity was obtained as a function of applied shear stress. The procedure of SSS
test for pre-shear stress determination described in chapter 4.1.1.1.1, and the
parameter input in this SSS procedure was specified in Table 4.4. After the
measurement, sample left on geometry and the Pelite plate was cleaned and water
bath was turned off. The instrument was powered off after use.
Table 4.4 Parameters for steady state shear test on E45 cream
Conditioning Step
Pre-Shear Yes Shear 50 Duration 5

stress (Pa) (min)
Variables Shear stress (Pa) 10-300
4.1.2.1.2 Continuous Shear Stress Ramp (up and down)
The continuous ramp test was applied in order to study the thixotropic property of
E45 cream, where the shear stress increased from 10 Pa to 150 Pa during ramping
up and then reduced to its original value of 10 Pa during ramping down step. The
procedure of calibration, zero gap setting and mapping could be referred to chapter
4.1.1.1.1. The created measurement program was specified in Table 4.5.
146
Table 4.5 Parameters for continuous shear stress ramp test on E45 cream
Conditioning Step

(Pa) (min)
Ramp up Variables Shear stress 10-150

(Pa)
Ramp down Variables Shear stress 150-10

(Pa)
4.1.2.1.3 Dynamic Oscillatory Stress Sweep
The accuracy of previous obtained LVER of E45 cream was further confirmed by
conducting a new dynamic oscillatory stress sweep (OSS) after a pre-shear step.
The procedure could refer to chapter 4.1.1.1.1 and parameters are specified in
Table 4.6.
Table 4.6 Parameters for new linear viscoelastic range (LVER) determination for E45
cream characterisation
Conditioning Step

(Pa) (min)
Oscillatory Stress Sweep Step
Variables Oscillatory stress (Pa) 0.1-1000
Controlled variable 1 Hz
4.1.2.1.4 Dynamic Oscillatory Frequency Sweep
The analysis of time-dependent non-Newtonian flow behaviour of E45 cream was

conducted using dynamic frequency sweep (OFS) measurement. The procedure
of calibration, zero gap setting and mapping in the measurement procedure were
147
introduced in chapter 4.1.1.1.1. Then an oscillatory frequency sweep program was
created and parameter inputs are specified in Table 4.7. The amplitude, which is
the oscillatory stress, was controlled at 4 Pa (the result from new OSS
measurement).
Table 4.7 Parameters for oscillatory frequency sweep on E45 cream
Conditioning Step
Temperature (°C ) 25

(Pa) (min)
Oscillatory Frequency Sweep Step
Variables Oscillatory frequency (Hz) 0.01-1000
Controlled variable Oscillatory stress (Pa) 4
4.1.2.2 Results and Analysis

Rheological behaviour of E45 cream under steady state shear
Viscosity profile of E45 cream was eventually achieved by carrying out rotational
shear test on E45 cream after pre-shear for removing history structure and
equilibrium for realizing zero shear condition. Apparently from Figure 4.4, viscosity
of E45 cream presents an overall decrease trend with the increased shear stress
ranging from 10 Pa to 300 Pa, which indicating a shear thinning behaviour of flow.
When the shear stress was lower than 40 Pa, viscosity of E45 cream kept constant
at approximately 3×105 Pa·s. After exceeding a yield stress, it started to decrease.
When applied shear stress was over 50 Pa, a dramatically sharp drop of viscosity
within a small stress range (50-60 Pa) was witnessed, indicating the shear thinning
behaviour. The 2nd Newtonian plateau refers to a gradual decrease of viscosity with
the shear stress over 60 Pa.
As stated previously in the preliminary test for E45 characterisation, wall slip may
happen in this situation leading to an inaccurate interpretation of E45 rheological
behaviour. Also, researchers pointed out that wall slip usually manifests itself,
giving lower viscosity and lower yield stress when changing to a smaller sized
geometry or sudden breaks witnessed in flow curves, especially for those
148
dispersions consisting of large droplets coupled with smooth surface and low flow
dimensions (Saarinen et al., 2014). Thus in this report, the following analysis in
respect to rheological measurements are specified that a 40 mm cone and plate
geometry was consistently applied with a measuring gap of 57 mm for all creams.
In addition to that, maximum viscosity of E45 that characterised in this project was
approximately 105 Pa·s which is similar to that obtained from a study where a
limiting viscosity for a cream was more than 104 and the values of yield stress were
reasonable which line in between 10 Pa and 100 Pa (Kwak et al., 2015).
1000000.00
100000.00
10000.00
1000.00
Viscosity Pa∙S
100.00
10.00
1.00
0.10
0.01
10 100 1000
shear stress Pa
Figure 4.4 Steady state shear test on E45 cream, where viscosity varied as a function of
shear stress ranging from 10 Pa to 300 Pa
Viscosity profile, which illustrates the flow and deformation of E45 cream when
subjecting to external shear, macroscopically reveals microstructure change of the
system. During lower shear stress range (below 40 Pa), the presence of 1st
Newtonian plateau reflects the stable three-dimensional gel structure or matrix of
E45 cream was formed by interacting forces between droplets, which is strong
enough to support cream and resist the external force. In addition, carbomer, a
high-molecular polymer, is used as thickener in the formula of E45. The cross-
linking of polymer chains also contributes to the structural network (Siemes et al.,
2018). Continuously increasing the external stress, microstructure of cream
gradually rearranged, where the aggregated structures, droplets and polymer
chains began to break down, deform and disentangle, thus presenting as a
decrease trend of viscosity (García et al., 2018). As the arrangement of droplets
149
completely aligned with the flow, shear thinning behaviour was witnessed, which
enables the application of cream product to skin.
Normally shear thinning behaviour will happen after the shear stress exceeds a
yield value, which is known as yield stress τF. With the definition of flow onset for
yield stress, the value is determined from the maximum of viscosity profile η max
from some literatures (Choi et al., 2015). While regarding to the flow curve of E45
cream, it is easier to define τF as the end of 1st Newtonian plateau. In the study of
primary skin feeling test, some researchers correlated that with yield stress,
indicating that a cream needed a higher shear stress to flow will be rated higher in
terms of spreadability. This information for E45 cream was recorded for further
comparing with lab-made mimic creams.
2nd Newtonian plateau started when the viscosity decreased to 10-1 Pa·s at shear
stress of 300 Pa, which is usually correlated to the secondary skin feeling, that is,
the cream is expected to show a low viscosity during high shear stress or shear
rate range, for achieving a better absorption capacity perceptible on the skin after
application and the end-of-use feeling (Kwak et al., 2015). A suggested shear rate
γ̇ for this assessment is 500 s-1, which corresponds to a shear stress of nearly 300
Pa for E45 cream. Thus for E45 cream, the viscosity of less than 0.1 Pa·s at high
shear rate γ̇ = 500 s−1 was displayed, which is similar to the test creams with
decent secondary skin feeling (viscosity of 0.02~0.4 Pa·s at shear rate γ̇ = 500 s−1 )
in the project of Bekker et al (Bekker et al., 2013). The step decrease (break in
curve) is witnessed in 2nd Newtonian plateau for all viscosity curves of E45. The
microstructure variations may contribute to this phenomenon, among which the re-
entanglement of polymer molecules of carbomer supplied the most.
Thixotropic property of E45 cream
Thixotropic property refers to the time-dependent shear thinning behaviour, where

a material exhibits decrease of viscosity or shear stress under constant shear rate
over time. In addition, thixotropic behaviour holds the responsibility for not
achieving microscopic reversibility of the stress-strain rate plot, therefore resulting
a hysteresis loop (Petrovic et al., 2010). Referring to the hysteresis loop test of E45
illustrated in Figure 4.5, ramp up step illustrated its shear tinning behaviour where
the decay of viscosity with increasing the shear rate; while the backward trend of
ramp down descending process does not retrace the original path, where the
structure gradually recovered and rebuilt. Therefore, a hysteresis loop is formed
as seen in the rheogram, the area of which indicates the degree of thixotropy and
150
the energy required to break down this thixotropic structure. Besides, the yield
stress τF of 54.12 Pa could be obviously acquired from the stress-rate curve,
which is similar to that obtained in previous steady state shear measurement.
160
120
Shear Stress /Pa
(3.80E-04, 54.12)
80
40
Ramp Up
Ramp Down
0
0 100 200 300 400 500
Shear Rate /s⁻¹
Figure 4.5 Shear ramp test on E45 cream for determination of hysteresis loop, where
shear stress ramped up and down as a function of shear rate
Rheological behaviour of E45 cream under oscillatory sweep
A modified oscillatory amplitude sweep was carried out on E45, where the sample
was pre sheared and equilibrium for a certain time, in order to obtain a reliable
LVER range. The result did not present large different from the preliminary one,
displaying a LVER range from 0.1 to 10 Pa during which storage modulus and
loose modulus were independent with oscillatory stress (result not shown in
diagram). Thus the oscillatory of 4 Pa could be applied as the critical strain for the
following oscillatory frequency sweep.
Dynamic oscillatory test is a common way for investigating the viscoelastic

properties of materials. As for E45 cream, when subjecting to a constant oscillatory
stress, the change of storage modulus G’ and loss modulus G’’ were recorded as
a function of angular frequency, the result of which is presented in the log mode
rheogram (Figure 4.6). G’ and G’’ of E45 cream exhibited a qualitatively similar
behaviour over the measured frequency range, nearly independent of frequency,
which agrees with the results for cream-like products (Sanz et al., 2017). Also,
storage modulus G’ is always greater than loss modulus G’’ during this frequency
range, indicating a structured solid domain system of E45 cream. However, during
151
lower frequency range, where longer period (duration of time) of one cycle applied,
G’ and G’’ presented a tendency of meeting together. In another words, E45 cream
may present like a liquid viscoelastic material at low frequencies.
Modulus as a function of frequency could be a sound explanation for interpreting

the microstructure of a viscoelastic material, when the amplitude applied is
confined in LVER. This is normally known as small amplitude oscillatory sweep
(SAOS), where the moduli are only dependent on frequency but not the strain or
stress (Luan et al., 2017). As for E45 cream, the SAOS result presented a well-
structured gelled system. In additions to the strong gel phase formed by the
interaction between water and bilayers of fatty amphiphiles and anionic surfactants,
the support from entangled long chain polymer (carbomer) also contribute to
maintain the structure against external force.
10000
1000
G'/G'' /Pa
100
G'
G''
10
0.01 0.1 1 10 100 1000
ang.frequency /rad s⁻¹
Figure 4.6 Oscillatory frequency sweep on E45 cream, where G’ and G’’ varied as
function of angular frequency from 0.01 rad s-1 to 1000 rad s-1 at controlled oscillatory
stress of 4 Pa
4.2 Droplet Size Distribution (DSD) Analysis

Droplet size distribution of E45 cream was studied using Mastersizer 3000
(Malvern Instruemnts Ltd, UK) combined with a wet sample dispersion unit, Hydro
EV.
4.2.1 Experimental Procedure

Solid state E45 cream was treated before the experiment. The preparation
procedure could refer to chapter 3.6.3.3.2 introducing measuring procedure of
152
preparation for solid-like cream sample. Specified for E45 cream, the
measurement procedure was carried out as follow.
1. Half teaspoon amount of E 45cream, nearly 2 g, was added into a beaker,

followed by adding hot water at around 50°C. The mixture was
homogenized using a stir and heater, where the temperature was set as
70°C. This is recorded as sample A. Sample B was prepared by adding 2%
of SLES in sample A, followed by a well mixing. They were characterised
in terms of droplet size distribution separately by the same measuring
procedure.
2. Meanwhile, power on Mastersizer 3000 and open the software. Instrument
cell cleaning was carried out regularly, so there is no need to do this step
every time before test, unless as required.
3. Set up a new SOP (standard operation procedure) for E45 cream
measurement. Details of important parameter settings are displayed in
Table 4.8. Refractive index of material was measured as mixed paraffin oils
as they are specified in the recipe of E45.
Table 4.8 Details of SOP applied in droplet size analysis for E45 Cream
Accessory (Hydro EV) Material (Mixed Paraffin Dispersant (Water)

Oils)
Stirrer Speed 1500 Refractive 1.466 Refractive 1.33

(rpm) Index index
Ultrasound Mode None Particle 0.89

Density
Analysis
Model General Sensitivity Normal Scattering Mie

purpose model
Measurement Sequence
Number of 5 Background measurement duration 10

measurement (red/blue) (seconds)
Obscuration limits 0.1- Sample measurement duration 10

(%) 20 (red/blue) (seconds)
153
4. After the mixture was visually observed to be homogenized, 3mL diluted
sample was pipetted into the dispersion unit containing 500 mL pure
degassed water which is used as dispersant.
5. Then start the measurement, follow the procedure induction of the software.
While measuring, obscuration bar was monitored within 5% to 15% by
changing the amount of injected sample.
6. When finished, a cleaning step as default in the software was carried out
by following the induction. Power off the instrument after use.
4.2.2 Results and Conclusions

The volume density of droplets was measured as a function of corresponding
droplet size as a result of droplet size distribution test. Sample A that prepared by
homogenized dissolving E45 cream in hot water before the test, the DSD of which
is presented in Figure 4.7 in red curve. It can be concluded that droplets of E45
presents a bimodal distribution, but based on the calculation of accumulative
volume density, that nearly 86.85 (v/v) of droplets were sized between 11.2 to 272
µm and less than 13% (v/v) small droplets with sizes below 10 µm. Besides, the
maximum of the curve corresponds to the largest population of droplets with
diameter of 51.8 µm, and the narrow distribution of the larger modal indicated that
most droplets in E45 cream are in equal size.
51.8, 7.2
E45 without sles
6 E45 cream+2%SLES
volume density %
11.2, 0.8 272, 0

0
0.01 0.1 1 10 100 1000 10000
droplet size um
Figure 4.7 Comparison of the droplet size distribution curves between sample A of pre-
treated E45 cream, and sample B of pre-treated E45 cream with additional 2%wt of
SLES
154
Sample B was made by adding 2% of SELS in sample A, followed by a well mixing,
which presents a completely different mode of distribution compared to that of
sample A. This value can only be applied as a qualitative indicator for the following
research as E45 was purchased from the store instead of freshly made,
flocculation or aggregation may occur in the system, leading to an inaccurate
exhibition of the microstructure. As can be seen from the blue curve of DSD for
sample B, adding 2% of SLES caused a shift to smaller droplet diameters and
broaden the size distribution. And a multimodal mode was detected. As suggested
from other study that an increase in the large size droplets reveals that the
interactions between flocculated oil droplets are sufficiently strong and/or
coalescence has occurred (Perlekar et al., 2012). Thus in a reversed way. 2% of
SLES in the sample may cause deflocculating of oil droplets in E45 cream,
resulting in average smaller droplets but an unstable system with a broader droplet
distribution.
4.3 Differential Scanning Calorimetry (DSC) Analysis

Thermodynamic property of E45 cream was analysed with the help of differential
scanning calorimetry (DSC) measurement, where TzeroTM DSC 2500 system (TA
Instrument) was applied.
4.3.1 Experimental Procedure

Measuring procedure for E45 cream could refer to chapter 3.6.2.2 introducing
preparation procedure of DSC measurement on mimic creams. The specific
measurement step for E45 cream is present as below.
1. Weigh 5-10 mg of E45 cream into the alumina pan (pre-weighed with
lid) and record data, followed by hermetically sealed with lid using Tzero
sample encapsulation press kit. This is used as sample cell
2. Seal another empty alumina pan with lid using the press kit. This is used
as reference cell.
3. Power on the instrument and check the availability of nitrogen supply.
Then open the TRIOS software. Input required parameters, including
pan weight and sample weight. Select Autosampler mode.
4. A scanning method was preliminarily created for E45 cream.
(1) Ramp up: Heating up E45 sample from -30 °C to 100 °C at a
constant heating rate of 5 ºC min-1
155
(2) Isothermal: Take an equilibration step where the sample was
isothermal at 100°C for 3 minutes.
(3) Ramp down: Cool down the sample from 100°C to the start point
which is -30 °C, with the cooling rate of 5 ºC min-1
(4) Isothermal: Equilibrate the sample at 20 °C for 3 minutes.
(5) Mark the cycle.
4.3.2 Results and Conclusions

5.07 mg sample of E45 was prepared weighed for the DSC test, the thermogram
is displayed as in Figure 4.8. As can be seen, the ice-melting peak was found
around zero degree centigrade, and another transition witnessed during
endothermal period was at temperature around 55 °C. Also, sample degradation
was found when heating over 90 °C, this may also because the instrument
malpractice. During cooling, a crystallisation point was found nearly 20 °C.
Figure 4.8 DSC thermogram of E45 cream (screen shot directly from the software)
4.4 Summary of Chapter 4

Commercialized E45 cream was characterised in terms of flow property, droplet
size distribution and thermal properties, aiming to provide a guidance for the
following preparation of mimic creams. When using 40 mm cone and plate
geometry, E45 was confined to a gap of 57 mm for rheological measurements
presenting shear thinning behaviour subjecting to increased shear stress, and
showing an apparent viscosity of 3×105 Pa·s with a yield stress of approximate 50
Pa. A solid domain viscoelastic behaviour was observed with the help of oscillatory
156
frequency sweep. No G’/G’’ crossover point is witnessed in SAOS reveals that no
frequency-invariant solid-to-liquid transition happened within the measuring range,
and it probably happens when the cream subjecting to larger amplitude or longer
period of oscillating. A bimodal mode of droplet size distribution was witnessed,
with droplets ranging from 10 µm to 100 µm with a narrow mode, presenting a
relatively stable system in spite of possibility of flocculation of droplets during its
shelf life. As for DSC result, no obvious transition was witnessed, only a melting
point was witnessed at around 55 °C. Mimic creams were then prepared using key
components in the formulation of E45 cream, including white soft paraffin, light
liquid paraffin, cetyl alcohol (CA) and glycerol monostearate (GM), incorporating
with lab-available sodium lauryl ether sulfate (SLES).
157
Chapter 5. Variation of Mimic Creams Prepared
with Different Emulsifying System
Characterisations of E45 cream in terms of its flow and thermal properties were
carried out and introduced in previous chapter, where a standard rheological
behaviour of cream-like products were achieved, giving reference for the following
mimic cream preparation and analysis. Formulating mimic creams with different
concentrations of surfactant systems incorporating mixed paraffin oils in water will
be introduced in this chapter, then desired formulations were determined in terms
of their rheological behaviours and thermodynamic properties when comparing to
standard E45 cream.
5.1 Explorer Formulation of Mimic Creams

5.1.1 First Trial of Cream Formulation without Sodium Lauryl
Ether Sulfate (SLES), Using a Homogenizer
In the first trial of cream preparation, only cetyl alcohol (CA) was applied as
surfactant for emulsifying mixed paraffin oils in water. However, as visually
observed from the appearance of the product (Figure 5.1), a heterogeneous
mixture was displayed where two phase were separated.
Figure 5.1 Appearance of mimic cream prepared in the first trial, using CA as the sole
surfactant and a homogenizer for mixing
A homogenised product with smooth texture in appearance is the preliminary
requirement for the preparation of a desired cream. Thus it could be deducted from
the failure of this trial that, only applying one type of fatty alcohol, cetyl alcohol
(C16), in this mixed paraffin oils with water system is unable to realize expected
emulsifying effect. Ionic or anionic surfactants were considered to be applied as
collaboration with fatty alcohol for achieving better emulsification (Terescenco et
158
al., 2018b). Another potential problem that led the production to failure could be
the selection of mixing unit. Although homogenizer provided strong turbulence and
high speed of shearing for preparing ultrafine emulsions, the efficiency was greatly
reduced by the contrast large size of vessel and its limited bulk mixing function.
Therefore, the homogenizer that used was unable to fully break down the oil phase
and water phase into small droplets for the following emulsification and stabilisation
by surfactants and emulsifiers.
5.1.2 Second Trial of Cream Formulation with Sodium Lauryl

Ether Sulfate (SLES), Using an Overhead Stirrer
Based on the first trial of preparation, in addition to cetyl alcohol (CA), SLES was
applied in the emulsifying system which is added in the aqueous phase. An
overhead stirrer was applied, equipped with a pitched blade turbine with six blades
as the impeller, resulting axial flow while the rotation.
Visually observed from the appearance of prepared product, shown in Figure 5.2,
a smooth and rich texture cream with a certain degree of firmness was obtained.
However, compared to commercial E45 cream, the prepared mimic cream was
witnessed to be thinner and easier to flow.
Figure 5.2 Appearance of mimic cream prepared in the second trial, using CA and SLES
as surfactants and a stirrer with pitched blade turbine for mixing
A steady state shear was carried on the mimic cream in order to get a general idea
about its rheological property. After pre-sheared under 70 Pa for 5 min followed by
an equilibrium of 55 minutes, the mimic cream was sheared from 10 Pa to 300 Pa,
resulting a viscosity profile as a function of shear stress. The Ostwald curve was
obtained, where three stages are displayed in the profile. The viscosity showed
independence with low shear stress, then behaved shear thinning property after
exceeding the yield stress, followed by a gradually decrease in the 2nd Newtonian
159
plateau. The comparison is schematically presented in Figure 5.3 with
representative rheological curve of E45 and mimic cream.
1000000
27.38, 3.65E+05
100000 51.3, 8.14E+04

25.06, 1.87E+05
10000 79.24, 2.19E+04
1000
Viscosity /Pa S
100
E45
10 1st Newtonian Plateau of

E45
mimic cream
1
1st Newtonian Plateau of
mimic cream
0.1
1 10 100 1000
Shear Stress /Pa
Figure 5.3 Comparison of representative flow behaviour between E45 cream and
mimic cream that emulsified by SLES and cetyl alcohol, where viscosity varied as a
function of shear stress ranging from 5 Pa to 300 Pa
Green line and purple line with dot represented for the 1 st Newtonian Plateau for
mimic cream and E45 cram separately, where the average viscosities of them were
in the same magnitude, indicating similar rigidity of mimic cream and E45 when at
rest. Both of mimic cream and E45 presented sharply drop of viscosity within short
shear stress range, when exceeding a certain yield stress, showing shear thinning
behaviour. The comparable data between E45 and mimic cream was summarised
in Table 5.1.
Shear stresses at the end of 1st Newtonian Plateau for mimic cream and E45 were
25.06 Pa and 27.38 Pa respectively which are similar, however, a transition region
between this point and the start of plunge for mimic cream was apparently longer
than that for E45 cream. Thus compared to E45 cream, more stress was required
for spreading out the mimic cream to the skin. In addition to that, mimic cream
failed to reach as low viscosity during higher shear stress range as the E45 cream,
showing a poor end-of-use in terms of absorption capacity perceptible on skin.
Comparison data was summarised in table.
160
Table 5.1 Results of steady state shear measurement for E45 and mimic cream
containing SLES and CA
Shear
Shear
stress at Average Dynamic
stress at
end of 1st viscosity at 1st viscosity at
Product onset of
Newtonian Newtonian shear stress of
plunge
Plateau Plateau (Pa·s) 300 Pa (Pa·s)
(Pa)
(Pa)
E45 cream 27.38 3.288×105 51.30 <0.211
Mimic
cream
containing 25.06 1.704×105 79.24 0.407
SLES and
CA
As a conclusion, the preliminary prepared mimic cream presents decent property

in terms of rheological behaviour under steady state shear, compared to E45
cream. Also, SLES as an ionic surfactant is vital in the emulsifying system for
complete the preparation of cream product, without which, agglomerates were
separated out (Kumari et al., 2018).
5.2 Formulation_Ⅰ of Cream Formulation, Using a

Simplified Configuration
5.2.1 Appearance of Mimic Creams in Formulation_Ⅰ
After the preparation, creams were transferred into 50 ml wide-opened jars where
they were rested for 20 min before subjecting to rheological tests. Appearances of
prepared creams were presented in Figure 5.4, where the corresponding weight
concentrations of surfactants were specified. Three components that involved in
the emulsifying system, sodium laureth sulfate (SLES), cetyl alcohol (CA) and
glycerol monostearate (GM), was classified as anionic surfactant (SLES) and fatty
alcohols (CA and GM). In order to be simplified, a nomenclature was created to
correlate surfactant components with their weight concentrations, that is, cream
containing [SLES, CA, GM] with the weight concentration wt% of [x,x,x]. For
example, cream [0,6,6] refers to the cream containing 0 wt% of SLES, 6 wt% of
cetyl alcohol (CA) and 6 wt% of glycerol monostearate (GM).
Visually observing the appearance of creams after preparation, those containing

no SLES displaying separated phases were identified to be failed preparation,
which is shown on orange background. This further proved the result obtained in
the second trial of preparation. It is noticeable, however, that higher concentration
161
of fatty alcohols (CA and GM) led to the conversion of small agglomerates to a
larger lump, and less water separated out.
SLES /%wt CA GM
(wt%) (wt%)
0 2 4 6
6 6
6 2
2 6
2 2
Figure 5.4 Appearances of mimic creams prepared in Formulation_Ⅰ
The presence of appropriate consistency and texture is the fundamental of a semi-

solid cream. Mimic creams showed on purple background were visually
determined to be desired cream products, especially those formulated with CA-to-
GM ration of 3:1, where 6 wt% CA and 2 wt% GM applied, are desired, namely
cream [2,6,2], [4,6,2] and [6,6,2], exhibiting smooth texture and seemly reasonable
rigidity. Increasing the concentration of fatty alcohols, creams with 6 wt% CA and
6 wt% GM were obtained (red background). These over-stiff products contained
crystals that were separated out. On the contrary, reduce the fatty alcohols in the
system had a tendency to result in fluid products with undesired low consistency.
Referring to creams formulated with 2 wt% CA and 2 wt% GM, they were very thin
and also bubbles were involved. Thus as preliminary deducted, that gel structure
was not fully established during cooling due to the lack of fatty alcohols (Deyab,
2019). Further rheological measurements will be applied to give the evidence and
explanation.
162
5.2.2 Rheological Characterisation of Mimic Creams in
Formulation_Ⅰ
Rheological measurements allow to translate the qualitative properties of skin feel

to quantitative evaluation of how the material responds to stress and strain (Bekker
et al., 2013). Mimic creams were analysed with different types of measurements
including steady state shear for viscosity profile analysis and dynamic oscillatory
for viscoelastic property investigation. Creep test was also conducted as the
additional information for viscoelasticity evaluation.
5.2.2.1 Steady State Shear
After rest in the storing jar for 20 minutes, mimic creams were analysed using
AR2000 rheometer for the study of their flow properties, using 40 mm cone and
plate geometry. Proper amount of cream sample was confined in the measuring
gap of 57 mm, followed by another equilibrium for 20 min before carrying out steady
state shear measurement. Also, the equilibrium time was proved to be reasonable
for sample to relax, as highly reproducible data was achieved. Figure 5.5 illustrated
flow properties of 12 creams which were allocated into four groups, where their
viscosities change dependent on shear stress from 5 Pa to 300 Pa at 25 °C was
obtained.
It has been suggested in the literature that, if yield stress exists, the typical steady
state shear viscosity curve for an emulsion presented in logarithm scale is roughly
divided into three stages: 1st Newtonian plateau where viscosity is constant at low
shear stress, shear thinning as shear stress increase, 2nd Newtonian plateau where
the sample undergo high shear stress. This is known as Ostwald curve (Blanco-
Díaz et al., 2018, Graziano et al., 1979). A three-dimensional gel structure or matrix
that established in the semisolid system was witnessed according to 1st Newtonian
plateau, where the cream remain its body and behaves like solid under small shear
forces, such as product on shelf or during transportation (Blanco-Díaz et al., 2018).
With the shear stress increasing by different processes such as mechanical mixing,
pumping, or rubbing until the critical stress level is exceeded, the matrix structure
will be destroyed, where the viscosity drops dramatically, and the cream body
becomes thinner and easier to flow. This critical stress is generally defined as yield
stress. Continuously increasing the shear stress leads to the cream with lower
163
viscosity behaving like fluidic emulsion state, which is presented as the gradually
decrease of viscosity in 2nd Newtonian plateau (Moresi et al., 2001).
Figure 5.5 Flow profiles of 12 mimic creams prepared in the Formulation_Ⅰ, using
simplified configuration, where viscosity varied as a function of shear stress from 5 Pa
to 300 Pa
Parallel compared between four rheograms, only when the combination of 6 wt%
cetyl alcohol (CA) and 6 wt% glycerol monostearate (GM) (cream [x, 6, 6]) or that
of 6 wt% CA and 2 wt% GM (cream [x, 6, 2]) formulated in the emulsifying system,
viscosity profiles behaved following Ostwald curve. When 6 wt% CA and 6 wt%
GM involved in the system, change of SLES concentration from 2 wt% to 6 wt%
had little effect on flow properties of creams in terms of average viscosity of 1×106
Pa·s at 1st Newtonian plateau, yield stress of over 100 Pa and shear thinning
behaviour. Many literatures explained the reason for the presence of yield stress
in emulsion products, some of which ascribed it to the formation of three-
dimensional network structure by the involvement of some polymeric thickening
agent or stabilizers (Oppong et al., 2006, Nelson and Ewoldt, 2017). As for the
preparation of creams in semisolid-state, gel phase will form when ionic surfactant
and fatty alcohols coexist in the system, therefore achieving self-bodied emulsion
(Strathclyde, 1990). Yield stress of product, which determines consumers’ initial
feel when applying the cream on skin, should be in an appropriate range. Thus the
sufficient amount of yield stress presented to avoid flow against its own gravity,
164
should not cause difficulties in the distribution of creams on skin. These creams
presented almost twice yield stress as E45, indicating undesired rigidity behaved.
The 2nd Newtonian plateau was not obviously obtained for [2, 6, 6], [4, 6, 6] and [6,
6, 6]. While it is worth to mention that, the dynamic viscosity at 300 Pa of these
creams were greater than that of E45 cream, indicating high rigidity of cream
bodies at high shear. As suggested in literatures that, those excess fatty
amphiphiles applied in the system, which did not participate in forming hydrophilic
gel phase along with ionic surfactants, build up hydrophobic gel phase contributing
for the undesired increase of consistency and viscosity, and the phase is
crystallized out upon cooling procedure (Kónya et al., 2003). This also help explain
the crystals witnessed in cream [2, 6, 6], [4, 6, 6] and [6, 6, 6].
By decreasing the concentration of glycerol monostearate from 6%wt to 2%wt,

cream [2, 6, 2], [4, 6, 2] and [6, 6, 2] were prepared. In general, their viscosities at
1st Newtonian plateau were one magnitude smaller than those containing 6%wt
glycerol monostearate, exhibiting less stiffness texture. Also, the viscosity profile
presented a more pronounced Ostwald curve for every cream, although details of
each stage differed between creams. It can be found that, increasing the
concentration of SLES from 2 wt% to 6 wt% in the cream system [x, 6, 2] leads to
cream of lower 1st plateau viscosity and yield stress, which is obviously presented
in Figure 5.6. The limited apparent viscosity at 1 st Newtonian plateau was
calculated by averaging the dynamic viscosities during the low shear plateau range,
displaying in the figure for each cream, where the value of cream containing 2 wt%
SLES was nearly double that of cream containing 4 wt% SLES, and four times
larger than that of cream with 6%wt SLES. And 4 wt% SLES in the system led to
a cream with limited viscosity twice larger than 6 wt% SLES did.
In terms of yield stress, different literatures presented with different definitions,

such as the value of onset flow (end of 1st Newtonian plateau) where the maximum
of viscosity is achieved (Mangal and Sharma, 2017) and the average value
between that and onset of plunged shear thinning (Zhu et al., 2005). Here, the yield
stress was analysed base on the onset of flow and the onset of plunge. Table 5.2
summarises the key flow parameters related for each cream, which provided data
for the flow curve interpretation.
165
3.0E+05 50
limited apparent viscosity
2.64E+05
yield stress
2.5E+05
40
2.0E+05
yield stress /Pa

Viscosity /Pa.s
30
1.5E+05 1.39E+05
20
1.0E+05
6.00E+04
10
5.0E+04
1.0E+01 0
2,6,2 4,6,2 6,6,2
Composition of emulsifying system, weight concentration of
[SLES,CA,GM]
Figure 5.6 Respective comparison of average of limit viscosity and corresponding yield
stress among mimic creams formulated with varied emulsifying system
Table 5.2 Key parameters derived from viscosity profiles of creams containing 6 wt%
CA and 2 wt% GM with varied concentrations of SLES
Product [SLES, CA, GM]

[2, 6, 2] [4, 6, 2] [6, 6, 2]
(wt%)
Shear stress at end of 1st

15.83±0.02 12.59±0.00 5±0.01
Newtonian Plateau (Pa)
Average viscosity at 1st

Newtonian Plateau 2.64×105 1.39×105 6.00×104
(Pa·s)
Shear stress at onset of

79.34±0.095 50.07±0.00 25.12±0.05
plunge (Pa)
Dynamic viscosity at
shear stress of 300 Pa 1.00±0.23 0.65±0.50 0.40±0.013
(Pa·s)
166
The rheological properties of semisolid creams have a close relationship with their
microstructures, thus the effect of change of SLES concentration on the rheological
behaviour for creams may due to the microstructure altered. It has been studied
that ionic surfactant involved in the system greatly promote the formation of
interlamellarly fixed water at the expense of bulk water than non-ionic ones, plus
that more water fixed as bulk water will lead to a product with higher yield stress
(Rønholt et al., 2012). As the interlamellarly fixed water and bulk water are in
dynamic equilibrium state in the microstructure system, more ionic surfactant in the
system, product with lower yield stress will be formulated (Kónya et al., 2003). In
addition, from previous study where the 2% w/w and 3% w/w of Eucarol AGE/EC
were formulated in creams separately, the amount of interlamellarly fixed water
increased when 3% w/w of this ionic surfactant formulated. This also indicates that
cream formulated higher quantity of ionic surfactant tends to possess lower yield
stress. Also, in the study of Grewe et al., it has been found that increasing anionic
surfactant sodium dodecyl sulfate (SDS) mass fraction in SDS/cetyl alcohol (CA)
mixture caused the decrease in viscosity (Grewe et al., 2015). However, in the
emulsifying system containing 6 wt% CA and 6 wt% GM, the change of SLES
concentration from 2 wt% to 6 wt% has little effect on creams in terms of their flow
behaviour. This may be attributed to that, the change of SLES concentration was
not sufficient to alter the microstructure of creams containing higher amount of fatty
amphiphiles.
Within measured stress range, creams containing 2 wt% cetyl alcohol in the system
showed no 1st Newtonian plateau and yield stress, only displaying shear thinning
behaviour with considerably low viscosity range, which implied that no or weaker
structural matrix formed in these creams. This indicates that cetyl alcohol is an
essential excipient as fatty amphiphile in this system. Besides, compared to
creams with 2 wt% cetyl alcohol and 2 wt% glycerol monostearate, 6 wt% glycerol
monostearate involved in the formulation helped increase the limiting viscosity. It
can be seen from cream [2, 2, 6] and [4, 2, 6] that, the dynamic viscosity reached
the magnitude of ten to the fourth during low shear range.
Shear thinning behaviour is an important attribute of creams, which is normally

linked with the spreadability and distribution of products on skin (Kwak et al., 2015).
Steady state shear test simulates the condition when the cream is being spread on
skin in rotational motion, where all 12 creams showed shear thinning behaviour
regardless whether yield stress presented or not. The rate of shear thinning is also
interpreted as the shear sensitivity of products, which reveals how fast the cream
167
will be sheared to a thin layer (Calero et al., 2013). Regarding to six creams
containing 6 wt% cetyl alcohol that presented acceptable viscosity profiles, similar
rate of shear thinning was witnessed during which the viscosity sharply dropped.
Thus there is no big difference of shear sensitivity between these creams; also
they all presented rapid shear thinning when the external shear exceeds the critical
value.
5.2.2.2 Oscillatory Sweep
Viscoelastic materials exhibit both viscous and elastic behaviour making time
dependent mechanical response, thus the consistency properties of creams were
analysed using small strain rheological tests in which the structure of cream system
is guaranteed not to be destroyed. Based on the results of preliminary steady state
shear test, creams formulated with 6 wt% CA and 2 wt% GM that showed
appropriate and desired rheological attributes, were further studied to figure out
their elasticity and viscosity using oscillatory sweep measurements, where the
viscoelasticity of a material is modelled by the combination of in-phase storage
modulus, G’, and loss modulus, G’’. Because the valid characterisation has to be
carried out in the linear viscoelastic (LVE) region, oscillatory strain sweep was
preliminary applied for its determination. Then a value with in this range was
selected for the following oscillatory frequency sweep.
In the oscillatory strain sweep, certain amount of cream samples was confined
within a 40 mm cone-plate geometry at a measuring gap of 57 mm and sinusoidally
tested with strain cyclically varied from 0.01% to 1000% at a constant frequency of
1 Hz. 20 minutes of equilibrium time was set for cream to fully relax before the
measurement. Every cream was proper loaded and measured at least duplicate
with the identical operation at 25 ℃. Referring to the results of strain sweep for
cream [x, 6, 2] presented in Figure 5.7, moduli of creams showed similar
behaviours as a function of %strain. Linear viscoelastic behaviour was found
during small strain amplitudes, where elastic modulus, G’, and loss modulus, G’’,
remained fairly constant as strain increased and elastic response was
predominantly displayed due to G’>G’’. Continuously increasing the strain, both of
G’ and G’’ exhibited a drop after yielding. A crossover point of moduli was
witnessed in every rheogram, indicating the point when G’=G’’, after which, G’’ was
over G’ revealing a viscous dominated system.
168
[SLES,CA,GM] of [2,6,2]
10000
G'
1273.254 G''
1000
G', G'' /Pa
100 LVER
10
τy=33.926 Critical strain

1
0.737 1 5.106
0.001 0.01 0.1 10 100
strain (%)
10000
G'
G''
1000
591.542
G', G'' /Pa
100 LVER
10
τy=24.125 Critical strain

1
0.730 1 8.992
0.001 0.01 0.1 10 100
strain (%)
10000
G'
G''
1000
998.696
G', G'' /Pa
100 LVER
10
Critical strain
1
0.900 8.292
0.001 0.01 0.1 1 10 100
strain (%)
Figure 5.7 Oscillatory strain sweep on mimic creams formulated with 6 wt% CA and 2
wt% GM with varied concentration of SLES, where G' and G'' varied as function
of %strain ranging from 0.01 to 100
169
The limit of linear viscoelastic region is needed to be defined, as below that value
the storage modulus, G’, and loss modulus, G’’, are independent of applied strain
amplitude at a fixed frequency and fully describe elastic response and viscous
response, resulted stress as a fundamental sinusoidal wave. When being
obviously witnessed to departure the plateau, G’ and G’’ cannot represent entirely
elastic or viscous contributions, because they start altering with the strain and the
resulting sinusoidal is in distorted form. Thus, the conventionally defined G’ and G’’
as fundamental coefficients are not applicable in the nonlinear regime. Compared
to the loss modulus, G’, the storage modulus, G’, is more often recorded for the
determination of LVE range (Calero et al., 2013).
The limit yield point of G’ is correlated to the end of LVE region. In some literatures,
beyond that G’ significantly drops beyond the plateau. This yield value is calculated
from the intersection of horizontal line of the behaviour of G’ during low strain range
with power law representing behaviour of G’ during large strain range (Dinkgreve
et al., 2016). Some others define the point only based on the linear plateau of G’.
Here in this study, the yield value is determined as a critical strain, 𝛾𝐶 , when
storage modulus dropped 10% from the plateau. Then the corresponding yield
stress, 𝜏𝑦 , was calculated by 𝜏𝑦 = 𝐺 ′ 𝛾𝐶 (Dimock et al., 2000).
During oscillatory strain sweep (OSS) test, as increasing the strain, the structural
network decays. When the experiment time of oscillation for recovery is not enough
compared to the relaxation time of the degradation, the sample may not recover.
This results in the nonlinear viscoelasticity of the sample (Nguyen et al., 2015).
The initial linear plateau of LVE was determined as a regime from the lowest
applied strain to the point where the maximum G’ occurred, then %strain
corresponding to 90% of the plateau value was recorded as critical strain. Linear
plateau for creams with 2 wt%, 4 wt% and 6 wt% SLES were at same range from
0.01% to 0.252% at frequency of 1 Hz, during which the intact structure was
presented for each of them and creams all behaved like solids. As can be seen in
the figure, the critical strain, yield stress and defined LVE region were presented.
Thus a value of 0.2% strain, from the LVE range, was selected as the amplitude
for the following oscillatory frequency test. This value is small enough to ensure
that the behaviour of viscoelastic is within linear region, and the measured stress
is proportional to the applied strain.
170
The crossover points were also indicated in the rheograms, indicating the condition
when G’ equalled to G’’ at a specific strain, normally interpreted as flow point or
flow stress, 𝜏𝑓 . The strain of crossover point was calculated by solving
simultaneous equations of exponential trend lines for G’ and G’’, followed by
interpolation to calculated corresponding modulus. Before the flow stress, G’ was
over G’’, indicating a solid domain system, whereas viscous predominated in the
system when strain increased beyond the point. In the transition region between
yield point, 𝜏𝑦 , and flow point, 𝜏𝑓 , storage moduli were higher than loss moduli of
three creams, suggesting that, although the structure of each cream was destroyed
and started to break down, they still displayed in solid state. And it is worth of
noticing that, as increasing the SLES concentration from 2 wt% to 6 wt%, the
difference between G’ and G’’ during LVE and transition region gradually
decreased, implying that cream [2, 6, 2] behaved more elastic predominant.
Some literatures compared the elastic yield stress obtained in oscillatory strain
sweep to the dynamic yield stress obtained from steady state, indicating that
dynamic yield stress is much larger than the elastic yield value (Mahaut et al.,
2008). Similar result was found in this study, except that the departure of two yield
stresses between creams with varied concentrations of SLES were small. Besides,
it is still under debate among researchers that whether the yield stress obtained
from steady state shear test is suitable for predicting the stability of product as the
microstructure destroyed during test (Dinkgreve et al., 2016).
Oscillatory frequency sweep test was carried out for each cream. The results in
Figure 5.8 presented storage modulus (G’), loss modulus (G’’) and complex
viscosity (ƞ*), of cream [2, 6, 2], [4, 6, 2] and [6, 6, 2] separately, as a function of
frequency (Hz), at the constant amplitude of 0.2% strain. It can be observed that,
G’, G’’ and ǀƞ*ǀ, were presented qualitatively similar trend as frequency rising from
0.01 Hz to 100 Hz, where G’ and G’’ slowly or greatly increased and complex
viscosity decreased. In addition, storage moduli (G’) of three creams were always
greater than loss moduli (G’’) over the whole range of measured frequency,
suggesting that elasticity domain the linear viscoelastic behaviour of all creams.
This indicates creams are prepared as viscoelastic solids.
Comparing dynamic sweep rheograms for three creams in parallel, the departure
of G’’ from G’ is witnessed to be smaller as increased amount of anionic surfactant
SLES involved in the system, which gives an assumption that, if being swept at
this constant strain for longer time, namely further decrease the frequency, cream
171
[6, 6, 2] has greater possibility or first priority to show viscous behaviour superior
than elasticity when G’’ over G’. This is in line with the previous steady state results
in which cream [6, 6, 2] shows lower consistency and smaller yield stress
compared to other two creams [2, 6, 2] and [4, 6, 2]. Loss modulus G’’ represents
the viscous component of the mechanical response of a material. When a load is
applied for a long period of time or periodically and the material must resist
structure failure, the viscous energy dissipation will impart superior mechanical
performance (Pouget et al., 2012). Besides, it is interesting to notice that, beyond
the frequency of 10 Hz, loss modulus G’’ of cream [4, 6, 2] and [6, 6, 2] gradually
levelled off while that of [2, 6, 2] still showed increasing. Also, complex viscosity
ǀƞ*ǀ exhibits a decrease trend as the frequency increase for three creams, which is
also an indicator for shear thinning behaviour.
172
100000 100000
10000 10000
G', G'' /Pa
1000 1000
|ƞ*| /Pa.s
100 100
G'
10 G'' 10
|ƞ*|
1 1
0.01 0.1 1 10 100
Frequency /Hz
[SLES,CA,GM] of [4,6 2]
100000 100000
10000 10000
G', G'' /Pa
1000 1000
|ƞ*| /Pa.s
100 100
G'
10 G'' 10
|ƞ*|
1 1
0.01 0.1 1 10 100
Frequency /Hz
100000 100000
10000 10000
G', G'' /Pa
1000 1000
|ƞ*| /Pa.s
100 100
G'
10 10
G''
|ƞ*|
1 1
0.01 0.1 1 10 100
Frequency /Hz
Figure 5.8 Oscillatory frequency sweep on mimic creams formulated with 6 wt% CA
and 2 wt% GM with varied concentration of SLES, where G', G'' and |η*| varied as a
function of frequency ranging from 0.01 Hz to 100 Hz
173
Cox-Merz rule describes the situation for some specific materials when their
behaviour of steady shear viscosity η(𝛾̇ ) versus shear rate is consistent with that
of complex viscosity versus angular frequency |η∗ |(𝜔) . However, as shown in
rheogram (Figure 5.9), where the comparison between representative dynamic
viscosity profile obtained from steady state shear and complex viscosity profile
obtained from oscillatory frequency sweep for cream containing 2 wt% SLES, 6 wt%
CA and 2 wt% GM is presented. The Cox-Merz rule is not applicable for the cream
[2, 6, 2], due to the presence of large departure between two flow curves,
where|η∗ |(𝜔) was superior to|η∗ |(𝜔) during the whole measured range. Similar
trend was found for cream [4, 6, 2] and [6, 6, 2] as well (data not shown).
1000000
Dynamic viscosity, complex viscosity /Pa.s
100000
10000
1000
100
10
steady shear viscosity η(γ ̇ )

1
complex viscosity|η* |(ω)
0.1
0.00001 0.001 0.1 10 1000
Shear rate /s⁻¹, ang.frequency /rad s⁻¹
Figure 5.9 Comparison between steady shear viscosity and complex viscosity
respectively varied as a function of shear rate and angular frequency, for cream
containing 2 wt% SLES, 6 wt% CA and 2 wt% GM
The reason for this non-match result may attribute to the magnitudes of stress
applied in steady state measurement, which is so large that the well-established
intermolecular and intramolecular bonds of material were disrupted when the
critical stress is exceeded, thus the dynamic viscosity was measured at different
equilibrium structure of material which is different from the original state (Dogan et
al., 2013). While in dynamic sweep test, no significantly structural change in the
system because the imposed strain is small enough. Thus the viscosity in general,
resistance against deformation, measured in nonlinear steady state is at variance
174
with that in linear dynamic state. Therefore, it is well explained the situation when
the curve of complex viscosity as a function of angular frequency is above that of
shear viscosity as a function of shear rate.
It has been acknowledged from steady state shear tests that, in the system where
6 wt% cetyl alcohol and 2 wt% glycerol monostearate was applied, the change of
the concentration of anionic surfactant SLES has effect on the rheological
behaviours of creams. This is further proved from dynamic oscillatory frequency
results. Figure 5.10 clearly reveals the differences of storage modulus, G’, and loss
modulus, G’’, responding to the varied frequency between creams formulated with
different concentrations of SLES ranging from 2%wt to 6%wt. Different from steady
state shear test where the difference of apparent viscosity among creams is
significant, the storage modulus, G’, representing the elastic contribution of creams
behaved similar within small variation.
50000
G'-cream [2, 6, 2] G''-cream [2, 6, 2]
G'-cream [4, 6, 2] G''-cream [4, 6, 2]
G'-cream [6, 6, 2] G''-cream [6, 6, 2]
G', G'' /Pa
5000
500
0.01 0.1 1 10 100
Frequency /Hz
Figure 5.10 Comparison of storage and loss moduli among creams containing 6 wt% CA
and 2 wt% GM with varied concentration of SLES, where storage and loss moduli
varied as a function of frequency ranging from 0.01 Hz to 1000 Hz
However, it could be noticed that the rate at which storage modulus increase with
frequency varied between creams. Compared to the trend of storage modulus, G’,
(blue triangle) of cream [6, 6, 2] rising over the range of frequency, that (blue
square) of cream [2, 6, 2] is slower, namely, the dependence of G’ on frequency
175
for cream [6, 6, 2] is greater than that for cream [2, 6, 2]. As there is no
macromolecular polymer such as thickening agent in the formulation of creams in
the formulation, the characterisation of viscoelastic properties ascribed to the
crystalline gel network formed by the ionic surfactant and fatty amphiphiles
(Salehiyan et al., 2018). Small strains in the linear dynamic sweep has little chance
to cause this network fully destroyed, thus a weaker microstructure originally
formed in the cream is more likely reflected as more rapid growth of G’ over
frequency (Rønholt et al., 2014). Loss modulus, G’’, varying with frequency also
provided the same evidence. As G’’ measured the dissipated energy which is
transformed from the friction heat producing when a material flows, G’’ behaviour
of cream formulated with 6 wt% SLES was displayed higher than that of the other
two creams, indicating larger energy dissipation happened in the system. Because
almost equal energy was stored, referring to little difference of G’ between creams,
the microstructure of cream with 6 wt% SLES collapsed the most, thereby
exhibiting a less structured system.
Loss tangent (tan δ) which is the tangent of phase angle, also known as dissipation
factor, is defined as the proportion of loss modulus, G’’, to storage modulus, G’ (tan
δ=G’’/G’). Lower value of tan δ indicates an elastic dominant viscoelastic material,
and higher tan δ represents a material of viscous domain (Ha et al., 2015). The
comparison of loss tangents dependant on frequency for three creams containing
different SLES concentrations is portrayed in Figure 5.11, where all creams
presented a decrease trend of tan δ, valued below 1, as frequency rising (shorter
time duration), thereby revealing predominantly elastic nature. With the increase
of SLES concentration in the formulation, tan δ dependence of frequency is
approaching value of 1, indicating a more viscous response. This supplementary
demonstrates that larger amount of ionic surfactant SLES involved in cream
system containing 6%wt cetyl aocohol and 2 wt% glycerol monostearate leads to
a more viscous domain system.
176
0.8
cream [2, 6, 2]
cream [4, 6, 2]
cream [6, 6, 2]
0.6
Dissipation factor (tan δ)
0.4
0.2
0.01 0.1 1 10 100
Frequency /Hz
Figure 5.11 Comparison of dissipation factor among creams containing 6 wt% CA and 2
wt% GM with varied concentration of SLES, where dissipation factor varied as a
function of frequency ranging from 0.01 Hz to 1000 Hz
5.2.2.3 Creep and Recovery
Creep-recovery test was carried out, in order to further analyse the viscoelastic
behaviour of creams and support the results of oscillatory sweep measurement.
Creams formulated with 2 wt%, 4 wt% and 6 wt% SLES together with 6 wt% CA
and 2 wt% GM was characterised using creep test respectively, where each cream
sample was subject to constant stress of 10 Pa within linear viscoelastic region for
30 minutes, followed by a recovery step for another 30 minutes when the applied
stress was removed. The resulted compliance for every cream was plotted as a
function of time, illustrated in Figure 5.12. It can be seen that creep compliance
and recovery raised when the concentration of SLES in the cream increasing from
2 wt% to 6 wt%. However, all creams exhibited similar response courses under the
stress within the time range, where instantaneous deformation, primary creep and
secondary creep were observed during the creep process followed by
instantaneous elastic and secondary elastic recovery, indicating their viscoelastic
properties.
The creep compliance, ratio of resulted strain to the applied stress, reveals the
softness of the material. That is, cream of stronger structure will behave higher
compliance during creep and a weaker structured cream is related to a lower J(t)
value (Sanz et al., 2017). Referring to the creep-recovery rheogram of creams,
177
cream formulated with 2 wt% SLES obviously showed the lowest J(t) compared to
cream containing 4 wt% and 6 wt% SLES, suggesting a robust structural network
formation and reinforcement induced by less amount of ionic surfactant in the
system containing 6 wt% CA and 2 wt% GM.
8
cream [2, 6, 2]
7 cream [4, 6, 2]
6 cream [6, 6, 2]
5
J /10⁻³Pa⁻¹
1
Stress Applied Stress Removed
0
0 500 1000 1500 2000 2500 3000 3500 4000
Time /s
Figure 5.12 Comparison of the compliance (J) response among creams containing 6
wt% CA and 2 wt% GM with varied concentration of SLES, where compliance varied as
a function of time. Curve illustrated with mean values, and standard deviations were
0.0002 1/Pa for 2 wt% SLES involved, 0.0002 1/Pa for 4 wt% SLES involved, 0.0003 1/Pa
for 6 wt% SLES involved
The typical creep-recovery curve of semisolid material is illustrated in Figure 5.13,

which is identified as instantaneous elastic deformation (OA), primary creep (AB)
and secondary creep (BC), followed by a fully elastic recovery (CD) of AB, partially
recovery (DE) from BC and irreversible residual. And the creep-recovery curve is
usually interpreted with a mechanical model, frequently as the generalized Kelvin-
Voigt model which is a Maxwell unit in series with several Voigt units, which is
illustrated in Figure 5.14.
Relating the resultant creep curve to the mechanical model, the instantaneous
elastic deformation of OA is associated with the Maxwell spring which is uncoupled
in Voigt unit, representing the elasticity and rigidity of the gel network. In molecular
aspect, this reveals the primary bonds, such as ionic bonds, which are stronger
and stretching elastically. The AB curve bending downwards indicates the
178
viscoelasticity of the material and could be interpreted by the series of Voigt units,
where the weaker secondary bonds in part of gel network are breaking and
rebuilding when subjecting to stress and then removed. This delayed elastic
response arises due to the operation internal viscous forces represented by the
dashpots coupled in Voigt units. The residual dashpot in series with Voigt units
gives rise to the Newtonian flow in BC region, indicating the viscous deformation
of the dispersion in liquid medium (Dolz et al., 2008). During recovery phase within
time interval 30min≤t≤60 min when the stress is removed, three regions are
observed, including instantaneous recovery in CD segment, which is
corresponding to the uncoupled spring, followed by the retardant recovery in DE
segment, which is the partially recovered from AB due to the Kelvin-Voigt units.
The residual compliance is a permanent deformation which is unrecoverable, due
to the uncoupled dashpot.
Secondary creep Instantaneous recovery

D
B
Retardant recovery
Compliance
Primary creep
A
Residual compliance
Instantaneous deformation
O
Time
Figure 5.13 Typical plot of compliance varied as a function of time in a creep-recovery
test for a viscoelastic material
η1 ηi
1 τ0
G0 G1 Gi η0
Figure 5.14 Mechanical model for interpretation of creep-recovery result
179
5.2.3 Droplet Size Distribution Analysis of mimic creams in
Formulation_Ⅰ
Droplet size distribution (DSD) analysis was carried out on three creams
respectively with [SLES, CA, GM] of [2, 6, 2], [4, 6, 2] and [6, 6, 2] at various mixing
speed of 500 rpm, 700 rpm and 900 rpm separately. Also, the DSD of creams are
studied at different mixing time (3 min, 5 min, 10 min, 15 min and 20 min). All the
figures presented the distribution in log-normal mode which will give a better idea
of the distribution. Figure 5.15 shows the droplet size distribution of three mimic
creams after being mixed 10min at 500rpm. As can be seen, one mode is detected
in each cream. Besides, when the concentration of SLES increased from 2 wt% to
6 wt%, the population of large droplets decreased, and the maximum point of their
size distribution curve was shifted to smaller values.
Figure 5.15 Comparison of droplet size distribution among creams containing 6 wt%
CA, 2 wt% GM with varied concentrations SLES, where volume density varied as a
function of diameter. Mean values are presented in curve for each cream.
Larger size droplets indicates stronger attractive interactions exists between

flocculated oil droplets (Udomrati et al., 2013). This indicates that in the formulation
where less SLES involved, the attractive interactions between oil droplets are
weaker. In another words, stronger repulsive forces were presented in the system
containing lower concentration of ionic surfactant. For the microstructure of O/W
semisolid cream, oil droplets are stabilised by monomolecular film and multilayers
of lamellar liquid crystals, instead one monomolecular of surfactant, and this multi-
180
layered interfacial film, which brings repulsive electrostatic forces, steric forces and
hydrational forces, contributes to the increase of consistency and stability of the
system (Eccleston, 1997). Combined with rheological results obtained above,
where cream formulated with 2 wt% presented higher consistency and higher
yields stress compared to that with 6 wt%, giving the evidence that the interfacial
film between droplets are stronger enough to protect them from coalescence. Also
according to micelle nucleation theory, with the increase of SLES, more micelles
are formed in the emulsion, thus the droplet size will be smaller.
Creams were also examined under a polarized light microscope one day after
preparation, under a magnification of ×64, where The Axioplan 2 imaging
microscope was applied (Zeiss, Germany). Samples were prepared by smearing
tiny amount of creams on to microscope slides, with glass cover slips on top. Figure
5.16 presents the photomicrographs of cream system containing 2, 4, 6 wt% SLES
combining with 6 wt% CA and 2 wt% GM respectively. The emulsifying system with
6 wt% SLES contained much smaller droplets than the other two systems. And the
difference of droplet size between creams formulated with 4 wt% and 6 wt% SLES
is not significant. This relatively agreed with the rheology result.
(a) (b)
(c)
Figure 5.16 Microscopic observation of mimic creams containing 6 wt% CA, 2 wt% GM
with varied concentrations of SLES
181
5.2.4 Thermodynamic Properties of Mimic Creams in
Formulation_Ⅰ
The thermodynamic properties of creams were analysed using differential

scanning calorimetry (DSC) experiments, with the help of a Q2000 DSC system
(TA Instrument). Samples of creams were weighed into the alumina pan. Then the
pans were hermetically sealed, as well as the reference (air). The measurement
was performed by heating the sample from 25 °C to 90 °C at a rate of 3 °C min-1,
equilibrating at 90 °C f or 3 min, followed by a backward cooling procedure to -
20 °C at the same scan speed. After the equilibrium at -20 °C for another 3 min,
the cream was heated up back to 25 °C. Therefore, thermos-diagrams of creams
were obtained. Similar method was applied to study thermal properties of pure
ingredients, such as mixed paraffin oils, SLES, CA and GM. The information of
melting points and crystallisation points of them was expected to be acquired, also
the differences between creams formulated with different emulsifying systems.
Figure 5.17 displayed the differential scanning calorimetry thermograms of

ramping circle between room temperature and 80 °C for CA and GM, and that for
paraffin oils and SLES are respectively displayed in Figure 5.18 and Figure 5.19.
4 cetyl alcohol
glycerol monostearate
Heat Flow (Normalized) Q(W/g)
-2
-4
20 25 30 35 40 45 50 55 60 65 70 75 80
Temperature T (°C)
Figure 5.17 DSC thermogram of cetyl alcohol and glycerol monostearate
182
0.3 white soft paraffin
liquid liqiud paraffin
0.2
Heat Flow (Normalized) Q (W/g)

0.1
-0.1
-0.2
-0.3
20 25 30 35 40 45 50 55 60 65 70 75 80
Temperature T (°C)
Figure 5.18 DSC thermogram of light liquid paraffin and white soft paraffin
There is no ndotherm peak showed in this range for light liquid paraffin. Cetyl
alcohol showed an endotherm peaking at 50 °C with a shoulder from 45 to 55 °C,
representing for the melting of the crystals. The melting of glycerol monostearate
crystals witnessed at higher temperature at around 65 °C. The thermogram of
SLES indicated that water existed in the sample, as ice-melting peak was
witnessed at around zero degree. Also crystallisation was observed at 1°C.
Figure 5.19 DSC thermogram of sodium lauryl ether sulfate (screen short directly from
software)
183
DSC scan of creams formulated with different concentrations of SLES in system
are compared in Figure 5.20. In this emulsifying system, where SLES as ionic
surfactant and cetyl alcohol combined with glycerol stearate being used as fatty
amphiphiles, with the increase of SLES concentration from 2 to 6 wt%, the
temperature of endotherm peak decrease from around 58 to 52 °C. It has been
studied that, as the formation of liquid crystals above transition temperature and
gel phase below this temperature is rapid, the gel structure will be formed soon
after preparation (Ribeiro et al., 2004, Zhang et al., 2017a). As only one endotherm
peak was presented in each cream thermogram, it cannot be concluded that, there
has a trend by which high-temperature gel endotherm diminishes and low-
temperature crystalline endotherm develops. However, combined with the results
of rheological test, with high concentration of surfactant used in the system, the
limiting value of viscosity and yield stress decreased, this could be explained as
the conversion of gel networks to an isotropic phase and cream system becomes
more mobile.
-0.15
Heat Flow Q(W/g)
-0.20
2,6,2
4,6,2
6,6,2
-0.25
30 40 50 60 70
Temperature T (°C)
Figure 5.20 Comparison of thermal behaviour among creams containing 6 wt% CA, 2
wt% GM with varied concentrations SLES, where heat flow varied as a function of
temperature ranging from 25 °C to 90 °C
5.3 Complementary Rheology Study of Creams

Formulated in Formulation_Ⅱ
From the visually observation from the appearances of formulated mimic creams
formulated in Formulation_ Ⅰ, it has been found that cetyl alcohol, as a fatty
amphiphile, played an essential role in the formulation of well-structured cream
184
product. Further analysis was made, by characterising mimic creams formulated
with varied concentration of cetyl alcohols in Formulation_Ⅱ.
The effect of changing concentration of fatty alcohols on the rheological behaviour

of cream system was studied using steady state rotational measurement. Two
emulsifying systems were studied, where 2%wt SLES and 4%wt SLES were
involved separately. Concentration of cetyl alcohol was increased from 5%wt to
7%wt, with the amount of glycerol monostearate at constant of 2%wt. Key data
was presented in Table 5.3.
Table 5.3 Key parameters derived from viscosity profiles of cream containing 2 wt%
SLES and 2 wt% GM with varied concentrations of CA

[2, 5, 2] [2, 6, 2] [2, 7, 2]
(wt%)

12.56±0.03 15.83±0.02 25.06±0.018

1.67×105 2.64×105 2.69×105
Newtonian Plateau (Pa·s)

50±0.015 79.34±0.095 125.6±0.09
plunge (Pa)
(Pa·s)
In the cream system containing 2 wt% of SLES, the average dynamic viscosities
at 1st Newtonian plateau of during low stress range were in the same magnitude.
Shear thinning behaviour was witnessed in every cream, but initiating at different
critical stress which could be refer to the shear stresses at the end of 1 st Newtonian
plateau. Thus although there is no big difference of initial consistency between
creams formulated with different concentrations of CA, their resistances to
structural deformation was varied. This is more obviously found according to the
shear stress at the onset of significant drop, where the stress value of cream
containing 7 wt% CA (125.6±0.09 Pa) was more than twice that of cream
containing 5 wt% CA (50±0.015 Pa). Thus larger amount of cetyl alcohol involved
tends to form a stronger structural configuration which required larger external
force to destroy (Okamoto et al., 2016).
185
Table 5.4 Key parameters derived from viscosity profiles of cream containing 4 wt%
SLES and 2 wt% GM with varied concentrations of CA

[4, 5, 2] [4, 6, 2] [4, 7, 2]
(wt%)

12.56±0.01 12.57±0.014 31.55±0.03

Newtonian Plateau 1.02×105 1.39×105 9.41×105
(Pa·s)

62.95±0.04 50.04±0.057 250.6±0.06
plunge (Pa)
(Pa·s)
As seen from Table 5.4, in the system where 4 wt% of SLES was applied, slightly
unexpected results were presented, where no significant difference of steady state
rheological behaviour between cream systems containing 5 wt% and 6 wt% cetyl
alcohol. However, a notable enhancement of consistency and yield stress was
presented when its concentration increased to 7 wt%. The rheological result may
be attributed the microstructural nature of creams. Part of fatty amphiphiles will
form hydrophilic gel phase cooperating with ionic surfactants, while the excessive
amount of that establish hydrophobic phase which contributes most to the higher
consistency of cream product (Okamoto et al., 2016). Thus in the system where
more SLES involved, the available sites for combination of cetyl alcohol to form
hydrophilic gel phase were increased, thus although the same increment of cetyl
alcohol from 2 wt% to 6 wt% was presented in two cream system containing 2 wt%
and 4 wt% SLES respectively, the presence of SLES may affect the amount of
hydrophobic phase, thereby contributing to different rheological behaviour in
different systems.
186
Mimic creams were prepared with surfactant systems of varied compositions,
followed by characterisation with the help of rheology, droplet size distribution
analysis and DSC, aiming to provide a guidance for the following study of bio-
creams containing biosurfactants instead. As a result, systems of 6 wt% cetyl
alcohol and 2 wt% of glycerol monostearate cooperating with various
concentrations of sodium lauryl ether sulfate (SLES) ranging from 2 wt% to 6 wt%,
namely cream [2SLES, 6CA, 2GM], [4SLES, 6CA, 2GM] and [6SLES, 6CA, 2GM],
exhibited desired rheological behaviours in comparison with E45 cream, especially
for cream [4SLES, 6CA, 2GM] where a smooth and rich texture was witnessed
from the appearance. The exhibited average apparent viscosity at 1 st Newtonian
plateau was 1.39×105 Pa.s with a yield stress of over 50 Pa which is in the same
magnitude as that of E45, when rheological measurements were conducted using
the same geometry (40 mm cone-plate with a measuring gap of 57 m). Elastic
domain viscoelastic was witnessed for all creams, where G’ was higher than G’’
over the whole frequency range from 0.01 Hz to100 Hz. Apart from that, it showed
that increasing concentration of SLES in this system led to a decrease in viscosity
and yield stress, where apparent viscosity before yield stress was 2.64×105 Pa.s
for cream containing 2 wt% of SLES, while that was only 6×104 Pa.s for cream with
6 wt% SLES. The same trend was confirmed by the result of oscillatory and creep
test. In addition, endotherm peak of creams decreased with the increased
concentration of SLES, indicating a more thermal stable system containing SLES
of 2 wt% compared to 6 wt%. In terms of the droplet size distribution analysis,
higher concentration of SLES involved resulted in a system with smaller sized
droplets. Cream [4SLES, 6CA, 2GM] was selected as a standard for bio-cream
formulation. After determination of the formulae, effect of various manufacturing
procedures on creams were then studied.
187
Chapter 6. Variation of Creams Prepared with
Different Processes
Different compositions of surfactant systems were applied in cosmetic cream
formulations and the optimal formulations were determined from previous chapter.
In order to further analyse effects of changing production process, including mixing
speed, mixing time and cooling procedure, on the property of formulated product,
mimic creams containing 6 wt% of cetyl alcohol (CA) and 2 wt% of glycerol
monostearate (GM) respectively with 2, 4, 6 wt% of sodium lauryl ether sulphate
(SLES) in mixed paraffin oils/water system were prepared under various
manufacturing processes.
6.1 Effect of Mixing Time on Cream Formulation During

Heating Procedure
The effect of different heating procedure on the performance of mimic cream was
studied, where the creams were heated and mixed for varied mixing duration
ranging from 3 min to 20 min at constant mixing speed, followed by being
characterized to determine the corresponding droplet size distributions (DSD) with
the help of Mastersizer 3000. The droplet size distributions of mimic creams [2, 6,
2], [4, 6, 2], and [6, 6, 2] being mixed at 500 rpm for 3 min, 5 min, 10 min, 15 min
and 20 min are shown in Figure 6.1, where the volume density (%) was plotted as
the function of droplet size (µm).
It can be seen that all creams being mixed at different speed for various time
presented unimodal distribution with a population of droplets with a mean diameter
approximately ranging from 1 µm to 10 µm. For different systems where different
concentrations of surfactants were involved, there is no significant effect of
homogenizing duration on the distribution of droplet size, only despite that for
cream containing 2 wt% of SLES where an obvious decrease of droplet size was
witnessed after 20 min of mixing. During the mixing process at high temperature,
no significant droplet size change was displayed, indicating that the microstructure
was well formed within very short time. The reason for this may because the
concentration of the mixed surfactant system (SLES, CA and GM) exceeds the
CMC value, and a stable and rigid crystalline phase was formed at the beginning
of emulsification (Kumari et al., 2018).
188
12 3min 12 Cream [4, 6, 2] 3min
Cream [2, 6, 2] 5min
5min
10
Volume Density /%
10 10min 10min
15min
Volume Density /%
15min
8 20min 8 20min
6 6
4 4
2 2
0 0
0.1 1 10 100 1000 0.1 1 10 100 1000
Diameter /μm Diameter /μm
12 Cream [6, 6, 2] 3min

5min
10 10min
15min
Volume Density /%
8
20min
6
0
0.1 1 10 100 1000
Diameter /μm
distribution of creams containing 6 wt% CA and 2 wt% GM with varied concentrations
of SLES, at controlled mixing speed of 500 rpm
D [3,2] values of droplets in cream systems being mixed at 500 rpm for different
mixing duration were summarised in Table 6.1, where mean values were
calculated based on five replicated measurements with standard deviations
attached. It clearly proved the similarity of droplet sizes when creams being mixed
for different times during heating procedure, which is roughly agreed with the
observation from distribution curves.
Table 6.1 Sauter mean diameter D[3, 2] in cream containing 6 wt% CA and 2 wt% GM
with varied concentrations of SLES, being mixed at 500 rpm at ifferent mixing time.
The value is presented as mean value ± standard deviation
Mixing Time at 500 rpm D3,2 of cream [SLES wt%, CA wt%, GM wt%] (μm)
(min)
[2, 6, 2] [4, 6, 2] [6, 6, 2]
3 8.93±0.088 3.48±0.039 3.93±0.152
5 8.63±0.204 4.17±0.072 3.86±0.211
10 9.01±0.551 4.43±0.111 4.21±0.106
15 8.26±0.055 4.67±0.118 3.73±0.184
20 5.82±0.056 4.85±0.011 2.84±0.104
189
As shown in Figure 6.2, similar conclusion could be obtained from the situation
when mixing speed at 700 rpm, where no apparent change of droplet size
distribution with varied mixing time ranging from 3 minutes to 20 minutes. For
cream containing 2 wt% of SLES, the unimodal distribution displayed a slightly
movement to smaller droplet size with increase of mixing time, which is consistent
with previous result at mixing speed of 500 rpm.
15 3min 15 Cream [4 6, 2] 3min

Cream [2, 6, 2] 5min
5min
12 10min
Volume Density /%
12 10min
Volume Density /%
15min 15min
20min 20min
9 9
6 6
3 3
0 0
0.1 1 10 100 1000 0.1 1 10 100 1000
Diameter /μm Diameter /μm
15 Cream [6, 6, 2] 3min

5min
12 10min
Volume Density /%
15min
9 20min
0
0.1 1 10 100 1000
Diameter /μm
distribution of creams containing 6 wt% CA and 2 wt% GM with varied concentrations
of SLES, at controlled mixing speed of 700 rpm
Table 6.2 compares Sauter mean diameter D3,2 of each cream, homogenized at
700 rpm and 900 rpm for various time, which quantitatively presented that the
average droplet size was not largely altered during mixing duration within 20
minutes. As for the results at 700 rpm, similar to that at 500 rpm, except for cream
containing 2 wt% and 4 wt% SLES where nearly less than 1µm decrease of droplet
size was witnessed from 3 min to 20 min mixing, droplets in cream [4, 6, 2] were
measured with an average diameter of 4.43±0.09 µm during 20 minutes mixing.
While increasing the mixing speed to 900 rpm, droplet size showed more sensitive
to the mixing time, where the decrement of average droplet size of nearly 2 µm
was witnessed within 20-minute duration for every cream.
190
Table 6.2 Sauter mean diameter D [3, 2] in cream containing 6 wt% CA and 2 wt% GM
with varied concentrations of SLES, respectively being mixed at 700rpm and 900rpm at
different mixing time. The value is presented as mean value ± standard deviation
Mixing Time at D3,2 of cream [SLES wt%, CA wt%, GM wt%] (μm)

700rpm (min) [2, 6, 2] [4, 6, 2] [6, 6, 2]
3 6.41±0.089 4.43±0.033 4.57±0.136
5 6.25±0.046 4.57±0.073 3.96±0.014
10 5.46±0.027 4.32±0.034 4.06±0.004
15 5.33±0.0717 4.46±0.137 3.66±0.005
20 5.86±0.189 4.35±0.024 3.5±0.021
Mixing Time at D3,2 of cream [SLES wt%, CA wt%, GM wt%] (μm)

900rpm (min) [2, 6, 2] [4, 6, 2] [6, 6, 2]
3 4.11±0.015 4.14±0.038 4.08±0.315
5 3.94±0.023 4±0.057 3.38±0.029
10 3.7±0.006 3.6±0.076 3.02±0.053
15 3.88±0.004 3.44±0.020 2.9±0.021
20 3.58±0.028 2.93±0.062 2.56±0.006
More cream systems containing different concentrations of surfactants were

prepared for analysing the effect of mixing time on microstructural property of
cream in terms of droplet size distribution. They further agreeded with the previous
obtained argument that a unimodal shape of droplet size distribution was formed
at very early stage (mixing for 3 minutes) and it was not significantly affected by
the mixing time during heating process, indicating that within certain stirring speed
range, the mixing time is not a key parameter for cream formulation during heating.
191
6.2 Effect of Mixing Speed on Cream Formulation During
Heating Procedure
Model creams were stirred at different speed while heating, followed by droplet
size analysis to study the effect of stirring speed on the microstructure of the
system. Figure 6.3 illustrates the distribution of droplet size in a representative
cream containing 2 wt% of SLES, 6 wt% of CA and 2 wt% of GM being mixed at
500 rpm for 3 min. The peak of unimodal distribution significantly moved towards
smaller diameter direction while increasing stirring speed from 500 rpm to 900 rpm,
indicating a significant decrease of average droplet size. During the coalescence
of emulsions, mixing is applied for both of dispersion and mass/heat transfer.
Higher mixing speed tends to minimize the droplet size, due to the resultant
turbulent flow and the enhancement of mixing effect (Boxall et al., 2012).
12
Cream [2, 6, 2]
500rpm
700rpm
9
900rpm
Volume Density /%
0
0.1 1 10 100 1000
Diameter /μm
Figure 6.3 Comparison of droplet size distribution among creams containing 6 wt% CA
and 2 wt% GM with 2 wt% SLES, respectively being mixed at 500 rpm, 700 rpm and 900
rpm for 3 minutes. Data presented as the mean value.
However, comparing the effect of mixing speed on cream formulation in different

systems where varied concentrations of surfactants involved, the degree of
influence varied. As the mixing time has little effect on the droplet size distribution,
mean value of D3,2 at each mixing time was calculated for different system,
presenting in Figure 6.4 as a function of mixing speed. In the system where 2 wt%
SLES involved, D3,2 values largely reduced with increasing mixing speed. While
for systems containing higher concentration of SLES, the average droplet size was
192
not greatly affected by the mixing speed. Also, at higher mixing speed of 900 rpm,
varied concentration of SLES showed small impact on D3,2 values of creams.
10
cream [2, 6, 2]
9 cream [4, 6, 2]
cream [6, 6, 2]
8
7
D[3, 2] /μm
1
500rpm 700rpm 900rpm
Mixing speed
Figure 6.4 Comparison of D [3, 2] values among creams containing 6 wt% CA and 2 wt%
GM with varied concentrations of SLES, respectively being mixed at 500 rpm, 700 rpm
and 900 rpm for 3 minutes. Data is presented with the standard deviation as error bar
6.3 Effect of Cooling Procedure on Cream Formulation

Cooling is a key process in the preparation of creams, during which ingredients of
dispersed phase will create three-dimensional gel structure to support cream body
and against minor stress caused deformation.
Based on cream [4, 6, 2] containing 4 wt% of SLES, 6 wt% of cetyl alcohol and 2
wt% of glycerol monostearate, different cooling procedures were carried out,
followed by mixing for 10 minutes at speed of 500 rpm. Table 6.3 summarises
different cooling procedures in the formulation.
193
Table 6.3 Parameters for cooling process, where mixing speed and mixing time are
specified
No. Mixing speed (rpm) Cooling duration (min)
A 200 20
B 200 5
C 300 10
D 200 10
E 0 10
The rheological properties of creams numbered A to E were analysed 20 minutes

after preparation, followed by steady state shear and oscillatory sweep
measurements. The viscosity profile of each cream prepared with different cooling
procedure was presented and compared in rheogram below (Figure 6.5), where
viscosity was plotted as function of shear stress in logarithmic coordinates. All
creams prepared with different cooling procedure showed 1 st Newtonian plateau
during low stress range followed by shear thinning behaviour when beyond yield
stress. From visually comparison, there is no big magnitude variation between
creams prepared different cooling process in terms of limiting values of viscosities
(1st Newtonian plateau). However, significant departure of yield stress was
discovered between different creams.
And important parameters related to the viscosity profile were quantitatively

summarised in Table 6.4, where key information was presented including average
limiting viscosity (η0), shear stress at end of 1st Newtonian plateau (τ0), shear stress
at onset of shear thinning plunge (τ1) and viscosity at shear stress of 300 Pa (η 300).
Yield stress (τy) was determined by averaging τ0 and τ1. Besides, the slope of shear
thinning (k) was calculated by joining the onset point of shear thinning and that of
2nd Newtonian plateau where the viscosity approaching level off.
194
1000000
200rpm/20min
200rpm/5min
300rpm/10min
100000
200rpm/10min
0rpm/10min
10000
Viscosity /Pa.s
1000
100
10
0.1
1 10 100 1000
Shear stress /Pa
Figure 6.5 Comparison of different cooling procedures on cream containing 6 wt% CA
and 2 wt% GM with 4 wt% SLES, where viscosity varied as a function of shear stress
ranging from 1 Pa to 300 Pa
Table 6.4 Key parameters derived from viscosity profile of cream containing 4 wt%
SLES with 6 wt% CA and 2 wt% GM formulated with different cooling procedure
A B C D E
Cooling
Procedure 200rpm 200rpm 300rpm 200rpm 0rpm
20min 5min 10min 10min 10min
×105 η0
0.68±0.19 1.76±0.39 2.31±0.53 1.40±0.11 0.55±0.10
(Pa.s)
τ0 (Pa) 3.98±0.001 10±0.001 15.85±0.002 7.94±0.001 3.16±0.003
τ1 (Pa) 12.59±0.002 39.81±0.001 63.10±0.002 39.81±0.002 15.85±0.001
τy (Pa) 8.29±0.001 24.91±0.001 39.48±0.002 23.88±0.001 9.51±0.002
η300 (Pa.s) 0.38±0.16 2.02±0.15 5.17±0.18 1.34±0.04 0.51±0.05
k -199.23 -194.05 -523.41 -1786.5 -421.69
195
For 10 min of cooling, both of the average of viscosity in 1 st plateau and the yield
stress of creams increased with the increase of mixing speed from 0 to 300 rpm.
Thus, in the cream system containing 4 wt% of SLES, 6 wt% of cetyl alcohol and
2 wt% of glycerol monostearate, within a certain time of cooling, higher mixing
speed will produce a more rigid cream. Also, as the yield stress is related to the
strength of three-dimensional microstructure of the creams, higher value of yield
stress indicates that the cream needs larger stress to initiate flow (Mahaut et al.,
2008). However, in terms of applicability of cream to the skin, the yield stress
should be controlled at a moderate value. A stronger gel structure of cream
systems refers to more contact surfaces, lower packing fraction and stronger
packing between particles (Rønholt et al., 2014), which could be achieved by
modify mixing speed during cooling procedure.
Referring to oscillatory sweep test, creams that formulated with different stirring
speed during 10-minute-cooling were oscillated sheared at a constant %strain from
0.1 Hz to 100 Hz, and the storage modulus was presented as a function of
frequency. Within the linear viscoelastic region, amplitude was small enough that
the structure of system kept intact during measurement. As can be seen from
Figure 6.6, higher mixing speed contributed to the formulation of more rigid
structure which responded with higher storage modulus indicating a distinctly
elastic predominant system (Colafemmina et al., 2020b).
When controlling the mixing speed at 200 rpm, longer mixing time led to production
of relatively less viscous cream product. Meanwhile, compared to being cooled for
10 minutes while mixing, the yield stress of cream sharply dropped by 2/3 from
23.88 to 8.29 Pa if extending cooling time to 20 min. This implies that a weaker
matrix structure formed and the cream is easier to flow at a small stress. In the
rheogram of oscillatory measurement shown in Figure 6.7, a relatively more elastic
domain system was obtained attributed to shorter time of stirring while cooling, at
a certain mixing speed of 200 rpm.
Cooling procedure is significant for cream preparation as gel formation by

surfactant molecules is generally controlled by thermodynamics. It has been
studied that cooling rate also largely affected the microstructure of gel formation,
where fast cooling procedure (quenched) resulted in higher elastic and viscous
moduli for system containing cetyltrimethylammonium chloride (CTAC) and
cetearyl alcohol in water, and the values were 4 times higher than the slow-cooling
procedure applied (Colafemmina et al., 2020b).
196
100000
300rpm/10min
200rpm/10min
0rpm/10min
G' /Pa 10000
1000
100
0.1 1 10 100
Frequency /Hz
Figure 6.6 Comparison of varied stirring speed during cooling procedure for 10 min on
cream containing 6 wt% CA and 2 wt% GM with 4 wt% SLES, where storage modulus
varied as a function of frequency ranging from 0.1 Hz to 100 Hz
100000
200rpm/5min
200rpm/10min
200rpm/20min
10000
G' /Pa
1000
100
0.1 1 10 100
Frequency /Hz
Figure 6.7 Comparison of varied stirring duration during cooling procedure at
controlled stirring speed of 200 rpm on cream containing 6 wt% CA and 2 wt% GM
with 4 wt% SLES, where storage modulus varied as a function of frequency ranging
from 0.1 Hz to 100 Hz
197
In this chapter, the effect of heating and cooling procedure on the performance of
creams are studied. As a result, during heating procedure, varied mixing duration
from 3 min to 20 min almost had no influence on the droplet size distribution of
cream [2SLES, 6CA, 2GM], [4SLES, 6CA, 2GM] and [6SLES, 6CA, 2GM], at varied
mixing speed of 500 rpm, 700 rpm and 900 rpm. However, higher mixing speed
led to average smaller droplets for all creams. Effect of cooling procedure were
analysed with the help of rheometer, coupled with 40 mm cone-plate geometry.
For the system of [4SLES, 6CA, 2GM], in the process where cooling duration set
for 10 min, higher mixing speed from 0 rpm to 300 rpm resulted in a more viscous
and rigid cream, while when comparing the mixing time during cooling of 5 min, 10
min and 20 min, at a constant mixing speed of 200 rpm, long-term stirring during
cooling procedure contributed to a less viscous cream with relatively lower yield
stress. For the following preparation of bio-creams, mixing at 500 rpm for 10
minutes was set for heating process, and then creams were stirred at 200 rpm
during cooling for another 10 minutes.
198
Chapter 7. Production of Bio-surfactants
Along with the mimic cream formulation, biosurfactants were produced through
microorganism cultivation, followed by structural analysis for their species
determination. This chapter will display the results related to biosurfactants
production, including sophorolipids (SLs) and mannosylerythritol Lipids (MELs).
7.1 Sophorolipids (SLs)

Media broth in every shake flask was transferred into one experimental glass
reagent bottle for the further extraction and purification. After standing for a few
hours, broth separated into different layers (Figure 7.1. a), including oil phase,
major SLs, media solution and the sedimentation. Due to the density difference in
SLs, some of them precipitated with cell pellet in the bottom (Figure 7.1. b).
Oil
SLs SLs Cell pellet
Sedimentation
Media
Broth
(a) (b)
Figure 7.1 Phase separation of media broth of sophorolipids production.
199
Following the procedure of isolation and purification in section 3.1.3.2, where n-
Hexane was applied three times for residual oil removal, followed by product
extraction with equal volume of ethyl acetate, biosurfactants were then dried out to
get rid of solvents through rotatory evaporation (Dolman et al., 2017). The
appearance of fresh product right after rotary evaporator was shown in Figure 7.2
(a), which was similar to dark orange viscous syrup. Products from every batch of
rotary evaporation were transferred into 50mL plastic bottles and left in fume
cupboard for 24 hours for drying, as seen figure 7.2 (b) where the bio-surfactant
became solid-like and unable to flow. This was applied for further analysis and
application in bio-cream formulation.
(a) (b)
Figure 7.2 Appearance of extracted sophorolipids (a) right after rotary evaporation
and (b) after 24h dried in fume cupboard
50 mg L-1 SLs was produced from the fermentation, determined with the help of
gravimetric method (Dolman et al., 2017). HPLC was also carried out for measuring
the concentration of SLs. The sample preparation and characterisation method of
that was introduced in in section 3.1.3.3.
The result of HPLC was not very clear, but in general, it can be seen that a nearly
flat baseline was obtained (Figure 7.3). Also, too many sharp peaks are witnessed,
indicating highly impurity of the product. Even though the peaks are sharp enough
to be witnessed which means HPLC can be used for detecting sophorolipid, there
is not a standard to be compared with, so it is difficult to identify the fractions that
each peak stands for.
200
Figure 7.3 Result of HPLC measurement of sophorolipids
7.1.1 Structural Analysis of Sophorolipids (SLs)

Mass spectroscopy was preliminary applied to study the structure of produced
biosurfactants, where samples were prepared following the method introduced in
section 3.6.6.2. A representative mass spectrum of SLs was shown in Figure 7.4,
where detected ions with specific mass-to-charge ratios (m/z) were exhibited by
bars with their lengths indicating the relative abundance of ions.
The main peaks were at the m/z value of 705.32 and 733.32. As negative ion
electrospray was applied in the measurement, the real molecular mass for these
two peaks should be 706.32 and 734.32 respectively. It has been reported that
diacylated lactonic sophorolipid of C18:1 has the molecular mass of 687
(Khanvilkar et al. 2013). In addition, the molecular mass of acidic form is 18 more
than lactonic form (Dolman et al., 2019). Therefore, the structure with molecular
mass of 705.32 tends to be diacylated acidic sophorolipid of C18:1.
Regarding to the peak valued 733.32, which is almost 28 more than that of
diacylated acidic sophorolipid of C18:1, possible structure suggested for this
molecular mass is diacylated acidic sophorolipid of C20:1.
201
Diacylated acidic 733.3223
SLs with C18:1
Diacylated acidic
SLs with C20:1
687.3149
Diacylated
lactonic SLs Diacylated
with C18:1 acidic SLs
with C25:2
Figure 7.4 Representative mass spectrum of sophorolipids obtained from mass

spectrometry
Besides, another two peaks were also detected, corresponding to the real
molecular mass of 688.31 and 802.31. The former represents for diacylated
lactonic Sophorolipids of C18:1. As for the latter, it can be found that this structure
of SLs was unlikely to consist of a hydrophobic tail with 18 carbons (C18), as it was
92 higher than the molecular mass of diacylated acidic sophorolipid with C18:0
which has the maximum molecular mass among structures with C18. Thus, for the
peak at m/z of 801.31, diacylated acidic sophorolipid of C25:2 was assumed. As a
matter of fact, this structure of sophorolipid with long chain is kind of reasonable,
as the hydrophobic carbon source was rapeseed oil which contains almost 50%
erucic acid (C22).
From the result of mass spectroscopy, more acidic SLs were produced in the
fermentation than lactonic forms. One possible reason may because that, during
the fermentation, the pH of the media was not maintained at the optimal value. This
was also found in literature that, when the pH value drops to 2, more acidic form
of SLs was presented in the product (Dolman et al., 2017).
7.1.2 Surface Tension Analysis of Sophorolipids (SLs)

The surface activity of SLs was measured, using method referring to section 3.6.5.
Figure 7.5 illustrated the surface tension of SLs aqueous solutions at different
concentrations. Surface tension rapidly decreased with the increase of the
202
concentration of SLs solution, and gradually levelled off after reaching approximate
34.59 mN m-1, corresponding to a CMC value of 50 mg L-1.
The CMC of SL solution (50 mg L-1) is lower than that of SLs produced by
cultivating Candida Bombicola on a medium containing sugarcane molasses with
soybean oil (59.43 mg L-1) (Daverey and Pakshirajan, 2009), and glucose with
soybean dark oil (150mg L-1) (KIM et al., 2005). The difference of CMC value may
due to different structures of SLs that produced by cultivating the strain on different
substrates. In another aspect, the purification of SLs may also affect the result. In
previous study, the minimum surface tensions in crude and purified SL solutions
were nearly the same, which are 39 mN m-1 and 36 mN m-1 respectively. However,
the crude SLs mixtures showed a much higher CMC value of 130 mg L-1 compared
to the purified SLs (CMC of 10 mg L-1) (Otto et al., 1999).
80
surface tension/(mN·m-1)
70
60
50
40
30
0 30 60 90 120 150 180 210 240 270 300 330
Concentration of sophorolipid solutions/(mg·L-1)
Figure 7.5 Surface activity of SLs in water solution, where surface tension varied as a
function of the concentration of sophorolipids
203
7.2 Mannosylerythritol Lipids (MELs)
Shake flask fermentation and fed-batch fermentation were carried out for MELs
production separately. After 10 days of strain cultivation, orange beads were found
in the shake flasks of batch fermentation, shown in Figure 7.6 (a), and products
with disparate morphology were obtained from fed-batch fermentation, where
yellow gel-like aggregates were witnessed.
(a) (b)
Figure 7.6 Appearance of MELs products from (a) batch fermentation and (b) fed-batch
fermentation
7.2.1 Structural Analysis of MELs

Mass spectrometry (MS) was performed on MELs to determine whether the
product was MELs and analyse the structure composition. Sample preparation and
measuring procedure has been introduced in 3.6.6.2.
Figure 7.7 presents the MS result of the product, where many peaks are exhibited
on the positive mass spectrum of [M+H]+ ion. This indicates that the crude product
contains oils and fatty acids (peaks at m/z less than 500), and various structures
of biosurfactants.
204
Figure 7.7 Results of mass spectrometry of mannosylerythritol lipids
In order to identify peaks in details, MS analysis was carried out within smaller
specific mass-to-ratio ranges including m/z of 450-600, 600-750 and 750-1050,
among which the MS spectrum at m/z from 600-750 is shown in Figure 7.8.
MW: 670.4
MW: 696.4
MW: 656.4
Figure 7.8 Representative mass spectrum of mannosylerythritol lipids with m/z

ranging from 600 to 750
Three major ion peaks of the [M+H]+ ion at m/z 671 (671.36), 697 (697.37) and
657 (657.38) are presented, and the corresponding molecular weight was
approximately determined as 670.4, 696.4 and 656.4. The ion peak at m/z 671 can
205
be interpreted as resulting from (ME-4H+: 280) + 2*(acetyl group: 43) + (decylenic
acid-OH- : 153) + (decynoic acid-OH-: 151) + (H+:1). In comparison, the ion peak
at m/z of 697 presenting a molecular mass difference of 21 from the main peak,
which is possible due to the difference in fatty acid chain. Based on this calculation,
Table 7.1 summarise some interpretation of peaks that obtained according to other
papers, where the possible fatty acid chains were included (Beck et al., 2019,
Madihalli and Doble, 2019).
Table 7.1 Prediction of MELs structure and corresponding possible fatty acid chains
[M+H+] Molecular mass Possible Possible fatty

structure of MELs acids chain
combinations
535.2741 534.3 MEL-D C8:1-8:0
643.3460 642.3 MEL-A C8:1-10:2
657.3792 656.4 MEL-B/MEL-C C10:2-12:1/C10:1-

12:2/C8:1-14:2
671.3578 670.4 MEL-A C10:1-10:2/C8:1-

12:2
697.3735 696.4 MEL-A C10:2-12:2
713.3647 712.4 MEL-B/MEL-C C8:1-18:2/C12:1-

14:2/C12:1-
14:2/C10:2-16:1
731.3800 730.4 MEL-A C10:1-14:0/C12:0-

12:1/C8:1-16:0
895.6104 894.6 MEL-A C18:3-18:3
961.6177 960.6 MEL-A C20:1-20:0
As seen from the result, most peaks that has been analysed represents MEL-A.
However, in order to get deeper insight into the oil or fatty acid moiety in different
structures, LC-MS measurement can be taken into account. Besides, more purified
sample should be used for further analysis, where further oil extraction is needed.
206
7.3 Thermodynamic Properties of Sophorolipids and
MELs
As can be seen from Figure 7.9, during the DSC scanning from room temperature
to 90°C and then ramping down to -20°C, followed by a ramping up back to room
temperature, SLs did not show any obvious endothermic or exothermic peaks,
indicating a thermostability during the measured range. So wider temperature
range is suggested on thermal study of SLs. Different from SLs, of which no
thermal transition witnessed with DSC scan, MELs presented ice-melting peak
around zero degree, and another crystallisation peak exhibited at around zero
degree, which may due to water existence in the crude product, shown in Figure
7.10. But results indicated excellent thermal stable of biosurfactants when
subjecting to temperature variation.
0.2
0.15
0.1
0.05
Heat Flow /W/g
ramp up
0 equilibrium
ramp down
-0.05 equilibrium
-0.1
-0.15
-0.2
-0.25
-20 -10 0 10 20 30 40 50 60 70 80 90
Temperature /°C
Figure 7.9 DSC thermogram of sophorolipids, where heat flow varied as function of
temperature ranging from -20 °C to 90 °C
0.2
0.15
0.1
Heat Flow /W/g
0.05 ramp up
0 equilibrium
ramp down
-0.05 equilibrium
-0.1 ramp up
-0.15
-0.2
-0.25
-20 -10 0 10 20 30 40 50 60 70 80 90
Temperature /°C
Figure 7.10 DSC thermogram of mannosylerythritol lipids where heat flow varied as
function of temperature ranging from -20 °C to 90 °C
207
In chapter 7, results of biosurfactant production were exhibited, mainly forcused on
their structural analysis. Sophorolipids (SLs) were prepared using shake flask
fermentation, and the fermentation technology was referenced from Dolman et al.
in our group (Dolman et al., 2017), where 50 mg L-1 of SLs was produced in a batch.
The structural analysis showed that diacylated acidic SLs of C18:1, diacylated
acidic SLs with C20:1 and diacylated lactonic SLs with C18:1 were found as main
peaks in mass spectrum. Also SLs that produced presented the ability to reduce
water surface tension from 72 to 34.02 mN m-1, with a critical micelle concentration
of around 50 mg L-1. Mannosylerythritol lipids (MELs) were prepared in shake-flask
fermentation, using similar procedure as that applied for SLs. More peaks were
observed as a result of the mass spectroscopy measurement of extracted MELs,
where MEL-A predominated. SLs and MELs were then formulated into bio-creams
without further purification in this study, for providing the information of cream
formulation with biosurfactants instead of synthetic ones.
208
Chapter 8. Production of bio-creams using
Continuous Configuration in
Formulation_Ⅲ
As concluded from previous study, including formula selection and manufacturing
process optimization, desired mimic creams with good performance compared to
standard E45 were produced with Formulation_Ⅲ using continuous configuration.
In this chapter results of bio-creams formulated with bio-surfactants and vegetable
oils were presented, and they were compared to those mimic creams in terms of
their performance.
New nomenclatures of creams are applied in this chapter, where surfactants

applied in creams are specified. For example, creams formulated with SLES, SLs
and MELs combining with fatty alcohols (CA and GM) are named as cream [SLES,
CA, GM], [SLs, CA, GM] and [MELs, CA, GM] respectively. In addition to that,
corresponding concentrations of each surfactant component are specified along
with their names. For example, cream [2SLs, 6CA, 2GM] referring to a bio-cream
formulated with 2 wt% SLs, 6 wt% CA and 2 wt% GM. Simplified, CA and GM are
elided, it turns to be cream [2SLs, 6, 2].
8.1 Reformulation of Mimic Creams Using Continuous

Configuration
Creams, [2SLES, 6, 2], [4SLES, 6, 2] and [6SLES, 6, 2] were reproduced using
continuous configuration, with the same manufacturing process applied in
Formulation_Ⅰ. Then they were initially analysed using steady state shear tests
after being prepared, in order to eliminating discrepancy caused by different
configurations.
Rotational shear tests were performed to obtain the viscosity profile for each cream
ranging from shear stress of 5 Pa to 300 Pa, using the same measuring procedure
as that being used in the analysis for Formulation_Ⅰ. Their viscosity profiles were
illustrated and compared respectively between two batches in Figure 8.1. It can be
seen that, viscosity profiles of mimic creams in Formulation_Ⅲ (line with solid filled
circle) greatly coincided with that in Formulation_Ⅰ (line with no filled circle),
especially for 1st Newtonian plateau, yield stress and shear thinning behaviour.
209
Cream [2, 6, 2] Cream [4, 6, 2]
1.0E+06 1.0E+06
1.0E+05 1.0E+05
1.0E+04 1.0E+04
viscosity /Pa.s
viscosity /Pa.s
1.0E+03 1.0E+03
1.0E+02 1.0E+02
1.0E+01 First Batch 1.0E+01
First Batch
1.0E+00 Third Batch 1.0E+00
Third Batch
1.0E-01 1.0E-01
1 10 100 1000 1 10 100 1000
Shear Stress /Pa Shear Stress /Pa
Cream [6, 6, 2]
1.0E+06
1.0E+05
1.0E+04
viscosity /Pa.s
1.0E+03
1.0E+02
1.0E+01
First Batch
1.0E+00
Third Batch
1.0E-01
1 10 100 1000
Shear Stress /Pa
Figure 8.1 Comparison of flow profile among creams formulated in Formulation_Ⅰ

using simplified configurations and that in Formulation_Ⅲ using the continuous one
Using simplified configuration, creams were crashed quenched by immersing the

beaker into a pot filled with large amount of cold water and the temperature was
cooled down to room temperature by 10 minutes. However, as for continuous
configuration, freshly cold water was continuously conveyed to the container jacket
for cooling and the duration was still set as 10 minutes, resulting in lower cooling
speed compared to the simplified configuration. But this difference did not cause
big effects on cream performance, this may due to the small quantity production of
the cream in lab scale, and the only difference in cooling rate was too small to
affect the production (Rønholt et al., 2014). Although mimic creams prepared in
Formulation_Ⅲ presents similar rheological behaviours as previous batch, freshly
produced mimic creams using continuous configuration were applied for further
comparison with bio-creams.
8.2 Creams Formulated with Bio-surfactants in Mixed

Paraffin Oils/Water System
In replacement of SLES, different concentrations of sophorolipids (SLs) and
mannosylerythritol lipids (MELs) were respectively formulated into the emulsifying
210
system containing 6 wt% cetyl alcohol (CA) and 2 wt% glycerol monostearate (GM),
incorporating with mixed paraffin oils and water. Details of recipes of formulation
could be referred from group P2 and P3 in Table 3.7 (section 3.4.2).
8.2.1 Appearance of Creams

Pictures of bio-creams were shown in Figure 8.2, where the composition of each
emulsifying system were specified corresponding to each cream. When having SLs
in the formulation, creams presented rigid appearance with self-bodying structure,
whereas creams formulated with MELs were less viscous. Simply from observation
of cream appearances, higher concentration of MELs in the system resulted in a
thinner product, which is in consistent with mimic creams formulated with SLES.
While the opposite effect was found in creams containing SLs instead, where more
structured product was obtained with higher concentration of SLs involved.
Mixed Paraffin oils
CA GM
Sophorolipids (wt%)
(wt%) (wt%)
2 4 6
Mannosylerythritol lipids (wt%) 6 2
2 4 6
Figure 8.2 Appearance of bio-creams formulated with 6 wt% CA and 2 wt% GM

respectively incorporated with varied concentrations of SLs and MELs, in mixed paraffin
oils-water system
8.2.2 Rheological Properties of Creams

Rheological measurements were applied to analyse the flow and deformation
behaviour of bio creams formulated with SLs and MELs separately, where
rotational shear, oscillatory sweep and creep-recovery tests were conducted.
211
Non-linear rotational shear test was preliminary performed on creams, using the
same sample preparation, sample loading and measuring procedure as introduced
in section 3.6.1.3.2. Figure 8.3 illustrates the viscosity profiles of creams [2SLs, 6,
2], [4SLs, 6, 2], [6SLs, 6, 2] containing 2 wt%, 4 wt% and 6 wt% SLs respectively
incorporating with same amount of fatty alcohols for stabilising mixed paraffin oils
in water, where viscosities varied with increasing shear stress from 1 Pa to 30 Pa.
Three bio-creams formulated with SLs all clearly showed decreased viscosity trend
as the shear stress increased, indicating shear thinning behaviour which is a
property of good cream in terms of spreadability and distribution ability (Malkin,
2013). In addition, it is interesting to notice that, the slope of shear thinning
behaviour of each cream varied to that obtained from mimic creams. When beyond
the yield stress, a viscosity drop was presented, followed by a gradually slow
decrease which includes a short plateau, then another sharp decrease of viscosity
was displayed. The reason for this may due to the multiple structure of crud SLs,
where the ring shaped lactonic form and opened acidic form co-existed in the
product, forming various structure of micelles.
Before reaching the yield stress, the viscosity behaviour of cream as a function of
shear stress is usually introduced as the 1 st Newtonian plateau, presented as
viscosity levelling off during low shear stress range if accurate measurements were
conducted (Tatar et al., 2017). As stated in previous chapter, rheological
measurements in this work may be influenced by wall slip phenomenon. However,
as absolutely same procedure was maintained and reduplications were carried out,
rheological data could be sufficient for the comparison between different creams
with varied surfactant systems. For flow profiles of cream [4SLs, 6, 2] and [6SLs,
6, 2], the corresponding zero viscosity was calculated as an average and displayed
in the figure. Cream containing 6 wt% SLs presented higher zero viscosity (1.17×
105 Pa·S) than that containing 4 wt% SLs (4.35×104 Pa·S). However, for cream
[2SLs, 6, 2], no plateau was witnessed, but it exhibited same curve trend of shear
thinning behaviour as other two creams. Thus, it is assumed that cream [2SLs, 6,
2] may reach zero viscosity when decreasing the shear stress below 1 Pa. In this
study during the measuring range, the limit viscosity of cream containing 2 wt%
SLs was determined as the apparent viscosity at 1 Pa (6.33×103 Pa·S).
212
The existence of the 1st Newtonian plateau reflects the formation of well-
established three-dimensional microstructure in the self-bodying cream, thereby
resulting a product with a solid appearance at rest (Ahmadi et al., 2020). This helps
explain the different appearance of three creams showed in Figure 8.3, where
creams containing 4 wt% and 6 wt% SLs clearly performed with solid state when
compared to that with 2 wt% SLs.
Figure 8.3 Comparison of flow behaviour among creams containing 6 wt% CA and
2wt% GM with varied concentrations of SLs in mixed paraffin oils-water system,
where viscosity varied as a function of shear stress ranging from 1 Pa to 300 Pa
From the viscosity profile as a function of shear stress, a bio-surfactant, SLs, were
proved to be a feasible substitution of chemically synthesized surfactant SLES. As
introduced in chapter 5.2.1, when no ionic surfactant (SLES) involved in the
formulation containing 6 wt% CA and 2 wt% GM, the product displayed
unhomogenized appearance where water was greatly separated from cream.
While 2 wt% SLs was able to contribute to the formulation of a homogenised cream,
even though it showed less viscous. Increase the concentration of SLs facilitated
the production of a more desired cream showing higher viscosity and yield stress,
exhibiting an opposite effect compared to SLES that higher concentration of SLES
resulted in a more viscous system. This may due to the non-ionic nature of SLs.
As reported in literatures, higher concentration of non-ionic surfactant contributes
to formation of more rigid system (Penkina et al., 2020).
213
Another biosurfactant, MELs, were applied to replace SLES for cream formulation.
The same characterisation regarding to viscosity profile determination was
conducted as that of SLs, the results is shown in Figure 8.4. These bio creams
displayed shear thinning behaviours within shear stress range from 1 Pa to 300 Pa.
Nevertheless, the limiting viscosities of creams at shear stress of 1 Pa were
unexpected lower than that of creams containing SLs. MELs were introduced as a
better emulsifier in the literatures, and compared to that, SLs work better on the
aspect of reducing the surface or interfacial tension (Xu et al., 2019). Thus, MELs
were expected to behave better in the formulation of creams. But this may due to
different micellar structure that formed when MELs were involved in the system, as
reported in literatures that MELs tended to self-assemble and form vesicles, which
is different from SLs or SLES. Also, a plateau was witnessed during shear thinning
range of every cream, which was in the same situation as cream containing SLs.
1.0E+04 Mixed Paraffin Oils

Cream [2MELs, 6, 2]
2.28E+03 Cream [4MELs, 6, 2]
Cream [6MELs, 6, 2]
1.75E+03
1.0E+03
2.22E+02
1.0E+02
viscosity /Pa.s
1.0E+01
1.0E+00
1.0E-01
0.1 1 10 100 1000
Shear Stress /Pa
Figure 8.4 Comparison of flow behaviour among creams containing 6 wt% CA and
2wt% GM with varied concentrations of MELs in mixed paraffin oils-water system,
where viscosity varied as a function of shear stress ranging from 1 Pa to 300 Pa
214
8.2.2.2 Oscillatory Sweep
Oscillatory strain sweep (OSS) test was performed to determine the LVE range.
Same procedure was applied in the analysis for bio-creams, where the prepared
sample was subject to increased oscillatory strain, strain% ranging from 0.0001 to
1000, while keeping the frequency as constant of 1 Hz. For the result of OSS,
variations of G’ and G’’ were displayed as the function of the increased %strain,
displaying in logarithmic coordinates. Then a strain% was selected among plateau
values that presented on the G’ (γ) curve, usually during low amplitude range. G’
and G’’ as a function of increased strain for bio-creams containing SLs is shown in
Figure 8.5. In every rheogram, the yield point of G’ was displayed as 90% of the
plateau value, and the crossover point was calculated using the method introduced
in section 5.2.2.2. Based on the result of OSS for bio creams, the strain% of 0.01
was selected as the constant amplitude for the further OFS test. The value is also
suited for bio-creams containing MELs. Before the cross-over point where G’
equalled to G’’, the elastic behaviour dominated the viscous one (G’>G’’) for all six
bio-creams, indicating a certain rigidity if the product is solid with relatively high
viscosity during medium or high shear rate range (Mahaut et al., 2008). While for
creams presented low-viscosity behaviour in shear thinning and the 2nd Newtonian
plateau, they still showed G’>G’’ in LVE range which indicated their gel-like
consistency and certain firmness when at rest, despite that the gel structure was
weak (Pan et al., 2018).
215
Cream [SLs, CA, GM] of [2, 6, 2]%wt (Mixed Paraffin Oils)
1.0E+05
1.0E+04
G', G'' /Pa 1.0E+03
1.0E+02
1.0E+01
1.0E+00 G'
1.0E-01 G''
1.0E-02
0.001 0.01 0.1 1 10
strain(%)
1.0E+05
Cream [SLs, CA, GM] of [4, 6, 2]%wt (Mixed araffin Oils)
1.0E+04
1.0E+03
G', G'' /Pa
1.0E+02
1.0E+01
1.0E+00 G'
1.0E-01 G''
1.0E-02
0.001 0.01 0.1 1 10
strain(%)
Cream [SLs, CA, GM] of [6, 6, 2]%wt (Mixed Paraffin Oils)

1.0E+05
1.0E+04
1.0E+03
G', G'' /Pa
1.0E+02
1.0E+01
G'
1.0E+00
1.0E-01 G''
1.0E-02
0.001 0.01 0.1 1 10
strain(%)
Figure 8.5 Oscillatory strain sweep on bio-creams formulated with 6 wt% CA and 2
wt% GM with varied concentration of SLs, where G' and G'' varied as function
of %strain ranging from 0.01 to 10
216
Oscillatory frequency sweep (OFS) test was then carried out, where cream
samples were sheared under sinusoinal oscillatory strain at a constant value of
0.01% with the frequency increased from 0.01 to 100 Hz. As Figure 8.6 presented,
where SLs was applied in the formulation, the result is displayed in form of storage
modulus, G’, loss modulus, G’’, and complex viscosity, |ƞ*|, varying as a function
of frequency, for cream containing different concentrations of SLs. The complex
viscosity for all bio-creams decreased as the frequency increasing, demonstrating
shear thinning behaviour of creams, which complemented results obtained from
non-linear rotational shear test (Sanz et al., 2017).
For SLs involved bio-creams, except cream with emulsifying system [SLs, CA, GM]
of weight concentration of [2, 6, 2], where G’ and G’’ intersected at certain
frequencies, the other two creams displayed gel-like character with elastic behavior
dominated over the measured frequency range (G’>G’’). This was also winessed
for bio-creams containing MELs. As described in literatures (Mahaut et al., 2008),
for stable dispersions or gels, trend of G’ is often greater than G’’ and both of them
show almost parallel lines increasing with the frequency rise, which is comparable
to that indicated by bio-creams.
The network structure built in the dispersion is the reason for G’ and G’’ response
against frequency during LVE range, which is usually in the form of physical
network, and vice versa, G’-curve and additionally G’’-curve could help confirm
whether a gel-like structure is formed in the cream product (Wang and Marangoni,
2016). The three-dimentional gel network was established by interaction forces
which is mainly due to the intermolecular forces based on physical-chemical bonds
(secondary bonds). This type of bonds generally show lower energy than chemical
bonds (primary bonds) contributing to intramolecular forces (Kónya et al., 2003).
OFS test could be applied to study the strength of internal structure by comparing
the G’ -value at a low frequency, but not able to distinguish the type of network,
as both of inermolecular and intramolecular forces result in relatively constrant
structural strength during LVE range of cream products (Zhao et al., 2013).
217
1.0E+05 Cream [SLs, CA, GM] of [2, 6, 2]%wt 1.0E+05
1.0E+04 1.0E+04
|ƞ*| /Pa.s
G', G'' /Pa
1.0E+03 1.0E+03
1.0E+02 1.0E+02
G'
1.0E+01 1.0E+01
G''
|n*|
1.0E+00 1.0E+00
0.01 0.1 1 10 100
Frequency /Hz
1.0E+04 1.0E+04
|ƞ*| /Pa.s
G', G'' /Pa
1.0E+03 1.0E+03
1.0E+02 1.0E+02
G'
1.0E+01 G'' 1.0E+01
|n*|
1.0E+00 1.0E+00
0.01 0.1 1 10 100
Frequency /Hz
1.0E+04 1.0E+04
|ƞ*| /Pa.s
G', G'' /Pa
1.0E+03 1.0E+03
1.0E+02 1.0E+02
G'
1.0E+01 G'' 1.0E+01
|n*|
1.0E+00 1.0E+00
0.01 0.1 1 10 100
Frequency /Hz
GM with varied concentration of SLs, where G', G'' and |ƞ*|varied as function of
frequency ranging from 0.01 Hz to 100 Hz
218
Althoug both of bio-creams formulated with SLs and MELs showed that G’ was
greater than G’’ within the frequency range, the degree of curves (G’ and G’’)
between them was different. For MELs incorporated bio-creams, G’ - and G’’- curve
nearly presented as parallel straight lines and probably no likelihood of crossing
over with each other at any point. However, relatively obvious curvature was found
for G’ and G’’ responsed by cream containing SLs, resulting the convex G’ curve
and and concave G’’ curve, and the curvature increased when lower concentration
of SLs was in the system. As a result, the tendency of G’- and G’’-curve meeting
at certain frequencies was witneesed in the rheogram of cream containing 2 wt%
SLs, and two regions near crossover points were illustrated in Figure 8.7.
[SLs, CA, GM] of [2, 6, 2] wt% [SLs, CA, GM] of [2, 6, 2] wt%
1.0E+03 1.0E+04
G', G'' /Pa

G', G'' /Pa
G' G'
G'' G''
1.0E+02 1.0E+03
0.01 0.1 10 100
Frequency /Hz Frequency /Hz
Figure 8.7 Specific oscillatory frequency range of oscillatory frequency sweep test for
SLs-involved cream, including the range between 0.01 and 0.1 (left) and that between
10 and 100 (right), showing crossover of G' and G''
During low frequency range from 0.01 Hz (ω≈0.0628 rad s-1) to 0.1 Hz (ω≈0.628
rad s-1), the cream sample was exposed to very slow motion, and responsed long-
term behavior which helped characterise its internal structural strengthe when at
rest (Pan et al., 2018). As can be seen from Figure 8.7 (left rheogram), the average
curve of G’ was dominant that of G’’, but the overlaps of error bars indicated that
G’ and G’’ probably crossed over with each other before reaching the frequency of
0.06 Hz (ω≈0.4 rad/s). Thus, during with low frequency range, that is, long-term
oscillation, frequency sweep teset indicated that cream [2SLs, 6, 2] behaved
between liquid and gel-like, suggesting the long-term storage unstability. Another
crossover point was found during high frequency range from 10 to 100 Hz (right
rheogram in Figure 8.7), approximately around 8 Hz after which G’’ was greater
219
than G’, indicating the cream behaved as a viscoelastic liquid at higher frequencies.
This may because of sample degradation and measuring inherent problems (Pan
et al., 2018).
Steady state rotational test (SSS) was previously applied to determine the “yield
stress” for analysing the structural network built in cream when at rest, thereby
evaluating the consistency of sample. This was realised in osillatory frequency
sweep (OFS) as well, where G’, and if necessary, along with G’’, were analysed at
low frequencies. But they were not in the same meauring range and just
complementing each other. For bio-creams involved MELs, although viscosity
profiles from SSS showed no yield stress of creams within the measured shear
stress range, suggesting no network structure established, storage moduli
response against frequency presented that G’ was predominant, thus indicating
gel-like structure and certain stability of creams.
As seen from Figure 8.8 and 8.9. Cream containing 6%wt of SLs presented higher
G’ compared to that containing 4 wt% and 2 wt% of SLs, showing a higher stability
and rigid gel network. However, higher concentration of MELs involved in the
formulation led to a weaker gel structured cream, showing lower G’-values against
frequencies compared to creams with lower concentration of MELs. The reason for
this may because the difference of micelles or liquid crystals structure formed by
MELs and SLs molecules, leading to different effects on rheological behaviour of
creams (Kelleppan et al., 2018, Worakitkanchanakul et al., 2009).
220
1.0E+05 Mixed Paraffin Oils
G'-cream [6SLs, 6, 2] G''-cream [6SLs, 6, 2]
G', G'' /Pa 1.0E+04
1.0E+03
1.0E+02
0.01 0.1 1 10
Frequency /Hz
Hz among bio-creams containing 6 wt% CA and 2 wt% GM with varied concentrations
of SLs in mixed paraffins-water system
1.0E+04
Mixed Paraffin Oils
G'-cream [2MELs, 6, 2] G''-cream [2MELs, 6, 2]
G', G'' /Pa
1.0E+03
1.0E+02
0.01 0.1 1 10
Frequency /Hz
Figure 8.9 Comparison of G' and G'' as function of frequency ranging from 0.01 Hz to
10 Hz among bio-creams containing 6 wt% CA and 2 wt% GM with varied
concentrations of MELs in mixed paraffins-water system
221
Results of creep and recovery test that carried out on bio-creams containing SLs
and MELs are shown in Figure 8.10 and 8.11 respectively. As introduced in the
creep results for creams having SLES in the system, a primary creep and
secondary creep are expected to be found in the creep compliance response under
the stress as function of time, especially primary creep that represented by spring
element indicating a system showing elastic behaviour (Dogan et al., 2013). While
for bio-cream formulated with 2 wt% SLs, only secondary creep region dominates,
indicating a viscous liquid behaviour. However, with the increase of SLs
concentration, secondary creep range was presented, as seen the creep curve of
bio-cream containing 4 wt% and 6 wt% SLs in the system. Therefore, higher
concentration of SLs in the system resulted in a more elastic behaved product,
which is the desired property in semi-solid system.
0.3
Mixed Paraffin Oils
Cream [2SLs, 6, 2]
Cream [4SLs, 6, 2]
0.25
Cream [6SLs, 6, 2]
1.8
0.2
1.5
J /Pa⁻¹
1.2
0.15
J /10⁻² Pa⁻¹
0.9
0.6
0.1
0.3
0
0.05 0 1000 2000 3000 4000
Time /s
0
0 500 1000 1500 2000 2500 3000 3500 4000
Time /s
containing 6 wt% CA and 2 wt% GM with varied concentrations of SLs in mixed
paraffins-water system
For the system where MELs was incorporated with paraffin mixed oils in water, no
primary creep phenomena showed in all three bio-creams containing different
concentrations of MELs. Also during recovery process after 30-minutes stress
shear within LVE range, bio-creams showed no strain recovery. Thus it means that
MELs is not a good substitute surfactant of SLES in this formulation of cream
product with paraffin oils in water system containing 6 wt% cetyl alcohol and 2 wt%
222
glycerol monostearate, in terms of creep response, as they all behaved as viscous
liquid and no elasticity witnessed. This agree with the results obtained from steady
state shear and oscillatory sweep tests.
Mixed Paraffin Oils

10
6
J /Pa⁻¹
4 Cream [6MELs, 6, 2]
0
0 500 1000 1500 2000 2500 3000 3500 4000
Time /s
containing 6 wt% CA and 2 wt% GM with varied concentrations of MELs in mixed
paraffins-water system
8.2.3 Thermodynamic Properties of Creams

DSC measurement was carried out to characterise bio-creams formulated with
sophorolipids (SLs) and mannosylerythritol lipids (MELs) separately, results of
corresponding thermograms of SLs and MELs were respectively displayed in
Figure 8.12 and 8.13. No obvious difference was found upon heating curve for both
thermograms, where for bio creams containing different concentrations of SLs, a
melting point was found at around 36 °C, and similar for that of MELs. While upon
cooling down for creams with SLs, exothermal peaks were observed, and with an
increase of SLs concentration, crystallization temperature moved to lower
temperature, resulting in smaller supercooling temperature difference (difference
between melting point and the cooling crystallization temperature) and thus higher
solidification rate of the material (Zhang et al., 2017a). However, the DSC result
for creams formulated with MELs with mixed paraffin oils in water was unable to
provide pronounce information. Thus additional measurement is needed where
lower heating or cooling rate is suggested.
223
0.4
Mixed Paraffin Oils
0.3
0.2
Heat Flow /W/g

0.1
-0.1
-0.2
Cream [2SLs, 6, 2]
Cream [4SLs, 6, 2]
-0.3
Cream [6SLs, 6, 2]
-0.4
25 30 35 40 45 50 55 60 65 70
Temperature /°C
SLs in mixed paraffins-water system
0.2 Mixed Paraffin Oils
0.1
0
Heat Flow /mW/mg
-0.1
-0.2
-0.3 Cream [2MELs, 6, 2]

Cream [4MELs, 6, 2]
Cream [6MELs, 6, 2]
-0.4
25 30 35 40 45 50 55 60 65 70
Temperature /°C

MELs in mixed paraffins-water system
224
8.3 Creams Formulated in Vegetable Oils/Water System
As the demand for greener product, vegetable oils, coconut oil and vegetable
shortening were considered as the substitutions for mixed paraffin oils (light liquid
paraffin and white soft paraffin), with the same weight concentration. Chemically
synthesized surfactants SLES, biosurfactant SLs and MELs of 2 wt%, 4 wt% and
6 wt% were respectively incorporated with CA and GM, as the emulsifying system.
Recipes could be referred from Table 7 in section 3.4.2 (group C1-C3 and V1-V3).
8.3.1 Appearance of Creams

Mimic creams containing different concentration of SLES were preliminary
formulated with coconut oil and vegetable shortening separately, shown in Figure
8.14. Yellow products were formulated with vegetable shortening, while white ones
were those with coconut oils. No significant differences of consistency between
creams were witnessed, and all of them showed a rigid solid state after preparation.
Coconut Oil
CA GM
SLES (wt%) (wt%) (wt%)
2 4 6
6 2
2 4 6
SLES (wt%)
Vegetable Shortening
Figure 8.14 Appearance of mimic creams formulated involving SLES, respectively with
coconut oil and vegetable shortening in water, containing surfactant system of 6 wt%
cetyl alcohol and 2 wt% glycerol monostearate with varied concentrations of sodium
lauryl ether sulfate
225
Pictures of bio creams with coconut oil and vegetable shortening in water are
presented in Figure 8.15 and 8.16 separately. With nearly 27 wt% coconut oil in
the formulation, white semi-solid products were obtained, presenting different
appearance with different concentrations of bio-surfactants. When SLs of 2 wt%
was involved, less viscous emulsion were presented. Higher concentration of SLs
obviously resulted in a structured cream in solid state with higher rigidity. On the
contrary, the lower concentration of MELs involved, the higher stiffness of product
was made. But the cream was unacceptable due to the undesired hardness and
coarse appearance when 2 wt% of MELs was involved. With higher concentration
of MELs in the system, where 6 wt% applied, a smooth semi-solid cream with more
desired appearance was formulated.
Coconut Oil
CA GM
Sophorolipids (SLs) (wt%) (wt%) (wt%)
2 4 6
Mannosylerythritol lipids (MELs) (wt%) 6 2

2 4 6
with coconut oil in water, containing surfactant system of 6 wt% cetyl alcohol and 2
wt% glycerol monostearate with varied concentrations of sodium lauryl ether sulfate
Still, when vegetable shortening applied instead of coconut oil, colour of the
product turned to yellow, as seen in Figure 8.16. Products having SLs in the system
showed suitable rigidity from the appearance, as semi-solid cream. However,
these coarse-grained creams were not smooth as required. As for creams
containing MELs in the emulsifying system, products seemed to be worse based
on their appearance, as they presented as the aggregation of granules but not
226
homogenized creams with acceptable consistency. The analysis from the
appearances of creams was direct but not accurate, so further characterisation
was conducted to determine their properties qualitatively and quantitatively.
CA GM
Sophorolipids (wt%) (wt%) (wt%)
2 4 6
Mannosylerythritol lipids (wt%) 6 2

2 4 6
Figure 8.16 Appearance of bio-creams formulated involving SLs and MELs, respectively with
vegetable shortening in water
8.3.2 Rheological Properties of Creams

Series of rheological tests were carried out to study the flow and deformation of bio
creams formulated with vegetable oils, where viscosity profile was determined by
conducting rotational shear test (steady state shear/SSS), and viscoelasticity
behaviour was analysed with the help of oscillatory frequency sweep (OFS) and
creep test.
As previous introduced, the viscosity profile could be obtained by carrying out SSS
test, where cream sample was subject to shear stress ranging from 1 Pa to 300 Pa
and corresponding viscosity change was recorded. Characterisations were
conducted at 25 °C for every cream sample, same sample preparation was made
prior to the test and minimum in duplicate. Also, 40 mm cone-plate geometry was
227
applied, and creams were confined within a gap of 57 mm, which is consistent as
previous characterisation for mimic creams.
As mentioned in previous chapters, rheological results that obtained in this work

were applied as indices for the comparison between creams formulated with
different compositions of surfactants, and actual interpretation of flow properties
for individual cream system required more work to be done for further eliminating
wall depletion problem. Figure 8.17 and 8.18 represents the viscosity change of
mimic creams respectively formulated with coconut oil and vegetable shortening
emulsified by SLES as a function of shear stress. All creams presented the shear
thinning behaviour which is desired. There was no big difference of zero-shear
viscosity and yield stress between creams containing different concentrations of
SLES, and this was also found in viscosity profiles of creams having vegetable
shortening as oil content (Figure 8.18). However, as for vegetable shortening
formulated in creams, flow curves seemed to be largely affected by sample dryness
and wall slip phenomena, where prominent sudden breaks were observed
compared to those for creams formulated with coconut oil (Hatzikiriakos, 2012).
Even though, more SLES involved led to the production of less viscous cream,
which was in accordance with mixed paraffin oils involved system. Vegetable
shortening involved creams presented approximate one magnitude larger of zero
shear viscosity and yield stress value respectively than coconut oil involved creams
did (Figure 8.17). This may because the difference of physical property between
two vegetable oils (Chizawa et al., 2019).
The zero shear viscosity (limiting viscosity at shear stress of 5 Pa) for the system
of mixed paraffin oil incorporating with 4 wt% of SLES in water was 1.39×105 Pa.s,
a comparable value of 1×105 Pa.s was obtained for coconut oil/water/4 wt% SLES
system, indicating the potential of coconut application in the replacement of
paraffin mixed oils in terms of their rheological behaviour. As a matter of fact,
similar coconut oil and mixed paraffins showed same magnitude of G’ and G’’ trend
with varied frequency from 0.1 Hz to 100 Hz (data not shown).
228
Coconut Oils
1.0E+06
2.05E+05
1.08E+05
1.0E+05
5.46E+04
1.0E+04
viscosity /Pa.s
1.0E+03
1.0E+02
1.0E+01
Cream [2SLES, 6, 2]
1.0E+00
Cream [4SLES, 6, 2]
Cream [6SLES, 6, 2]
1.0E-01
0.1 1 10 100 1000
Shear Stress /Pa
and 2 wt% GM with varied concentrations of SLES in coconut oil-water system, where
viscosity varied as a function of shear stress ranging from 1 to 300 Pa
1.0E+07
2.76E+06
1.0E+06
2.48E+05
1.0E+05
1.30E+05
1.0E+04
1.0E+03
viscosity /Pa.s
1.0E+02
1.0E+01
1.0E+00
Cream [2SLES, 6, 2]
1.0E-01 Cream [4SLES, 6, 2]

Cream [6SLES, 6, 2]
1.0E-02
0.1 1 10 100 1000
Shear Stress /Pa
and 2 wt% GM with varied concentrations of SLES in vegetable shortening-water
system, where viscosity varied as a function of shear stress ranging from 1 to 300 Pa
229
As can be seen from Figure 8.19, for the system of coconut oil in water, bio creams
containing different concentrations of SLs, where 2 wt%, 4 wt% and 6 wt% applied,
showed generally shear thinning behaviour during the shear stress range from 1
Pa to 300 Pa, where the limit viscosity was at nearly 104 Pa·s for all creams. And
no obvious difference between viscosity profiles of them when different
concentration of SLs applied, but similar as that mentioned in the case where SLs
involved in the system of mixed paraffin oils in water, three stages plateau could
be witnessed, especially for cream [6SLs, 6, 2]. This obviously related to the
complex structures of SLs (Ankulkar and Chavan, 2019). As a result, bio creams
containing SLs as surfactant for emulsifying coconut oil in water behaved less
viscous with a relatively weak structural network.
1.0E+05
Coconut Oil
1.81E+04 1.67E+04
1.0E+04 1.65E+04
1.0E+03
viscosity /Pa.s
1.0E+02
1.0E+01
1.0E+00
Cream [6SLs, 6, 2]
1.0E-01 Cream [4SLs, 6, 2]
Cream [2SLs, 6, 2]
1.0E-02
0.1 1 10 100 1000
Shear Stress /Pa
Figure 8.19 Comparison of flow behaviour among bio-creams containing 6 wt% CA and
2 wt% GM with varied concentrations of SLs in coconut oil-water system, where
When vegetable shortening emulsified in water, with the help of different

concentrations of SLs mixed with CA and GM, all creams performed shear thinning
behaviour, where zero shear viscosity values were over 105 Pa·s, which can be
seen from Figure 8.20. However, predominant wall slip phenomenon seems affect
the result of system where 2 wt% SLs was involved, as the sudden break presented
(Barnes, 1995). This was found in the situation where SLES was applied with
vegetable shortening in water. But for comparison, higher concentration of SLs in
230
the cream resulted in more rigid cream with higher viscosity and yield stress, which
agreed with the results obtained for SLs being applied in mixed paraffin oils and
water system.
1.00E+07
9.25E+05
1.00E+06
2.71E+05
1.00E+05
1.28E+05
1.00E+04
viscosity /Pa.s
1.00E+03
1.00E+02
1.00E+01
Cream [6SLs, 6, 2]
1.00E+00
Cream [4SLs, 6, 2]
1.00E-01 Cream [2SLs, 6, 2]
1.00E-02
0.1 1 10 100 1000
Shear Stress /Pa
2 wt% GM with varied concentrations of SLs in vegetable shortening-water system,
where viscosity varied as a function of shear stress ranging from 1 to 300 Pa
Figure 8.21 represents effect of different concentrations of MELs on flow behaviour
of bio-creams with coconut oil. MELs performed better rheological behaviour in
coconut oil-in-water system, compared to SLs. All creams showed desired
viscosity profiles when subjecting to shear stress from 1 Pa to 300 Pa, presenting
desired shear thinning behaviours and reasonable zero shear viscosity.
Interestingly, although higher concentration of MELs involved made the bio-cream
become less viscous with lower yield stress, the trend was reversed during high
shear range and cream with 6 wt% of MELs became more viscous than 2 wt% of
that. But the difference of viscosity was very small at 300 Pa. This phenomenon
occurred may due to the dryness of sample while being measured at high shear
stress.
Vegetable shortening-in-water system containing MELs was presented in Figure

8.22, and very high zero viscosity was obtained during low shear range, indicating
undesired rigidity of the product, even though this result was not seemed in line
with their appearances. But viscosity profiles of all bio creams formulated with
231
vegetable shortening behaved not as good as that with coconut oil, which could be
correlated with aggregated clusters presented in those vegetable shortening-in-
water bio creams (Chizawa et al., 2019). Again, wall slip was obvious for the
formulation with vegetable shortening. Briefly summarised, from results of steady
state shear, coconut oil could be a promising alternative for mixed paraffin oils in
the formulation of cosmetic cream with SLES, CA and GM as the emulsifying
system, and even for bio creams incorporating SLs and MELs. However, as the
difference of physiochemical properties between vegetable shortening and mixed
paraffin oils or coconut oils, those creams formulated with vegetable shortening
failed to present desired performance, although wall slip phenomenon may exist
for these systems, comparison could be sufficiently made when consistent
measuring procedure was carried out using 40 mm cone-plate geometry at a
measuring gap of 57 mm.
1.0E+06
Coconut Oil
1.18E+05
1.0E+05
1.71E+04
1.0E+04
5.7E+03
viscosity /Pa.s
1.0E+03
1.0E+02
1.0E+01
1.0E+00 Cream [2MELs, 6, 2]
Cream [4MELs, 6, 2]
1.0E-01
Cream [6MELs, 6, 2]
1.0E-02
0.1 1 10 100 1000
Shear Stress /Pa
2 wt% GM with varied concentrations of MELs in coconut oil-water system, where
232
1.0E+07
2.14E+06
1.0E+06 6.39E+05
5.69E+05
1.0E+05
1.0E+04
viscosity /Pa.s
1.0E+03
1.0E+02
1.0E+01
Cream [2MELs, 6, 2]
1.0E+00 Cream [4MELs, 6, 2]
Cream [6MELs, 6, 2]
1.0E-01
0.1 1 10 100 1000
Shear Stress /Pa
2 wt% GM with varied concentrations of MELs in vegetable shortening-water system,
where viscosity varied as a function of shear stress ranging from 1 to 300 Pa
8.3.2.2 Oscillatory Frequency Sweep
Results of oscillatory frequency sweep (OFS) for creams were presented, where
storage modulus, G’, and loss modulus, G’’, changing with frequency was
measured. The test was conducted within linear viscoelastic range of every sample.
The LVE range was determined by carrying out oscillatory strain sweep tests
(OSS), and then a value of %strain was selected for the following OFS tests.
Figures 8.23 and 8.24 showed rheograms of strain sweep for the mimic cream
containing 6 wt% SLES and the bio cream containing 6 wt% MELs respectively
with coconut oil in water, which separately represented for the determination
of %strain for mimic creams and bio creams.
For mimic creams involving 6 wt% SLES in the system, storage modulus, G’, was
independent with increased strain, until reaching the yield strain, 𝛾𝑦 , at around
0.75%. During this low strain range, the curve of G’ was over G’’, indicating a solid
domain system. Moduli decreased with increasing the amplitude (strain), and a
crossover point of G’ and G’’ was witnessed in the rheogram. This point suggested
the transition of sample from gel-like structure to liquid-like structure (Awad et al.,
2011). Same trend of moduli dependence on %strain was achieved in the system
of bio-creams. But 𝛾𝑦 was smaller than that for mimic cream, which was less than
233
0.1% (0.0895% shown in the figure for the selected cream), indicating a less
viscous system. The amplitude was determined at strain of 0.1% for mimic creams,
and that of 0.01% for bio creams, with vegetable oils in water. The selected strains
were accordingly applied for other creams, as they were proved to be within their
LVE range.
Cream [SLES, CA, GM] of [6, 6, 2] (Coconut Oil)
1.0E+04
1.0E+03
1.0E+02
G', G'' /Pa
1.0E+01
1.0E+00 G'
G''
1.0E-01
0.001 0.01 0.1 0.7465 1 10 100
strain(%)
Figure 8.23 Oscillatory strain sweep on mimic creams containing 6 wt% CA and 2 wt%
GM with 6 wt% SLES in coconut oil-water system, where G'and G'' varied as function of
strain% ranging from 0.01 to 100
Cream [MELs, CA, GM] of [6, 6, 2] (Coconut Oil)

1.0E+03
1.0E+02
1.0E+01
G', G'' /Pa
1.0E+00
G'
1.0E-01
G''
1.0E-02
0.001 0.01 0.0895 0.1 1 10
strain(%)
Figure 8.24 Oscillatory strain sweep on bio-creams containing 6 wt% CA and 2 wt%
GM with 6 wt% MELs in coconut oil-water system, where G' and G'' varied as function
of strain% ranging from 0.01 to 100
234
Oscillatory frequency sweep was applied afterwards. As a result, the storage
modulus, G’, and loss modulus, G’’, of cream containing different concentrations
of SLES, SLs and MELs with vegetable oils in water are respectively shown in
Figure 8.25~8.30, as a function of frequency ranging from 0.01 to 100 Hz. In
general, all cream samples formulated with different concentration of surfactants
incorporated, with fatty alcohols in vegetable oils and water system, behaved as
structured gel, as G’ was higher than G’’ over the whole measured frequency range
at strain within linear region for every sample. The mechanical spectra, namely the
trends of G’ and G’’ changing with oscillatory frequency measured in LVE range,
were applied to illustrate the structural characters of samples.
5.0E+04 Coconut Oils
5.0E+03
G', G'' /Pa
5.0E+02
G'-cream [2SLES, 6, 2] G''-cream [2SLES, 6, 2]

5.0E+01
0.01 0.1 1 10 100
Frequency /Hz
wt% GM with varied concentrations of SLES in coconut oil-water system, where G', G''
and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz
235
5.0E+04
G', G'' /Pa 5.0E+03
5.0E+02

5.0E+01
0.01 0.1 1 10 100
Frequency /Hz
wt% GM with varied concentrations of SLES in vegetable shortening-water system,
where G', G'' and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz
1.0E+05 Coconut Oil

G'-cream [6SLs, 6, 2] G''--cream [6SLs, 6, 2]
G'--cream [4SLs, 6, 2] G''--cream [4SLs, 6, 2]
G'--cream [2SLs, 6, 2] G''--cream [2SLs, 6, 2]
1.0E+04
G', G'' /Pa
1.0E+03
1.0E+02
0.01 0.1 1 10 100
Frequency /Hz
GM with varied concentrations of SLs in coconut oil-water system, where G', G'' and
|ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz
236
1.0E+06
1.0E+05
G', G'' /Pa
1.0E+04
1.0E+03
1.0E+02
0.01 0.1 1 10 100
Frequency /Hz
GM with varied concentrations of SLs in vegetable shortening-water system, where G',
G'' and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz
Coconut Oil
5.0E+04
5.0E+03
G', G'' /Pa
5.0E+02
5.0E+01
0.01 0.1 1 10
Frequency /Hz
GM with varied concentrations of MELs in coconut oil-water system, where G', G'' and
|ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz
237
5.0E+06
5.0E+05
G', G'' /Pa
5.0E+04
5.0E+03
5.0E+02 G'-cream [2MELs, 6, 2] G''-cream [2MELs, 6, 2]

5.0E+01
0.01 0.1 1 10
Frequency /Hz
GM with varied concentrations of MELs in vegetable shortening-water system, where
G', G'' and |ƞ*|varied as function of frequency ranging from 0.01 to 100 Hz
When different concentrations of SLES were involved in the formulation where

coconut oil was applied, curves of G’ for every cream did not display huge
departure from each other, indicating a similarity in terms of gel strength. As
previous obtained from steady state shear test, zero shear viscosity and yielding
properties were not significantly affected by the concentrations of SLES (increasing
from 2 wt% to 6 wt%) when coconut oil was emulsified with water, which coincided
with the oscillation test. Even though, during lower oscillatory frequencies, less
SLES involved cream (2 wt%) displayed more obvious solid-structural properties
compared to higher ones did, indicating longer stability of system containing lower
concentration of SLES (Kelleppan et al., 2018). This is more obvious in the system
of vegetable shortening-in-water, as larger difference of G’ between creams with
varied concentrations of SLES is witnessed, especially at low frequencies,
although as previous steady state shear results pointed out that the flow behaviour
of vegetable shortening incorporated creams exhibited undesired performance.
The trends of G’ and G’’ of creams containing 2 wt%, 4 wt% and 6 wt% of MELs
was similar to that involved SLES instead, where increased MELs led to products
showing more viscous structural properties. Moreover, concentration of MELs had
a significant influence on the viscoelastic properties of creams, as seen from Figure
238
8.29 that G’ of cream [2MEL, 6, 2] are shifted one magnitude lower when 6 wt%
MELs was applied. But in the system where SLs participating, the effect of
surfactant concentration on rheological properties and characters was opposite
compared to MELs or SLES. From the Figure 8.27 and 8.28, it can be seen that,
moduli of SLs involved cream [2SLs, 6, 2], [4SLs, 6, 2] and [6SLs, 6, 2] suggested
that more SLs involved in the formulation contributed to the product with more
pronounced solid dominant structure and rigid gel-like behaviour. Again whether
for coconut oil or mixed paraffin mixed oils, the influence of surfactant
concentration on flow property is not significant, indicating the potential of altering
the formulation using vegetable oils (Salehiyan et al., 2018).
When coconut oil and vegetable shortening being emulsified in water, the system
with SLES showed good elastic behaviour in terms of creep test, where primary
creep was witnessed, and the creep response of cream containing SLES in
coconut oil-water system is similar to that in mixed paraffin oils-water system. This
is found in almost all rheological tests. And the reason may due to coconut oil has
similar physicochemical properties compared to the mixed paraffin oils
(Terescenco et al., 2018a). The representative result of creep test of cream
involving SLES with vegetable shortening in water is shown in Figure 8.31, where
all creams present elastic behaviour with the presents of primary creep and
recovered strain. In addition, 6 wt% SLES in the system greatly decrease the
rigidity of product as compliance sharply increased when compared to 2 wt% and
4 wt% involved.
Those MELs involved systems, when having coconut oil in water, performed well
in terms of viscoelastic property. As can be seen from Figure 8.32, all creams
showed good viscoelastic properties, and it showed similar effect as SLES, where
lower concentration of MELs or SLES in the system tends to result in a more rigid
cream with good elastic behaviour. From Figure 8.33, as for creams containing
SLs, with coconut oils in water, the result was similar to that with mixed paraffin
oils in water, where higher concentration of SLs had the potential to produce a
product exhibiting more obvious elasticity, especially for cream containing 2 wt%
of SLs, merely secondary creep was witnessed indicating a viscous system
(Nguyen et al., 2015).
239
Figure 8.31 Comparison of compliance as a function of time among mimic creams
containing 6 wt% CA and 2 wt% GM with varied concentrations of SLES in vegetable
shortening-water system

containing 6 wt% CA and 2 wt% GM with varied concentrations of MELs in coconut oil-
water system
240
Coconut Oil
1
Cream [2SLs, 6, 2]
0.9
Cream [4SLs, 6, 2]
0.8 Cream [6SLs, 6, 2]
8
0.7
6
0.6
J /10⁻² Pa⁻¹
J /Pa⁻¹
0.5
4
0.4
2
0.3
0.2 0
0 1000 2000 3000 4000
Time /s
0.1
0
0 500 1000 1500 2000 2500 3000 3500 4000
Time /s
containing 6 wt% CA and 2 wt% GM with varied concentrations of SLs in coconut oil-
water system
8.3.3 Thermodynamic Properties of Creams

DSC measurement was carried out and expected for investigating the thermal
properties of creams and the effect of changing surfactants on the performance of
cream. Creams formulated with vegetable oils (coconut oil and vegetable
shortening) respectively incorporated with SLES, SLs and MELs were heated up
from room temperature to 90°C at a rate of 3°C min -1, followed by cooling down
back to room temperature at the same rate. As can be seen in Figure 8.34 and
8.35, showing the DSC result of SLES and MELs separately applied in the cream
with vegetable shortening in water, although higher concentration of SLES leading
to a lower melting point and decrease in crystallisation temperature, change is
insignificant, so further investigation is needed in terms of procedure modification
of DSC (Zhang et al., 2017a). Similar, no obvious trend could be witnessed from
DSC result for creams containing MELs with vegetable shortening in water.
However, creams with MELs exhibited broader range of melting compared to those
with SLES, in the system of vegetable shortening in water, indicating higher
impurity of the system which may due to the multiple structure of MELs (Okamoto
et al., 2016).
241
0.3
Cream [2SLES, 6, 2]
Cream [4SLES, 6, 2]
0.2 Cream [6SLES, 6, 2]
Heat Flow /mW/mg 0.1
-0.1
-0.2
-0.3
25 30 35 40 45 50 55 60 65 70
Temperature /°C
Figure 8.34 DSC thermograms of mimic creams containing 6 wt% CA and 2 wt% GM
with varied concentrations of SLES in vegetable shortening-water system
varied concentrations of MELs in vegetable shortening-water system
242
As shown in Figure 8.36, in the system where SLs was involved, multiple
endothermic peaks were witnessed within temperature range between 30°C and
40°C, indicating inhomogeneous system with uninvolved component (Drzeżdżon
et al., 2019), but the it was different when 2 wt% SLs was involved where less
melting points existed. Glass transition was found for all three SLs-involved creams,
and 2 wt% SLs exhibiting a higher crystallisation temperature. However, further
DSC measurements are suggested by modifying the heating rate and temperature
range for giving more information in terms of thermal properties of creams and
correlating this to their microstructure. It could also help optimizing the formulation
process such as heating and cooling temperature control (Pivsa-Art et al., 2019).
varied concentrations of SLs in coconut oil -water system
243
In chapter 8, mimic creams and bio creams were preliminary prepared with mixed
paraffin oils with water, incorporating with sodium lauryl ether sulfate (SLES),
sophorolipids (SLs) and mannosylerythritol lipids (MELs) separately with 6 wt%
cetyl alcohol and 2 wt% glycerol monostearate. Rheological measurements were
carried out using a 40 mm cone-plate geometry and a constant measuring gap was
set as 57 mm, results of which were applied as indices for comparing the effect of
different surfactants on cream performances. For the system having SLs in the
formulation, creams were prepared with desired limiting viscosity which is in the
same magnitude as that of mimic creams. From results of oscillatory frequency
sweep tests, solid dominant viscoelasticity was witnessed for creams containing
SLs within the test frequency range from 0.01 to 100 Hz, presenting as G’ was over
G’’, even though there has a high possibility of the cross point of G’ and G’’ which
indicates a glass transition. It is interesting to observed that higher concentration
of SLs resulted in a more flexible cream system with relatively lower limit viscosity
and yield stress, which is in the opposite trend as that for SLES involved system.
This may due to the reason that SLs are non-ionic molecules, and sufficient higher
concentration in the system tend to form a well-structured system (Ren, 2017).
This was also witnessed from creep test, where compared to the system containing
2 wt% of SLs, significant primary creep was witnessed for the system containing 4
wt% SLs, indicating an elastic behaviour.
Creams were then prepared using vegetable oils such as coconut oil and vegetable
shortening as an alternative to mixed paraffin oils consisting of light liquid oil and
white soft paraffin, in order to provide the information of using vegetable oils for
formulating “greener” cosmetic creams. As a result, creams formulated with
coconut oil presented desired results, where creams were prepared with
reasonable consistency and self-bodying structure, both for mimic creams
containing SLES and bio creams formulated with biosurfactants. However,
vegetable shortening was not a desired substitute for cream preparation, due to
the unfavourable colour, granular texture and unexpected high yield stress in
comparison with other creams characterized in this work.
244
Chapter 9. Conclusion and Future Work
Human-friendly emulsions play a significant role in various industries, especially
for personal care products that closely related to people’s everyday life. As a key
component in their formulation, surfactant system is usually inevitable for
enhancing emulsification process during preparation and stabilizing microstructure
of the emulsion during shelf life (Akbari and Nour, 2018).
In this project, in order to provide standards for the formulation bio creams
containing different concentrations of biosurfactants such as sophorolipids (SLs)
and mannosylerythritol lipids (MELs), mimic creams were prepared consisting of
different concentrations of sodium lauryl ether sulfate (SLES), cetyl alcohol (CA)
and glycerol monostearate (GM) with mixed paraffin oils (white soft paraffin and
light liquid paraffin) in water. As a result, creams containing 6 wt% CA and 2 wt%
GM incorporating with varied concentrations of SLES were selected as standards
for bio-cream formulation by replacing SLES with SLs and MELs respectively. SLs
that produced by cultivating Candida bombicola in the medium containing
rapeseed oil, glucose, peptone and yeast extract in shake flask fermentation,
mixture of diacylated acidic SLs of C18:1, diacylated acidic SLs with C20:1 and
diacylated lactonic SLs with C18:1 was obtained after purification. And MELs were
secreted by Pseudozyma aphidis DSM 70725 and mainly MEL-A was isolated.
SLES as an anionic surfactant played key role in system of mixed paraffin oils in
water, without which cream was failed to form a homogenized structure showing
phase separate right after preparation (only 6 wt% CA and 2 wt% GM applied in
the formulation as surfactant system). From this aspect, when 2 wt% of SLs was
applied in the system with 6 wt% CA and 2 wt% GM, cream was successfully
formulated with consistent texture, although the limiting viscosity and
corresponding yield stress is relatively low compared to the system containing 2
wt% SLES instead and a viscous behaviour dominant the system from creep test
results. However, increasing concentration of SLs led to the formulation of more
desired creams with comparable consistency with mimic cream containing
same concentration of SLES. Thus, when SLs were applied in the formulation
with mixed paraffin oils in water, higher concentration incorporated has
potential to produce creams with desired performance. While when 2 wt% of
MELs was added to the system with fatty alcohols, less viscous product was
formulated with smooth texture and consistency but easier to flow,
presenting low limit viscosity and corresponding yield stress, which is also
proved with oscillatory sweep and creep
245
test. And higher concentration of MELs resulted in a worse cream system. Thus
for emulsifying mixed paraffin oils in water, MELs was not recommended
incorporating with 6 wt% CA and 2 wt% GM. Modification should be made in
altering surfactant system composition in terms of fatty alcohols. Unique molecular
structure of MELs is different from SLES and SLs which possesses one
hydrocarbon chain, MELs tend to self-assemble into vesicles (Morita et al., 2015).
Besides, it is interesting to find that the effect of different concentrations of SLES
on cream performance is the same as that of MELs in this study where 6 wt% CA
and 2 wt% GM involved in mixed paraffin oils/water system, while that was different
from what obtained from SLs. This could provide information for optimising the
composition of formulations.
Vegetable oils are capable of being the substitute for mixed paraffin oils in order to
prepare “greener” products. No big difference was found when same amount of
coconut oil was applied instead of mixed paraffin oils. This may because the
similarity of property between them. A frequency sweep indicated that G’ values
dependent of frequency of mixed paraffin oils and coconut oil are almost the same,
but vegetable shortening exhibiting an extremely high G’ compared to coconut oil
and paraffin mixed oils.
Apart from composition of formulation, manufacturing procedure also greatly

affects cream performance, especially cooling process where the microstructure of
semi-solid state was altered from lamellar phase to gel phase, reflecting as product
of flexible state to a structured body. From this work, in the system of 4 wt% SLES,
6 wt% CA and 2 wt% GM, increasing stirring speed during cooling within 10
minutes resulted in a more viscous and rigid cream, while longer stirring duration
at a constant speed of 200 rpm led to a reversed effect. And for heating procedure,
microstructure of creams remains unchanged after mixing for 3 minutes, and the
same droplet size distribution was observed for another 17 minutes. However,
higher mixing speed help formulating creams with small droplets dispersed in
continuous phase. Thus appropriate manufacturing procedure should be
determined in order to achieve specific type of products.
Rheology is an effective method for rapidly interpreting the flow behaviours of

cream products. In this study, rheological parameters were applied as indices for
comparing the performance of creams formulated with different concentrations of
surfactant systems and optimising the composition. From non-linear rotational test,
the limiting value of viscosity was determined by extrapolation of 1st Newtonian
246
plateau, and corresponding yield stress was selected as the initial point of shear
thinning, which highly agreed with the consistency and texture of the creams from
observation. However, compared to rotational test, oscillatory sweep test provides
more precise explanation of material response to tiny disturbance such as zero
shear viscosity, where the microstructure is not fully destroyed. As achieved from
this study, storage modulus, G’, presenting as solid domain behaviours, positively
supported results from steady state shear, along with loss modulus, G’’. Similar to
oscillatory sweep, creep test is applied for viscoelastic behaviour determination
provided same results as frequency sweep did, but more sophisticated and time-
consuming. However, it is applicable for the material showing delayed elasticity
that cannot be predicted with the help complex modulus, G* (Shibaev et al., 2019).
To summarize, sophorolipids (SLs) mixture of lactonic and acidic forms that

produced by cultivating Candida bombicola consuming glucose and rapeseed oil
as substrates, is promising for cream formulation in replacement of same amount
of anionic surfactant (sodium lauryl ether sulfate/SLES), incorporating with cetyl
alcohol (CA) and glycerol monostearate (GM) in mixed paraffin oils and water
system. Better performance of cream (appropriate stiffness with consistent texture)
could be realized when higher concentration of SLs is involved. However, for
mannosylerythritol lipids (MELs) (mainly MEL-A) that originated from Pseudozyma
aphidis DSM 70725 growing in the medium containing glucose and rapeseed, less
structured creams with higher mobility were produced, and higher concentration of
MELs incorporated, more dissatisfactory cream tends to be produced. Coconut oil
is a potential substitute for mixed paraffin oils in cream formulation. However,
although same amount of coconut oils applied in the formulation is able to produce
cream-like products, the texture and morphology may not be satisfied when same
manufacturing procedure was applied as that for mixed paraffin oils included, and
further modification of the formulae composition should also be taken into account.
Vegetable shortening may need pre-treatment or further modification for
eliminating undesired colour and granular texture of cream.
Still, further study could be conducted for improving and perfecting this project:
1. The interfacial tension of the surfactant system is worth of analysing. Because

mixture of liquid paraffin and white soft paraffin is not in liquid state at room
temperature, silicon oil could be an alternative for the study. As suggested, 0 wt%,
2 wt%, 4 wt%, 6 wt% and 10 wt% of SLES solution could be prepared. After
obtaining the dependence of interfacial tension on SLES concentration, different
247
concentration of cetyl alcohol could be added into silicon oil to get the
measurement of the interfacial tension between silicon oil (with cetyl alcohol) and
SLES solution.
2. Emulsification Index (EI) measurement should be carried out, for understanding

the emulsifying property of SLES. Two types of oils could be used in the
measurement: silicon oil and the mixture of two paraffin oils. Equal volume of oil is
mixed with different concentrations of SLES solutions (0 wt%, 2 wt%, 3 wt%, 4 wt%,
6 wt%), followed by a vortex for 2 min. After standing for 24 h, EI could be
calculated. The measurement could also be conducted at different temperatures,
for example, 25±2 °C, 40±2 °C, 55±2 °C and 70±2 °C.
3. Rheological measurement should take more caution of wall depletion which may
lead to inaccurate characterisation of actual flow property of materials, although it
is very common, and as a matter of fact that it cannot be fully eliminated. However,
in this project, all characterisations of creams were consistently applied 40 mm
cone-plate geometry with a measuring gap of 57 mm, and results was not largely
discrepant with that obtained from literatures, where a limiting viscosity of 104 Pa.s
for a cream and 103 Pa.s for a lotion (Kwak et al., 2015). And the values of yield
stress were reasonable which line in between 10 Pa and 100 Pa. Even though, in
order to further investigate the effect degree of wall slip on the results, a geometry
with roughed surface is suggested, and different size of geometry and mearing gap
are worth of trying with.
4. Further purification of biosurfactants is necessary, as biosurfactants applied in

the formulation were mixtures of different structures and forms. Large effect may
arise on cream performance when surfactants with structural differences are
applied. Thus structural separation of SLs and MELs could help investigate effect
of biosurfactants with unique structure on cream formulation.
5. When reliable results were obtained in lab scale, enlarging formulation scale in
a pilot scale is suggested, for better understanding influences of manufacturing
process on cream production and optimizing lab-scaled results. From this aspect,
economic friendly biosurfactants production with higher yield is required, for
facilitating the commercialization of bio-cream production in lab-scaled research.
248
References
Ade-Browne, C., Mirzamani, M., Dawn, A., Qian, S., Thompson, R., Glenn, R. & Kumari, H.
2020. Effect of ethoxylation and lauryl alcohol on the self-assembly of sodium
laurylsulfate: Significant structural and rheological transformation. Colloids and
Surfaces A: Physicochemical and Engineering Aspects, 124704.
Adu, S. A., Naughton, P. J., Marchant, R. & Banat, I. M. 2020. Microbial Biosurfactants in
Cosmetic and Personal Skincare Pharmaceutical Formulations. Pharmaceutics, 12,
1099.
Agneta, M., Zhaomin, L., Chao, Z. & Gerald, G. 2019. Investigating synergism and
antagonism of binary mixed surfactants for foam efficiency optimization in high
salinity. Journal of Petroleum Science & Engineering, 175, 489-494.
Agrawal, N., Maddikeri, G. L. & Pandit, A. B. 2017. Sustained release formulations of
citronella oil nanoemulsion using cavitational techniques. Ultrasonics
Sonochemistry, 36, 367-374.
Ahmadi-Ashtiani, H. R., Baldisserotto, A., Cesa, E., Manfredini, S. & Vertuani, S. 2020.
Microbial Biosurfactants as Key Multifunctional Ingredients for Sustainable
Cosmetics.
Ahmadi, D., Mahmoudi, N., Li, P., Tellam, J., Barlow, D. & Lawrence, M. J. 2020. Simple
creams, complex structures. Molecular Assemblies: Characterization and
Applications. ACS Publications.
Ahmed, T. M. 2019. Fatigue performance of hot mix asphalt tested in controlled stress
mode using dynamic shear rheometer. International Journal of Pavement
Engineering, 20, 255-265.
Aiza, Gay, Corpuz, Priyabrata, Pal, Fawzi & Banat] 2019. Effect of temperature and use of
regenerated surfactants on the removal of oil from water using colloidal gas
aphrons. Separation & Purification Technology.
Akbari, S. & Nour, A. H. 2018. Emulsion types, stability mechanisms and rheology: A review.
International Journal of Innovative Research and Scientific Studies, 1, 14-21.
Ali, Ebadi, Nayer, Azam, Khoshkholgh, Sima, Mohsen, Olamaee, Maryam & Hashemi 2017.
Effective bioremediation of a petroleum-polluted saline soil by a surfactant-
producing Pseudomonas aeruginosa consortium. Journal of Advanced Research.
Ali, M. F., Amin, D. & Reza, S. S. 2018. An investigation into surfactant flooding and
alkaline-surfactant-polymer flooding for enhancing oil recovery from carbonate
reservoirs: Experimental study and simulation. Energy Sources Part A Recovery
Utilization & Environmental Effects, 40, 1-12.
Almeira, N., Komilis, D., Barrena, R., Gea, T. & Sánchez, A. 2015. The importance of
aeration mode and flowrate in the determination of the biological activity and
stability of organic wastes by respiration indices. Bioresource technology, 196,
256-262.
Alsinan, M., Kwak, H., Marques, D. S. & Kaidar, Z. Identifying High-Performance EOR
Surfactants Through Non-Destructive Evaluation of the Phase Behavior
Microstructure. SPE/IATMI Asia Pacific Oil & Gas Conference and Exhibition, 2019.
Ananthapadmanabhan, K. 2019. Amino-Acid Surfactants in Personal Cleansing. Tenside
Surfactants Detergents, 56, 378-386.
Anburajan, L., Meena, B., Raghavan, R. V., Shridhar, D., Joseph, T. C., Vinithkumar, N. V.,
Dharani, G., Dheenan, P. S. & Kirubagaran, R. 2015. Heterologous expression,
purification, and phylogenetic analysis of oil-degrading biosurfactant biosynthesis
genes from the marine sponge-associated Bacillus licheniformis NIOT-06.
Bioprocess and Biosystems Engineering, 38, 1009-1018.
249
Ankulkar, R. & Chavan, M. 2019. Characterisation and Application Studies of Sophorolipid
Biosurfactant by Candida tropicalis RA1. Journal of Pure and Applied Microbiology,
13, 1653-1665.
Araújo, J. S. d. 2018. Produção de ramnolipídeos por Pseudomonas aeruginosa AP029-
GLVIIA usando glicose como substrato e aplicações. Brasil.
Arias, A., Macorra, J. C., Govindjee, S. & Peters, O. A. 2018. Correlation between
temperature-dependent fatigue resistance and differential scanning calorimetry
analysis for 2 contemporary rotary instruments. Journal of endodontics, 44, 630-
634.
Ashby, R. D. & Solaiman, D. K. 2019. Sophorolipids: Unique microbial glycolipids with vast
application potential. Microbial Biosurfactants and their Environmental and
Industrial Applications. CRC Press.
Aswal, A., Kalra, M. & Rout, A. 2013. Preparation and evaluation of polyherbal cosmetic
cream. Der Pharmacia Lettre, 5, 83-88.
Awad, T. S., Johnson, E. S., Bureiko, A. & Olsson, U. 2011. Colloidal Structure and Physical
Properties of Gel Networks Containing Anionic Surfactant and Fatty Alcohol
Mixture. Journal of Dispersion Science and Technology, 32, 807-815.
Bages-Estopa, S., White, D., Winterburn, J., Webb, C. & Martin, P. 2018. Production and
separation of a trehalolipid biosurfactant. Biochemical Engineering Journal, 139,
85-94.
Bai, L. & McClements, D. J. 2016. Formation and stabilization of nanoemulsions using
biosurfactants: Rhamnolipids. Journal of colloid and interface science, 479, 71-79.
Ballmann, C. & Müeller, B. 2008. Stabilizing Effect of Cetostearyl Alcohol and
Glycerylmonstearate as Co-emulsifiers on Hydrocarbon-free O/W Glyceride
Creams. Pharmaceutical development and technology, 13, 433-445.
Ban, T., Kawaizumi, F., Nii, S. & Takahashi, K. 2000. Study of drop coalescence behavior
for liquid–liquid extraction operation. Chemical engineering science, 55, 5385-
5391.
Banat, I. M., Franzetti, A., Gandolfi, I., Bestetti, G., Martinotti, M. G., Fracchia, L., Smyth,
T. J. & Marchant, R. 2010. Microbial biosurfactants production, applications and
future potential. Applied microbiology and biotechnology, 87, 427-444.
Banerjee, K., Thiagarajan, N. & Thiagarajan, P. 2019. Formulation and characterization of
a Helianthus annuus‐alkyl polyglucoside emulsion cream for topical applications.
Journal of cosmetic dermatology, 18, 628-637.
Bang, J.-H., Song, K. S., Lee, M. G., Jeon, C. W. & Jang, Y. N. 2010. Effect of critical micelle
concentration of sodium dodecyl sulfate dissolved in calcium and carbonate
source solutions on characteristics of calcium carbonate crystals. Materials
transactions, 1007121124-1007121124.
Bankole, M. T., Abdulkareem, S. A., Tijani, J. O., Ochigbo, S. S. & Roos, W. D. 2017.
Chemical oxygen demand removal from electroplating wastewater by purified
and polymer functionalized carbon nanotubes adsorbents. Water Resources &
Industry, 18, 33-50.
Barnes, H. A. 1995. A review of the slip (wall depletion) of polymer solutions, emulsions
and particle suspensions in viscometers: its cause, character, and cure. Journal of
Non-Newtonian Fluid Mechanics, 56, 221-251.
Barnes, H. A., Hutton, J. F. & Walters, K. 1989. An introduction to rheology, Elsevier.
Beck, A., Werner, N. & Zibek, S. 2019. Mannosylerythritol Lipids: Biosynthesis, Genetics,
and Production Strategies. Biobased Surfactants. Elsevier.
Bekker, M., Webber, G. & Louw, N. 2013. Relating rheological measurements to primary
and secondary skin feeling when mineral‐based and Fischer–Tropsch wax‐based
250
cosmetic emulsions and jellies are applied to the skin. International Journal of
Cosmetic Science, 35, 354-361.
Bera, A., Ojha, K. & Mandal, A. 2013. Synergistic Effect of Mixed Surfactant Systems on
Foam Behavior and Surface Tension. Journal of Surfactants & Detergents, 16, 621-
630.
Bertin, H., Estrada, E. D. C. & Atteia, O. 2017. Foam placement for soil remediation.
Environmental Chemistry, 14.
Bezerraa, K. G., Durvala, I. J., Silvab, I. A. & CG, F. 2020. Emulsifying Capacity of
Biosurfactants from Chenopodium Quinoa and Pseudomonas Aeruginosa UCP
0992 with Focus of Application in the Cosmetic Industry. CHEMICAL ENGINEERING,
79.
Bharali, P., Singh, S. P., Dutta, N., Gogoi, S., Bora, L., Debnath, P. & Konwar, B. K. 2014.
Biodiesel derived waste glycerol as an economic substrate for biosurfactant
production using indigenous Pseudomonas aeruginosa. RSC advances, 4, 38698-
38706.
Bhattachar, S. N., Risley, D. S., Werawatganone, P. & Aburub, A. 2011. Weak bases and
formation of a less soluble lauryl sulfate salt/complex in sodium lauryl sulfate (SLS)
containing media. International journal of pharmaceutics, 412, 95-98.
Bhosale, S. S., Rohiwal, S. S., Chaudhary, L. S., Pawar, K. D., Patil, P. S. & Tiwari, A. P. 2019.
Photocatalytic decolorization of methyl violet dye using Rhamnolipid
biosurfactant modified iron oxide nanoparticles for wastewater treatment.
Journal of Materials Science: Materials in Electronics, 30, 4590-4598.
Blanco-Díaz, E., Castrejón-González, E., Rico-Ramírez, V., Aztatzi-Pluma, D. & Díaz-Ovalle,
C. 2018. Polydispersity influence in rheological behavior of linear chains by
molecular dynamics. Journal of Molecular Liquids, 268, 832-839.
Bnyan, R., Khan, I., Ehtezazi, T., Saleem, I., Gordon, S., Neill, F. O. & Roberts, M. 2018.
Surfactant Effects on Lipid-Based Vesicles Properties. Journal of Pharmaceutical
Sciences, S0022354918300054.
Bonnin, L. 2019. Optimization of stability and rheological robustness of cosmetic salt-
containing lamellar gel phase emulsions.
Borah, S. N., Sen, S., Goswami, L., Bora, A., Pakshirajan, K. & Deka, S. 2019. Rice based
distillers dried grains with solubles as a low cost substrate for the production of a
novel rhamnolipid biosurfactant having anti-biofilm activity against Candida
tropicalis. Colloids and Surfaces B: Biointerfaces, 182, 110358.
Boxall, J. A., Koh, C. A., Sloan, E. D., Sum, A. K. & Wu, D. T. 2010. Measurement and
calibration of droplet size distributions in water-in-oil emulsions by particle video
microscope and a focused beam reflectance method. Industrial & engineering
chemistry research, 49, 1412-1418.
Boxall, J. A., Koh, C. A., Sloan, E. D., Sum, A. K. & Wu, D. T. 2012. Droplet size scaling of
water-in-oil emulsions under turbulent flow. Langmuir, 28, 104-110.
Boyer, H. C., Bzdek, B. R., Reid, J. P. & Dutcher, C. S. 2017. Statistical thermodynamic
model for surface tension of organic and inorganic aqueous mixtures. The Journal
of Physical Chemistry A, 121, 198-205.
Brummer, R. 2013. Rheology of Cosmetic Emulsions. Product Design and Engineering:
Formulation of Gels and Pastes, 51-74.
Bunet, R., Riclea, R., Laureti, L., Hotel, L., Paris, C., Girardet, J.-M., Spiteller, D., Dickschat,
J. S., Leblond, P. & Aigle, B. 2014. A single Sfp-type phosphopantetheinyl
transferase plays a major role in the biosynthesis of PKS and NRPS derived
metabolites in Streptomyces ambofaciens ATCC23877. PLoS One, 9, e87607.
Caffalette, C. A., Kuklewicz, J., Spellmon, N. & Zimmer, J. 2020. Biosynthesis and export of
bacterial glycolipids. Annual review of biochemistry, 89, 741-768.
251
Calero, N., Muñoz, J., Cox, P. W., Heuer, A. & Guerrero, A. 2013. Influence of chitosan
concentration on the stability, microstructure and rheological properties of O/W
emulsions formulated with high-oleic sunflower oil and potato protein. Food
Hydrocolloids, 30, 152-162.
Callaghan, B., Lydon, H., Roelants, S. L., Van Bogaert, I. N., Marchant, R., Banat, I. M. &
Mitchell, C. A. 2016. Lactonic Sophorolipids increase tumor burden in Apcmin+/-
mice. PloS one, 11, e0156845.
Callow, N. V., Ray, C. S., Kelbly, M. A. & Ju, L.-K. 2016. Nutrient control for stationary phase
cellulase production in Trichoderma reesei Rut C-30. Enzyme and microbial
technology, 82, 8-14.
Câmara, J., Sousa, M., Neto, E. B. & Oliveira, M. 2019. Application of rhamnolipid
biosurfactant produced by Pseudomonas aeruginosa in microbial-enhanced oil
recovery (MEOR). Journal of Petroleum Exploration and Production Technology, 9,
2333-2341.
Cantero del Castillo, J. 2019. Development of functional facial creams and their
manufacturing process.
Canu, R., Puggelli, S., Essadki, M., Duret, B., Menard, T., Massot, M., Reveillon, J. &
Demoulin, F. 2018. Where does the droplet size distribution come from?
International Journal of Multiphase Flow, 107, 230-245.
Caracciolo, A. B., Cardoni, M., Pescatore, T. & Patrolecco, L. 2017. Characteristics and
environmental fate of the anionic surfactant sodium lauryl ether sulphate (SLES)
used as the main component in foaming agents for mechanized tunnelling.
Environmental Pollution, 226, 94-103.
Caritá, A. C., de Azevedo, J. R., Buri, M. V., Bolzinger, M.-A., Chevalier, Y., Riske, K. A. &
Leonardi, G. R. 2020. Stabilization of vitamin C in emulsions of liquid crystalline
structures. International Journal of Pharmaceutics, 120092.
Castellano, S., Carrillo, L., Sheibat-Othman, N., Marchisio, D., Buffo, A. & Charton, S. 2019.
Using the full turbulence spectrum for describing droplet coalescence and
breakage in industrial liquid-liquid systems: Experiments and modeling. Chemical
Engineering Journal, 374, 1420-1432.
Chayabutra, C. & Ju, L. K. 2001. Polyhydroxyalkanoic acids and rhamnolipids are
synthesized sequentially in hexadecane fermentation by Pseudomonas
aeruginosa ATCC 10145. Biotechnology progress, 17, 419-423.
Chebbi, A., Hentati, D., Zaghden, H., Baccar, N., Rezgui, F., Chalbi, M., Sayadi, S. &
Chamkha, M. 2017. Polycyclic aromatic hydrocarbon degradation and
biosurfactant production by a newly isolated Pseudomonas sp. strain from used
motor oil-contaminated soil. International Biodeterioration & Biodegradation,
122, 128-140.
Chellapa, P., Ariffin, F. D., Eid, A. M., Almahgoubi, A. A., Mohamed, A. T., Issa, Y. S. &
Elmarzugi, N. A. 2016. Nanoemulsion for cosmetic application. European Journal
of Biomedical and Pharmaceutical Sciences, 3, 8-11.
Chen, W., Qu, Y., Xu, Z., He, F., Chen, Z., Huang, S. & Li, Y. 2017a. Heavy metal (Cu, Cd, Pb,
Cr) washing from river sediment using biosurfactant rhamnolipid. Environmental
Science and Pollution Research, 24, 16344-16350.
Chen, X., Feng, Q., Liu, W. & Sepehrnoori, K. 2017b. Modeling preformed particle gel
surfactant combined flooding for enhanced oil recovery after polymer flooding.
Fuel, 194, 42-49.
Chizawa, Y., Miyagawa, Y., Yoshida, M. & Adachi, S. 2019. Effect of crystallization of oil
phase on the destabilization of O/W emulsions containing vegetable oils with low
melting points. Colloids and Surfaces A: Physicochemical and Engineering Aspects,
582, 123824.
252
Choi, B., Loh, X. J., Tan, A., Loh, C. K., Ye, E., Joo, M. K. & Jeong, B. 2015. Introduction to in
situ forming hydrogels for biomedical applications. In-Situ Gelling Polymers.
Springer.
Chrzanowski, Ł., Ławniczak, Ł. & Czaczyk, K. 2012. Why do microorganisms produce
rhamnolipids? World Journal of Microbiology and Biotechnology, 28, 401-419.
Ciesielska, K., Roelants, S. L., Van Bogaert, I. N., De Waele, S., Vandenberghe, I.,
Groeneboer, S., Soetaert, W. & Devreese, B. 2016. Characterization of a novel
enzyme—Starmerella bombicola lactone esterase (SBLE)—responsible for
sophorolipid lactonization. Applied microbiology and biotechnology, 100, 9529-
9541.
Coelho, A. L. S., Feuser, P. E., Carciofi, B. A. M., de Andrade, C. J. & de Oliveira, D. 2020.
Mannosylerythritol lipids: antimicrobial and biomedical properties. Applied
Microbiology and Biotechnology, 104, 2297-2318.
Cohen, L., Martin, M., Soto, F., Trujillo, F. & Sanchez, E. 2016. The effect of counterions of
linear alkylbenzene sulfonate on skin compatibility. Journal of Surfactants and
Detergents, 19, 219-222.
Colafemmina, G., Palazzo, G., Mateos, H., Amin, S., Fameau, A.-L., Olsson, U. & Gentile, L.
2020a. The cooling process effect on the bilayer phase state of the CTAC/cetearyl
alcohol/water surfactant gel. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 124821.
Colafemmina, G., Palazzo, G., Mateos, H., Amin, S. & Gentile, L. 2020b. The cooling process
effect on the bilayer phase state of the CTAC/cetearyl alcohol/water surfactant
gel. Colloids and Surfaces A Physicochemical and Engineering Aspects, 597,
124821.
Colo, S. M., Herh, P. K., Roye, N. & Larsson, M. 2004. Rheology and the texture of
pharmaceutical and cosmetic semisolids. American Laboratory, 36, 26-35.
Coronel León, J., Manresa Presas, M. & Marqués Villavecchia, A. M. 2016. Lichenysin
production and application in the pharmaceutical field. Recent Advances in
Pharmaceutical Sciences VI, 2016, Research Signpost. Editors: Diego Muñoz
Torrero, Àngela Domínguez García & Ma. Ángeles Manresa Presas. ISBN: 978-81-
308-0566-5. Chapter 9, p. 147-163.
Coussot, P. 2005. Rheometry of pastes, suspensions, and granular materials: applications
in industry and environment, John Wiley & Sons.
Da Costa, F., Le Grand, F., Quéré, C., Bougaran, G., Cadoret, J. P., Robert, R. & Soudant, P.
2017. Effects of growth phase and nitrogen limitation on biochemical composition
of two strains of Tisochrysis lutea. Algal research, 27, 177-189.
da Rocha Junior, R. B., Meira, H. M., Almeida, D. G., Rufino, R. D., Luna, J. M., Santos, V. A.
& Sarubbo, L. A. 2019. Application of a low-cost biosurfactant in heavy metal
remediation processes. Biodegradation, 30, 215-233.
Dalili, D., Amini, M., Faramarzi, M. A., Fazeli, M. R., Khoshayand, M. R. & Samadi, N. 2015.
Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide
from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity.
Colloids and Surfaces B: Biointerfaces, 135, 425-432.
Damasceno, F. R., Cavalcanti-Oliveira, E. D., Kookos, I. K., Koutinas, A. A., Cammarota, M.
C. & Freire, D. M. 2018. Treatment of wastewater with high fat content employing
an enzyme pool and biosurfactant: technical and economic feasibility. Brazilian
Journal of Chemical Engineering, 35, 531-542.
Danley, R. L. 2002. Power compensation differential scanning calorimeter. Google Patents.
Dao, H., Lakhani, P., Police, A., Kallakunta, V., Ajjarapu, S. S., Wu, K.-W., Ponkshe, P., Repka,
M. A. & Murthy, S. N. 2018. Microbial stability of pharmaceutical and cosmetic
products. Aaps Pharmscitech, 19, 60-78.
253
Das, A. J. & Kumar, R. 2019. Production of biosurfactant from agro-industrial waste by
Bacillus safensis J2 and exploring its oil recovery efficiency and role in restoration
of diesel contaminated soil. Environmental Technology & Innovation, 16, 100450.
Dashtaki, S. R. M., Ali, J. A., Manshad, A. K., Nowrouzi, I. & Keshavarz, A. 2020.
Experimental investigation of the effect of Vitagnus extract on enhanced oil
recovery process using interfacial tension (IFT) reduction and wettability
alteration mechanisms. Journal of Petroleum Exploration & Production
Technology.
Daverey, A. & Pakshirajan, K. 2009. Production, characterization, and properties of
sophorolipids from the yeast Candida bombicola using a low-cost fermentative
medium. Applied biochemistry and biotechnology, 158, 663-674.
Daverey, A. & Pakshirajan, K. 2010. Sophorolipids from Candida bombicola using mixed
hydrophilic substrates: production, purification and characterization. Colloids and
Surfaces B: Biointerfaces, 79, 246-253.
David, O. A., David, D. O., Mogoase, C., Popescu, L. C., Giosan, C. & Pellegrino, A. 2019.
Psychological effects and brain correlates of a rose ‐ based scented cosmetic
cream. Journal of Sensory Studies, 34, e12536.
de Almeida, D. G., Brasileiro, P. P. F., Rufino, R. D., de Luna, J. M. & Sarubbo, L. A. 2019.
Production, formulation and cost estimation of a commercial biosurfactant.
Biodegradation, 30, 191-201.
De Andrade, C. J., De Andrade, L. M., Rocco, S. A., Sforça, M. L., Pastore, G. M. & Jauregi,
P. 2017. A novel approach for the production and purification of
mannosylerythritol lipids (MEL) by Pseudozyma tsukubaensis using cassava
wastewater as substrate. Separation and Purification Technology, 180, 157-167.
de Freitas Ferreira, J., Vieira, E. A. & Nitschke, M. 2019. The antibacterial activity of
rhamnolipid biosurfactant is pH dependent. Food Research International, 116,
737-744.
De Souza, P. M., Andrade Silva, N. R., Souza, D. G., Lima e Silva, T. A., Freitas-Silva, M. C.,
Andrade, R. F., Silva, G. K., Albuquerque, C. D., Messias, A. S. & Campos-Takaki, G.
M. 2018. Production of a Biosurfactant by Cunninghamella echinulata using
renewable substrates and its applications in enhanced oil spill recovery. Colloids
and Interfaces, 2, 63.
Delbeke, E. I., Everaert, J., Uitterhaegen, E., Verweire, S., Verlee, A., Talou, T., Soetaert,
W., Van Bogaert, I. N. & Stevens, C. V. 2016. Petroselinic acid purification and its
use for the fermentation of new sophorolipids. Amb Express, 6, 28.
Devaraj, S., Sabapathy, P. C., Nehru, L. & Preethi, K. 2019. Bioprocess optimization and
production of biosurfactant from an unexplored substrate: Parthenium
hysterophorus. Biodegradation, 30, 325-334.
Deyab, M. 2019. Effect of nonionic surfactant as an electrolyte additive on the
performance of aluminum-air battery. Journal of Power Sources, 412, 520-526.
Dickinson, E. 2019. Strategies to control and inhibit the flocculation of protein-stabilized
oil-in-water emulsions. Food Hydrocolloids, 96, 209-223.
Dimock, G. A., Lindler, J. E. & Wereley, N. M. Bingham biplastic analysis of shear thinning
and thickening in magnetorheological dampers. Smart Structures and Materials
2000: Smart Structures and Integrated Systems, 2000. International Society for
Optics and Photonics, 444-455.
Dinkgreve, M., Paredes, J., Denn, M. M. & Bonn, D. 2016. On different ways of measuring
“the” yield stress. Journal of non-Newtonian fluid mechanics, 238, 233-241.
Dogan, M., Kayacier, A., Toker, Ö . S., Yilmaz, M. T. & Karaman, S. 2013. Steady, dynamic,
creep, and recovery analysis of ice cream mixes added with different
concentrations of xanthan gum. Food and Bioprocess Technology, 6, 1420-1433.
254
Dolman, B. M., Kaisermann, C., Martin, P. J. & Winterburn, J. B. 2017. Integrated
sophorolipid production and gravity separation. Process Biochemistry, 54, 162-
171.
Dolman, B. M., Wang, F. & Winterburn, J. B. 2019. Integrated production and separation
of biosurfactants. Process Biochemistry, 83, 1-8.
Dolz, M., Hernandez, M. & Delegido, J. 2008. Creep and recovery experimental
investigation of low oil content food emulsions. Food Hydrocolloids, 22, 421-427.
Drakontis, C. E. & Amin, S. 2020a. Biosurfactants: Formulations, Properties, and
Applications. Current Opinion in Colloid & Interface ence, 48.
Drakontis, C. E. & Amin, S. 2020b. Design of Sustainable Lip Gloss Formulation with
Biosurfactants and Silica Particles. International Journal of Cosmetic ence.
Drzeżdżon, J., Jacewicz, D., Sielicka, A. & Chmurzyński, L. 2019. Characterization of
polymers based on differential scanning calorimetry based techniques. TrAC
Trends in Analytical Chemistry, 110, 51-56.
Du, C. A., Song, Y., Yao, Z., Su, W., Zhang, G. & Wu, X. 2019. Developments in in-situ
microbial enhanced oil recovery in Shengli oilfield. Energy Sources, Part A:
Recovery, Utilization, and Environmental Effects, 1-11.
Dubey, K., Charde, P., Meshram, S., Yadav, S., Singh, S. & Juwarkar, A. 2012. Potential of
new microbial isolates for biosurfactant production using combinations of
distillery waste with other industrial wastes. J. Pet. Environ. Biotechnol, 12, 1-11.
Eccleston, G. 1997. Functions of mixed emulsifiers and emulsifying waxes in
dermatological lotions and creams. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 123, 169-182.
Elshafie, A. E., Joshi, S. J., Al-Wahaibi, Y. M., Al-Bemani, A. S., Al-Bahry, S. N., Al-Maqbali,
D. & Banat, I. M. 2015. Sophorolipids production by Candida bombicola ATCC
22214 and its potential application in microbial enhanced oil recovery. Frontiers
in microbiology, 6, 1324.
Elshikh, M., Moya‐Ramírez, I., Moens, H., Roelants, S., Soetaert, W., Marchant, R. & Banat,
I. M. 2017. Rhamnolipids and lactonic sophorolipids: natural antimicrobial
surfactants for oral hygiene. Journal of applied microbiology, 123, 1111-1123.
Enayati, M., Gong, Y., Goddard, J. M. & Abbaspourrad, A. 2018. Synthesis and
characterization of lactose fatty acid ester biosurfactants using free and
immobilized lipases in organic solvents. Food chemistry, 266, 508-513.
Englerová, K., Nemcová, R. & Styková, E. 2018. Biosurfactants and their role in the
inhibition of the biofilmforming pathogens. Ceska a Slovenska farmacie: casopis
Ceske farmaceuticke spolecnosti a Slovenske farmaceuticke spolecnosti, 67, 107-
112.
Falbe, J. 2012. Surfactants in consumer products: Theory, Technology and Application,
Springer Science & Business Media.
Fan, L., Xie, P., Wang, Y., Huang, Z. & Zhou, J. 2018. Biosurfactant–protein interaction:
influences of mannosylerythritol lipids-A on β-glucosidase. Journal of agricultural
and food chemistry, 66, 238-246.
Faramarzi, S., Bonnett, B., Scaggs, C. A., Hoffmaster, A., Grodi, D., Harvey, E. & Mertz, B.
2017. Molecular dynamics simulations as a tool for accurate determination of
surfactant micelle properties. Langmuir, 33, 9934-9943.
Faria, N. T., Santos, M. V., Fernandes, P., Fonseca, L. L., Fonseca, C. & Ferreira, F. C. 2014.
Production of glycolipid biosurfactants, mannosylerythritol lipids, from pentoses
and D-glucose/D-xylose mixtures by Pseudozyma yeast strains. Process
Biochemistry, 49, 1790-1799.
Farias, J. M., Stamford, T. C. M., Resende, A. H. M., Aguiar, J. S., Rufino, R. D., Luna, J. M.
& Sarubbo, L. A. 2019. Mouthwash containing a biosurfactant and chitosan: An
255
eco-sustainable option for the control of cariogenic microorganisms.
International journal of biological macromolecules, 129, 853-860.
Farzad, R., Puttinger, S., Pirker, S. & Schneiderbauer, S. 2018. Investigation of droplet size
distribution for liquid-liquid emulsions in Taylor-Couette flows. Journal of
Dispersion Science and Technology, 39, 250-258.
Feng, J., Chen, Q., Wu, X., Jafari, S. M. & McClements, D. J. 2018. Formulation of oil-in-
water emulsions for pesticide applications: impact of surfactant type and
concentration on physical stability. Environmental Science and Pollution Research,
25, 21742-21751.
Feng, J., Shi, Y., Yu, Q., Sun, C. & Yang, G. 2016. Effect of emulsifying process on stability
of pesticide nanoemulsions. Colloids and Surfaces A: Physicochemical and
Ferreira, A., Vecino, X., Ferreira, D., Cruz, J., Moldes, A. & Rodrigues, L. 2017. Novel
cosmetic formulations containing a biosurfactant from Lactobacillus paracasei.
Colloids and Surfaces B: Biointerfaces, 155, 522-529.
Flick, E. 2001. Cosmetic and Toiletry Formulation Vol. 8., Noyes Publication. William
Andrew Publishing, LLC, New York.
Fonseca, G. G., Heinzle, E., Wittmann, C. & Gombert, A. K. 2008. The yeast Kluyveromyces
marxianus and its biotechnological potential. Applied microbiology and
biotechnology, 79, 339-354.
Fukuoka, T., Morita, T., Konishi, M., Imura, T. & Kitamoto, D. 2008. A basidiomycetous
yeast, Pseudozyma tsukubaensis, efficiently produces a novel glycolipid
biosurfactant. The identification of a new diastereomer of mannosylerythritol
lipid-B. Carbohydrate research, 343, 555-560.
Fukuoka, T., Yanagihara, T., Imura, T., Morita, T., Sakai, H., Abe, M. & Kitamoto, D. 2011.
Enzymatic synthesis of a novel glycolipid biosurfactant, mannosylerythritol lipid-
D and its aqueous phase behavior. Carbohydrate research, 346, 266-271.
Fukuoka, T., Yanagihara, T., Imura, T., Morita, T., Sakai, H., Abe, M. & Kitamoto, D. 2012.
The diastereomers of mannosylerythritol lipids have different interfacial
properties and aqueous phase behavior, reflecting the erythritol configuration.
Carbohydrate research, 351, 81-86.
Fuzhen, C., Jianwei, G., Hanqiao, J., Xue, Y. & Yuan, L. 2018. Laboratory evaluation and
numerical simulation of the alkali–surfactant–polymer synergistic mechanism in
chemical flooding. Rsc Advances, 8, 26476-26487.
Ganji, Z., Beheshti-Maal, K., Massah, A. & Emami-Karvani, Z. 2020. A novel sophorolipid-
producing Candida keroseneae GBME-IAUF-2 as a potential agent in microbial
enhanced oil recovery (MEOR). FEMS Microbiology Letters, 367, fnaa144.
Gao, Z., Li, D., Buffo, A., Podgórska, W. & Marchisio, D. L. 2016. Simulation of droplet
breakage in turbulent liquid–liquid dispersions with CFD-PBM: comparison of
breakage kernels. Chemical Engineering Science, 142, 277-288.
García-Cervilla, R., Romero, A., Santos, A. & Lorenzo, D. 2020. Surfactant-Enhanced
Solubilization of Chlorinated Organic Compounds Contained in DNAPL from
Lindane Waste: Effect of Surfactant Type and pH. International Journal of
Environmental Research and Public Health, 17, 4494.
García, M. C., Cox, P., Trujillo-Cayado, L., Muñoz, J. & Alfaro, M. C. 2018. Rheology,
microstructural characterization and physical stability of W/α-PINENE/W
emulsions formulated with copolymers. Colloids and Surfaces A: Physicochemical
and Engineering Aspects, 536, 125-132.
Gaur, V. K., Regar, R. K., Dhiman, N., Gautam, K., Srivastava, J. K., Patnaik, S., Kamthan, M.
& Manickam, N. 2019. Biosynthesis and characterization of sophorolipid
256
biosurfactant by Candida spp.: Application as food emulsifier and antibacterial
agent. Bioresource technology, 285, 121314.
Gein, S. V., Kuyukina, M. S., Ivshina, I. B., Baeva, T. A. & Chereshnev, V. A. 2011. In vitro
cytokine stimulation assay for glycolipid biosurfactant from Rhodococcusruber:
role of monocyte adhesion. Cytotechnology, 63, 559-566.
Gilbert, L., Picard, C., Savary, G. & Grisel, M. 2013. Rheological and textural
characterization of cosmetic emulsions containing natural and synthetic polymers:
relationships between both data. Colloids and Surfaces A: Physicochemical and
Göbbert, U., Lang, S. & Wagner, F. 1984. Sophorose lipid formation by resting cells of
Torulopsis bombicola. Biotechnology letters, 6, 225-230.
Goswami, T., Tack, F. M., McGachy, L. & Šír, M. 2020. Remediation of Aviation Kerosene-
Contaminated Soil by Sophorolipids from Candida bombicola CB 2107. Applied
Sciences, 10, 1981.
Graziano, F., Cohen, R. & Medalia, A. 1979. Rheology of concentrated suspensions of
carbon black in low molecular weight vehicles. Rheologica Acta, 18, 640-656.
Grewe, F., Ortmeyer, J., Haase, R. & Schmidt, C. 2015. Colloidal gels formed by dilute
aqueous dispersions of surfactant and fatty alcohol. Colloid process engineering.
Springer.
Gudiña, E. J., Fernandes, E. C., Teixeira, J. A. & Rodrigues, L. R. 2015. Antimicrobial and
anti-adhesive activities of cell-bound biosurfactant from Lactobacillus agilis
CCUG31450. RSC Advances, 5, 90960-90968.
Guilmanov, V., Ballistreri, A., Impallomeni, G. & Gross, R. A. 2002. Oxygen transfer rate
and sophorose lipid production by Candida bombicola. Biotechnology and
bioengineering, 77, 489-494.
Guo, C., Wang, J., Cao, F., Lee, R. J. & Zhai, G. 2010. Lyotropic liquid crystal systems in drug
delivery. Drug Discovery Today, 15, 1032-1040.
Guo, J. & Gao, Q. 2020. Enhancement of ethylbenzene removal from contaminated gas
and corresponding mechanisms in biotrickling filters by a biosurfactant from
piggery wastewater. Journal of Environmental Management, 277, 111411.
Guo, Y., Wang, Y., Zhang, C., Mu, Q., Li, X., Sun, Y., Dou, H. & Wang, Q. 2018. Blue-phase
liquid crystal display with insulating protrusion. Liquid Crystals, 45, 1585-1593.
Guzmán, E., Llamas, S., Fernández-Peña, L., Léonforte, F., Baghdadli, N., Cazeneuve, C.,
Ortega, F., Rubio, R. G. & Luengo, G. S. 2020. Effect of a natural amphoteric
surfactant in the bulk and adsorption behavior of polyelectrolyte-surfactant
mixtures. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 585,
124178.
Guzman, M. L., Marques, M. R., ME, M. E. O. & Stippler, E. S. 2016. Enzymatic activity in
the presence of surfactants commonly used in dissolution media, Part 1: Pepsin.
Results in Pharma Sciences, 6, 15-19.
Ha, H. J., Park, Y. J., An, J. H. & Kim, J. H. 2015. Influence of microstructure on dynamic
mechanical property of emulsion blend containing heterocoagulated composite
particles. Journal of Applied Polymer Science, 66, 1899-1909.
Habibi, A. & Babaei, F. 2017. Biological Treatment of Real Oilfield-Produced Water by
Bioaugmentation with Sophorolipid-Producing Candida catenulata.
Environmental Processes, 4, 891-906.
Hailu, S. L., Nair, B. U., Redi-Abshiro, M., Diaz, I. & Tessema, M. 2017. Preparation and
Characterization of Cationic Surfactant Modified Zeolite Adsorbent Material for
Adsorption of Organic and Inorganic Industrial Pollutants. Journal of
Environmental Chemical Engineering, 5, 3319-3329.
257
Haloi, S., Sarmah, S., Gogoi, S. B. & Medhi, T. 2020. Characterization of Pseudomonas sp.
TMB2 produced rhamnolipids for ex-situ microbial enhanced oil recovery. 3
Biotech, 10, 1-17.
Hamed, R., Alnadi, S. H. & Awadallah, A. 2020. The Effect of Enzymes and Sodium Lauryl
Sulfate on the Surface Tension of Dissolution Media: Toward Understanding the
Solubility and Dissolution of Carvedilol. AAPS PharmSciTech, 21, 146.
Han, X., Kurnia, I., Chen, Z., Yu, J. & Zhang, G. 2019. Effect of oil reactivity on salinity profile
design during alkaline-surfactant-polymer flooding. Fuel, 254, 115738.1-115738.9.
Hanano, A., Shaban, M. & Almousally, I. 2017. Biochemical, molecular, and transcriptional
highlights of the biosynthesis of an effective biosurfactant produced by Bacillus
safensis PHA3, a petroleum-dwelling bacteria. Frontiers in microbiology, 8, 77.
Hantal, G. r., Sega, M., Horvai, G. & Jedlovszky, P. l. 2019. Contribution of different
molecules and moieties to the surface tension in aqueous surfactant solutions.
The Journal of Physical Chemistry C, 123, 16660-16670.
Hartland, S. 2004. Surface and interfacial tension: measurement, theory, and applications,
CRC Press.
Hassan, K. A., Sani, Y. & Ajoke, O. Y. 2018. Preliminary Screening of Bio-surfactant
Producing Bacteria Isolated from an Oil Contaminated Soil. Asian Journal of
Biochemistry, Genetics and Molecular Biology, 1-9.
Hatzikiriakos, S. G. 2012. Wall slip of molten polymers. Progress in Polymer Science, 37,
624-643.
Hellström, L. H. O., Samaha, M. A., Wang, K. M., Smits, A. J. & Hultmark, M. 2014. Errors
in parallel-plate and cone-plate rheometer measurements due to sample underfill.
Measurement Science and Technology, 26, 015301.
Hentati, D., Chebbi, A., Hadrich, F., Frikha, I., Rabanal, F., Sayadi, S., Manresa, A. &
Chamkha, M. 2019. Production, characterization and biotechnological potential
of lipopeptide biosurfactants from a novel marine Bacillus stratosphericus strain
FLU5. Ecotoxicology and environmental safety, 167, 441-449.
Heyd, M., Franzreb, M. & Berensmeier, S. 2011. Continuous rhamnolipid production with
integrated product removal by foam fractionation and magnetic separation of
immobilized Pseudomonas aeruginosa. Biotechnology progress, 27, 706-716.
Hiemenz, P. C. 1986. Principles of colloid and surface chemistry, M. Dekker New York.
Hilares, R. T., Ahmed, M. A., de Souza Junior, M. M., Marcelino, P. R., da Silva, S. S. & dos
Santos, J. C. 2018. Beyond Ethanol: Contribution of Various Bioproducts to
Enhance the Viability of Biorefineries. Sustainable Biotechnology-Enzymatic
Resources of Renewable Energy. Springer.
Hiraoka, K. 2013. Fundamentals of mass spectrometry.
Höhne, G., Hemminger, W. F. & Flammersheim, H.-J. 2013. Differential scanning
calorimetry, Springer Science & Business Media.
Hong, I. K., Kim, S. I. & Lee, S. B. 2018. Effects of HLB value on oil-in-water emulsions:
Droplet size, rheological behavior, zeta-potential, and creaming index. Journal of
Industrial and Engineering Chemistry, 67, 123-131.
Hrůzová, K., Patel, A., Masák, J., Maťátková, O., Rova, U., Christakopoulos, P. & Matsakas,
L. 2020. A novel approach for the production of green biosurfactant from
Pseudomonas aeruginosa using renewable forest biomass. Science of The Total
Environment, 711, 135099.
Hu, X., Gong, H., Li, Z., Ruane, S., Liu, H., Pambou, E., Bawn, C., King, S., Ma, K. & Li, P. 2019.
What happens when pesticides are solubilized in nonionic surfactant micelles.
Journal of colloid and interface science, 541, 175-182.
Hu, Y. & Ju, L.-K. 2001. Purification of lactonic sophorolipids by crystallization. Journal of
258
Huang, J. & Ren, Z. H. 2020. Mechanism on micellization of amino sulfonate amphoteric
surfactant in aqueous solutions containing different alcohols and its interfacial
adsorption. Journal of Molecular Liquids, 316, 113793.
Huck-Iriart, C., Montes-de-Oca-Ávalos, J., Herrera, M. L., Candal, R. J., Pinto-de-Oliveira, C.
L. & Linares-Torriani, I. 2016. New insights about flocculation process in sodium
caseinate-stabilized emulsions. Food Research International, 89, 338-346.
Ibrahim, N., Raman, I. & Yusop, M. R. 2015. Effects of functional group of non-ionic
surfactants on the stability of emulsion. Malaysian J Anal Sci, 19, 261-267.
Imura, T., Hikosaka, Y., Worakitkanchanakul, W., Sakai, H., Abe, M., Konishi, M.,
Minamikawa, H. & Kitamoto, D. 2007. Aqueous-phase behavior of natural
glycolipid biosurfactant mannosylerythritol lipid A: sponge, cubic, and lamellar
phases. Langmuir, 23, 1659-1663.
Invally, K., Sancheti, A. & Ju, L.-K. 2019. A new approach for downstream purification of
rhamnolipid biosurfactants. Food and Bioproducts Processing, 114, 122-131.
Irfan-Maqsood, M. & Seddiq-Shams, M. 2014. Rhamnolipids: well-characterized
glycolipids with potential broad applicability as biosurfactants. Industrial
Jadhav, J. V., Pratap, A. P. & Kale, S. B. 2019. Evaluation of sunflower oil refinery waste as
feedstock for production of sophorolipid. Process Biochemistry, 78, 15-24.
Janek, T., Czyżnikowska, Ż., Łukaszewicz, M. & Gałęzowska, J. 2019. The effect of
Pseudomonas fluorescens biosurfactant pseudofactin II on the conformational
changes of bovine serum albumin: Pharmaceutical and biomedical applications.
Journal of Molecular Liquids, 288, 111001.
Jardak, K., Drogui, P. & Daghrir, R. 2016. Surfactants in aquatic and terrestrial environment:
occurrence, behavior, and treatment processes. Environ Sci Pollut Res Int, 23,
3195-3216.
Jiang, J., Zu, Y., Li, X., Meng, Q. & Long, X. 2020. Recent progress towards industrial
rhamnolipids fermentation: Process optimization and foam control. Bioresource
technology, 298, 122394.
Jiang, Y., Zheng, Z., Zhang, T., Hendricks, G. & Guo, M. 2016. Microencapsulation of
Lactobacillus acidophilus NCFM using polymerized whey proteins as wall material.
International journal of food sciences and nutrition, 67, 670-677.
Jiménez-Peñalver, P., Gea, T., Sánchez, A. & Font, X. 2016. Production of sophorolipids
from winterization oil cake by solid-state fermentation: optimization, monitoring
and effect of mixing. Biochemical engineering journal, 115, 93-100.
Jiménez‐Peñalver, P., Koh, A., Gross, R., Gea, T. & Font, X. 2020. Biosurfactants from
waste: Structures and interfacial properties of Sophorolipids produced from a
residual oil cake. Journal of Surfactants and Detergents, 23, 481-486.
Jimoh, A. A. & Lin, J. 2019a. Biosurfactant: A new frontier for greener technology and
environmental sustainability. Ecotoxicology and Environmental Safety, 184.
Jimoh, A. A. & Lin, J. 2019b. Enhancement of Paenibacillus sp. D9 lipopeptide
biosurfactant production through the optimization of medium composition and
its application for biodegradation of hydrophobic pollutants. Applied
biochemistry and biotechnology, 187, 724-743.
Jimoh, A. A. & Lin, J. 2019c. Heterologous expression of Sfp-type phosphopantetheinyl
transferase is indispensable in the biosynthesis of lipopeptide biosurfactant.
Molecular biotechnology, 61, 836-851.
Jing, H., Lu, L., Feng, Y., Zheng, J.-F., Deng, L., Chen, E.-Q. & Ren, X.-K. 2016. Synthesis,
Aggregation-Induced Emission, and Liquid Crystalline Structure of
Tetraphenylethylene–Surfactant Complex via Ionic Self-Assembly. The Journal of
Physical Chemistry C, 120, 27577-27586.
259
Joshi-Navare, K., Khanvilkar, P. & Prabhune, A. 2013. Jatropha oil derived sophorolipids:
production and characterization as laundry detergent additive. Biochemistry
research international, 2013.
Kandasamy, R., Rajasekaran, M., Venkatesan, S. K. & Uddin, M. 2019. New Trends in the
Biomanufacturing of Green Surfactants: Biobased Surfactants and Biosurfactants.
Next Generation Biomanufacturing Technologies. ACS Publications.
Kanouni, M., Rosano, H. & Naouli, N. 2002. Preparation of a stable double emulsion (W
1/O/W 2): role of the interfacial films on the stability of the system. Advances in
colloid and interface science, 99, 229-254.
Karlapudi, A. P., Venkateswarulu, T., Tammineedi, J., Kanumuri, L., Ravuru, B. K., ramu
Dirisala, V. & Kodali, V. P. 2018. Role of biosurfactants in bioremediation of oil
pollution-a review. Petroleum, 4, 241-249.
Karthick, A., Chauhan, M., Krzan, M. & Chattopadhyay, P. 2019. Potential of surfactant
foam stabilized by Ethylene glycol and Allyl alcohol for the remediation of diesel
contaminated soil. Environmental Technology & Innovation, 14.
Kaspchak, E., de Oliveira, M. A. S., Simas, F. F., Franco, C. R. C., Silveira, J. L. M., Mafra, M.
R. & Igarashi-Mafra, L. 2017. Determination of heat-set gelation capacity of a
quinoa protein isolate (Chenopodium quinoa) by dynamic oscillatory rheological
analysis. Food chemistry, 232, 263-271.
Katarzyna, Hąc-Wydro, Agnieszka, Mateja, Aleksandra, Ożóg, Paweł & Miśkowiec 2017.
Influence of metal ions on the aggregation of anionic surfactants. Studies on the
interactions between environmental pollutants in aqueous solutions. Journal of
Molecular Liquids.
Kaur, G., Wang, H., To, M. H., Roelants, S. L., Soetaert, W. & Lin, C. S. K. 2019. Efficient
sophorolipids production using food waste. Journal of Cleaner Production, 232, 1-
11.
Kelleppan, V. T., Moore, J. E., McCoy, T. M., Sokolova, A. V., Campo, L. d., Wilkinson, B. L.
& Tabor, R. F. 2018. Self-assembly of long-chain betaine surfactants: effect of
tailgroup structure on wormlike micelle formation. Langmuir, 34, 970-977.
Kezrane, I., Harouna, B. M., Hamadache, M., Benkortbi, O. & Amrane, A. 2020. Use of
hydrocarbons sludge as a substrate for the production of biosurfactants by
Pseudomonas aeruginosa ATCC 27853. Environmental Monitoring and
Assessment, 192, 1-16.
Khan, B. A., Akhtar, N., Khan, H. M. S., Waseem, K., Mahmood, T., Rasul, A., Iqbal, M. &
Khan, H. 2011. Basics of pharmaceutical emulsions: A review. African Journal of
Pharmacy and Pharmacology, 5, 2715-2725.
Kim, H.-S., Jeon, J.-W., Kim, S.-B., Oh, H.-M., Kwon, T.-J. & Yoon, B.-D. 2002. Surface and
physico-chemical properties of a glycolipid biosurfactant, mannosylerythritol lipid,
from Candidaantarctica. Biotechnology letters, 24, 1637-1641.
KIM, H.-S., KIM, Y.-B., LEE, B.-S. & KIM, E.-K. 2005. Sophorolipid production by Candida
bombicola ATCC 22214 from a corn-oil processing byproduct. Journal of
microbiology and biotechnology, 15, 55-58.
Kim, I.-Y., Nam, E.-H. & Shin, M.-S. 2020a. A Study on the Formation of Lamellar Liquid
Crystalline Using Skin Mimicking Surfactant. Journal of the Korean Applied Science
and Technology, 37, 484-495.
Kim, J.-H., Oh, Y.-R., Hwang, J., Jang, Y.-A., Lee, S. S., Hong, S. H. & Eom, G. T. 2020b. Value-
added conversion of biodiesel into the versatile biosurfactant sophorolipid using
Starmerella bombicola. Cleaner Engineering and Technology, 100027.
Kim, Y.-B., Yun, H. S. & Kim, E.-K. 2009. Enhanced sophorolipid production by feeding-rate-
controlled fed-batch culture. Bioresource technology, 100, 6028-6032.
260
Kim, Y. T., Kim, S. E., Lee, W. J., Fumei, Z., Cho, M. S., Moon, J. S., Oh, H.-W., Park, H.-Y. &
Kim, S. U. 2020c. Isolation and characterization of a high iturin yielding Bacillus
velezensis UV mutant with improved antifungal activity. Plos one, 15, e0234177.
Kinjo, Y., Takahashi, M., Hirose, N., Mizu, M., Hou, D.-X. & Wada, K. 2019. Anti-stress and
Antioxidant Effects of Non Centrifuged Cane Sugar, Kokuto, in RestraintStressed
Mice. Journal of oleo science, ess18198.
Kiran, G. S., Priyadharsini, S., Sajayan, A., Priyadharsini, G. B., Poulose, N. & Selvin, J. 2017.
Production of lipopeptide biosurfactant by a marine Nesterenkonia sp. and its
application in food industry. Frontiers in microbiology, 8, 1138.
Kiran, G. S., Thomas, T. A., Selvin, J., Sabarathnam, B. & Lipton, A. 2010. Optimization and
characterization of a new lipopeptide biosurfactant produced by marine
Brevibacterium aureum MSA13 in solid state culture. Bioresource technology, 101,
2389-2396.
Kirby, S. M., Anna, S. & Walker, L. 2017. Effect of surfactant tail length and ionic strength
on the interfacial properties of nanoparticle–surfactant complexes. Soft Matter,
10.1039.C7SM01806A.
Kitamoto, D., Morita, T., Fukuoka, T., Konishi, M.-a. & Imura, T. 2009. Self-assembling
properties of glycolipid biosurfactants and their potential applications. Current
Opinion in Colloid & Interface Science, 14, 315-328.
Koneva, A., Safonova, E., Kondrakhina, P., Vovk, M., Lezov, A., Chernyshev, Y. S. &
Smirnova, N. 2017. Effect of water content on structural and phase behavior of
water-in-oil (n-decane) microemulsion system stabilized by mixed nonionic
surfactants SPAN 80/TWEEN 80. Colloids and Surfaces A: Physicochemical and
Konishi, M. & Makino, M. 2018. Selective production of deacetylated mannosylerythritol
lipid, MEL-D, by acetyltransferase disruption mutant of Pseudozyma hubeiensis.
Journal of bioscience and bioengineering, 125, 105-110.
Konishi, M., Morita, T., Fukuoka, T., Imura, T., Uemura, S., Iwabuchi, H. & Kitamoto, D.
2018. Efficient production of acid-form sophorolipids from waste glycerol and
fatty acid methyl esters by Candida floricola. Journal of oleo science, 67, 489-496.
Kónya, M., Sorrenti, M., Ferrari, F., Rossi, S., Csóka, I., Caramella, C., Bettinetti, G. & Erős,
I. 2003. Study of the microstructure of o/w creams with thermal and rheological
methods. Journal of thermal analysis and calorimetry, 73, 623-632.
Korhonen, M., Lehtonen, J., Hellen, L., Hirvonen, J. & Yliruusi, J. 2002. Rheological
properties of three component creams containing sorbitan monoesters as
surfactants. International journal of pharmaceutics, 247, 103-114.
Koroleva, M., Tokarev, A. & Yurtov, E. 2015. Simulation of flocculation in W/O emulsions
and experimental study. Colloids and Surfaces A: Physicochemical and
Krasinski, A. 2013. A numerical model of droplets coalescence and drainage in fibrous
structures. Chem. Eng. Trans, 32, 1495.
Kreling, N., Zaparoli, M., Margarites, A., Friedrich, M., Thom é , A. & Colla, L. 2020.
Extracellular biosurfactants from yeast and soil–biodiesel interactions during
bioremediation. International Journal of Environmental Science and Technology,
17, 395-408.
Kügler, J. H., Muhle-Goll, C., Kühl, B., Kraft, A., Heinzler, R., Kirschhöfer, F., Henkel, M.,
Wray, V., Luy, B. & Brenner-Weiss, G. 2014. Trehalose lipid biosurfactants
produced by the actinomycetes Tsukamurella spumae and T. pseudospumae.
Applied microbiology and biotechnology, 98, 8905-8915.
261
Kukla, C., Duretek, I. & Holzer, C. Controlled shear stress method to measure yield stress
of highly filled polymer melts. AIP Conference Proceedings, 2016. AIP Publishing
LLC, 070006.
Kulik, V. & Boiko, A. 2018. Physical Principles of Methods for Measuring Viscoelastic
Properties. Journal of Applied Mechanics and Technical Physics, 59, 874-885.
Kulkarni, C. V. 2016. Lipid self-assemblies and nanostructured emulsions for cosmetic
formulations. Cosmetics, 3, 37.
Kumar, Sunil, Mandal, Ajay, Kar & Shranish 2016. The Synergistic Effect of a Mixed
Surfactant (Tween 80 and SDBS) on Wettability Alteration of the Oil Wet Quartz
Surface. Journal of Dispersion Science & Technology.
Kumari, R., Kakati, A., Nagarajan, R. & Sangwai, J. S. 2018. Synergistic effect of mixed
anionic and cationic surfactant systems on the interfacial tension of crude oil-
water and enhanced oil recovery. Journal of Dispersion ence and Technology, 1-
13.
Kuyukina, M. S., Ivshina, I. B., Baeva, T. A., Kochina, O. A., Gein, S. V. & Chereshnev, V. A.
2015. Trehalolipid biosurfactants from nonpathogenic Rhodococcus
actinobacteria with diverse immunomodulatory activities. New biotechnology, 32,
559-568.
Kwak, M.-S., Ahn, H.-J. & Song, K.-W. 2015. Rheological investigation of body cream and
body lotion in actual application conditions. Korea-Australia Rheology Journal, 27,
241-251.
Lade, R., Wasewar, K., Sangtyani, R., Kumar, A., Shende, D. & Peshwe, D. 2019. Dynamic
shear rheology of nanocomposite propellant suspension. Emerging Materials
Research, 8, 258-264.
Lal, S., Ratna, S., Said, O. B. & Kumar, R. 2018. Biosurfactant and exopolysaccharide-
assisted rhizobacterial technique for the remediation of heavy metal
contaminated soil: An advancement in metal phytoremediation technology.
Environmental Technology & Innovation, 10, 243-263.
Langenbucher, F. & Lange, B. 1970. Prediction of the application behaviour of cosmetics
from rheological measurements. Pharm Acta Helv, 45, 572-82.
Langley, C. A. & Belcher, D. 2012. Pharmaceutical Compounding and Dispensing,
Pharmaceutical Press.
Laville, R., Cicchetti, E., Garnier, S., Duroure, L., Manzone, G. & Borsotto, J. 2020. Plant
extract comprising sucrose esters as an active agent for use in cosmetic,
dermatological or nutricosmetic composition. Google Patents.
Le Roes-Hill, M., Durrell, K. A. & Kügler, J. H. 2019. Biosurfactants from Actinobacteria:
State of the Art and Future Perspectives. Microbial Biosurfactants and their
Environmental and Industrial Applications, 174-208.
Leal-Calderon, F., Schmitt, V. & Bibette, J. 2007. Emulsion science: basic principles,
Springer Science & Business Media.
Lee, B.-B., Chan, E.-S., Ravindra, P. & Khan, T. A. 2012. Surface tension of viscous
biopolymer solutions measured using the du Nouy ring method and the drop
weight methods. Polymer bulletin, 69, 471-489.
Li, J., Li, H., Li, W., Xia, C. & Song, X. 2016. Identification and characterization of a flavin-
containing monooxygenase MoA and its function in a specific sophorolipid
molecule metabolism in Starmerella bombicola. Applied microbiology and
Li, P. & Ishiguro, M. 2016. Adsorption of anionic surfactant (sodium dodecyl sulfate) on
silica. Soil science and plant nutrition, 62, 223-229.
262
Li, T., Wang, Y. & Dong, Y. 2012. Effect of solid contents on the controlled shear stress
rheological properties of different types of sludge. Journal of Environmental
Sciences, 24, 1917-1922.
Li, Y., Hu, J., Liu, H., Zhou, C. & Tian, S. 2020. Electrochemically reversible foam enhanced
flushing for PAHs-contaminated soil: Stability of surfactant foam, effects of soil
factors, and surfactant reversible recovery. Chemosphere, 260.
Li, Y., Hu, X., Liu, X., Zhang, Y. & Tian, S. 2017. Adsorption behavior of phenol by reversible
surfactant-modified montmorillonite: Mechanism, thermodynamics, and
regeneration. Chemical Engineering Journal, 334.
Li, Z., Dai, L., Wang, D., Mao, L. & Gao, Y. 2018. Stabilization and rheology of concentrated
emulsions using the natural emulsifiers quillaja saponins and rhamnolipids.
Journal of agricultural and food chemistry, 66, 3922-3929.
Lian, J., Zheng, S., Liu, C., Xu, Z. & Ruan, X. 2019. Investigation of microfluidic co-flow
effects on step emulsification: Wall contact angle and critical dimensions. Colloids
and Surfaces A: Physicochemical and Engineering Aspects, 580, 123733.
Liang, X., Zhang, M., Guo, C., Abel, S., Yi, X., Lu, G., Yang, C. & Dang, Z. 2014. Competitive
solubilization of low-molecular-weight polycyclic aromatic hydrocarbons
mixtures in single and binary surfactant micelles. Chemical Engineering Journal,
244, 522-530.
Lima, F. A., Santos, O. S., Pomella, A. W. V., Ribeiro, E. J. & de Resende, M. M. 2020. Culture
Medium Evaluation Using Low‐Cost Substrate for Biosurfactants Lipopeptides
Production by Bacillus amyloliquefaciens in Pilot Bioreactor. Journal of
Surfactants and Detergents, 23, 91-98.
Lin, W., Guo, C., Zhang, H., Liang, X., Wei, Y., Lu, G. & Dang, Z. 2016a. Electrokinetic-
enhanced remediation of phenanthrene-contaminated soil combined with
Sphingomonas sp. GY2B and biosurfactant. Applied biochemistry and
Lin, X., Qiu, X., Lou, H., Li, Z., Zhan, N., Huang, J. & Pang, Y. 2016b. Enhancement of
lignosulfonate-based polyoxyethylene ether on enzymatic hydrolysis of
lignocelluloses. Industrial Crops and Products, 80, 86-92.
Liu, C., Zhang, Y., Sun, S., Huang, L., Yu, L., Liu, X., Lai, R., Luo, Y., Zhang, Z. & Zhang, Z. 2018.
Oil recovery from tank bottom sludge using rhamnolipids. Journal of Petroleum
Science and Engineering, 170, 14-20.
Liu, F., Zhu, Z., Ma, C., Luo, X., Bai, L., Decker, E. A., Gao, Y. & McClements, D. J. 2016.
Fabrication of concentrated fish oil emulsions using dual-channel
microfluidization: Impact of droplet concentration on physical properties and lipid
oxidation. Journal of agricultural and food chemistry, 64, 9532-9541.
Liu, H.-f., Xu, J.-y., Wu, Y.-x. & Zheng, Z.-c. 2010. Numerical study on oil and water two-
phase flow in a cylindrical cyclone. Journal of Hydrodynamics, Ser. B, 22, 832-837.
Liu, S. & Li, D. 1999. Drop coalescence in turbulent dispersions. Chemical Engineering
Science, 54, 5667-5675.
Liu, X., Shu, Q., Chen, Q., Pang, X., Wu, Y., Zhou, W., Wu, Y., Niu, J. & Zhang, X. 2020.
Antibacterial Efficacy and Mechanism of Mannosylerythritol Lipids-A on Listeria
monocytogenes. Molecules, 25, 4857.
Liu, Y., Wu, Z., Zhao, B., Li, L. & Li, R. 2013. Enhancing defoaming using the foam breaker
with perforated plates for promoting the application of foam fractionation.
Separation and Purification Technology, 120, 12-19.
Louhasakul, Y., Cheirsilp, B., Intasit, R., Maneerat, S. & Saimmai, A. 2020. Enhanced
valorization of industrial wastes for biodiesel feedstocks and biocatalyst by
lipolytic oleaginous yeast and biosurfactant-producing bacteria. International
Biodeterioration & Biodegradation, 148, 104911.
263
Luan, A., Zielins, E. R., Wearda, T., Atashroo, D. A., Blackshear, C. P., Raphel, J., Brett, E. A.,
Flacco, J., Alyono, M. C. & Momeni, A. 2017. Dynamic rheology for the prediction
of surgical outcomes in autologous fat grafting. Plastic and Reconstructive Surgery,
140, 517-524.
Lukic, M., Pantelic, I. & Savic, S. 2016. An Overview of Novel Surfactants for Formulation
of Cosmetics with Certain Emphasis on Acidic Active Substances. Tenside
Surfactants Detergents, 53, 7-19.
Lydon, H. L., Baccile, N., Callaghan, B., Marchant, R., Mitchell, C. A. & Banat, I. M. 2017.
Adjuvant antibiotic activity of acidic sophorolipids with potential for facilitating
wound healing. Antimicrobial agents and chemotherapy, 61.
Ma, X.-j., Li, H., Shao, L.-j., Shen, J. & Song, X. 2011. Effects of nitrogen sources on
production and composition of sophorolipids by Wickerhamiella domercqiae var.
sophorolipid CGMCC 1576. Applied microbiology and biotechnology, 91, 1623-
1632.
Ma, X.-j., Li, H., Wang, D.-x. & Song, X. 2014. Sophorolipid production from delignined
corncob residue by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576
and Cryptococcus curvatus ATCC 96219. Applied microbiology and biotechnology,
98, 475-483.
Ma, X., Meng, L., Zhang, H., Zhou, L., Yue, J., Zhu, H. & Yao, R. 2020. Sophorolipid
biosynthesis and production from diverse hydrophilic and hydrophobic carbon
substrates. Applied Microbiology and Biotechnology, 104, 77-100.
Maazouz, A. 2020. Rheology and Flow of Complex Fluids: Fundamental and Experimental
Aspects. Available at SSRN 3639136.
Madihalli, C. & Doble, M. 2019. Microbial Production and Applications of
Mannosylerythritol, Cellobiose and Trehalose Lipids. Microbial Biosurfactants and
their Environmental and Industrial Applications. CRC Press.
Madihalli, C., Sudhakar, H. & Doble, M. 2020. Production and investigation of the physico-
chemical properties of MEL-A from glycerol and coconut water. World Journal of
Microbiology and Biotechnology, 36, 1-11.
Mahaut, F., Chateau, X., Coussot, P. & Ovarlez, G. 2008. Yield stress and elastic modulus
of suspensions of noncolloidal particles in yield stress fluids. Journal of Rheology,
52, 287-313.
Makkar, R. S., Cameotra, S. S. & Banat, I. M. 2011. Advances in utilization of renewable
substrates for biosurfactant production. AMB express, 1, 5.
Malkin, A. Y. 2013. Non-Newtonian viscosity in steady-state shear flows. Journal of Non-
Newtonian Fluid Mechanics, 192, 48-65.
Mandal, A., Kar, S. & Kumar, S. 2016. The synergistic effect of a mixed surfactant (Tween
80 and SDBS) on wettability alteration of the oil wet quartz surface. Journal of
Dispersion Science and Technology, 37, 1268-1276.
Mangal, S. & Sharma, V. 2017. Multi-parameter optimization of magnetorheological fluid
with high on-state yield stress and viscosity. Journal of The Brazilian Society Of
Mechanical Sciences And Engineering, 39, 4191-4206.
Manohar, C. & Narayanan, J. 2012. Average packing factor approach for designing micelles,
vesicles and gel phases in mixed surfactant systems. Colloids and Surfaces A:
Physicochemical and Engineering Aspects, 403, 129-132.
Marcelino, P. R. F., Gonçalves, F., Jimenez, I. M., Carneiro, B. C., Santos, B. B. & da Silva, S.
S. 2020. Sustainable Production of Biosurfactants and Their Applications.
Lignocellulosic Biorefining Technologies, 159-183.
Marquez, R., Forgiarini, A. M., Langevin, D. & Salager, J.-L. 2018. Instability of emulsions
made with surfactant–oil–water systems at optimum formulation with ultralow
interfacial tension. Langmuir, 34, 9252-9263.
264
Maxwell, R., Costache, M., Giarrosso, A., Bosques, C. & Amin, S. 2020. Optimizing
Interactions Between Soluble Silk Fibroin and Capryl Glucoside for Design of a
Natural and High‐Performance Co‐surfactant System. International Journal of
Cosmetic Science.
McClements, D. & Coupland, J. 1996. Theory of droplet size distribution measurements in
emulsions using ultrasonic spectroscopy. Colloids and Surfaces A:
McClements, D. J. & Gumus, C. E. 2016. Natural emulsifiers — Biosurfactants,
phospholipids, biopolymers, and colloidal particles: Molecular and
physicochemical basis of functional performance. Advances in Colloid & Interface
Science, 3-26.
McLafferty, F. W. 2012. Mass spectrometry of organic ions, Elsevier.
Merghni, A., Dallel, I., Noumi, E., Kadmi, Y., Hentati, H., Tobji, S., Amor, A. B. & Mastouri,
M. 2017. Antioxidant and antiproliferative potential of biosurfactants isolated
from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus
aureus strains. Microbial pathogenesis, 104, 84-89.
Metian, M., Renaud, F., Oberhänsli, F., Teyssié, J.-L., Temara, A. & Warnau, M. 2019.
Biokinetics of the anionic surfactant linear alkylbenzene sulfonate (LAS) in the
marine fish Sparus aurata: Investigation via seawater and food exposure
pathways. Aquatic Toxicology, 216, 105316.
Mezger, T. 2020. The rheology handbook: for users of rotational and oscillatory
rheometers, European Coatings.
Mezger, T. G. 2006. The rheology handbook: for users of rotational and oscillatory
rheometers, Vincentz Network GmbH & Co KG.
Minucelli, T., Ribeiro-Viana, R. M., Borsato, D., Andrade, G., Cely, M. V. T., de Oliveira, M.
R., Baldo, C. & Celligoi, M. A. P. C. 2017. Sophorolipids production by Candida
bombicola ATCC 22214 and its potential application in soil bioremediation. Waste
and biomass valorization, 8, 743-753.
Mitrinova, Z., Tcholakova, S. & Denkov, N. 2018. Control of surfactant solution rheology
using medium-chain cosurfactants. Colloids and Surfaces A: Physicochemical and
Mitru, D., Lucaciu, I., Nita-Lazar, M., Stoica, C., Gheorghe, S., Banciu, A. & Ionica, D. 2020.
Evaluation of surfactant removal efficiency in different municipal WWTPs in
Romania.
Mittal, K. L. & Shah, D. O. 2013. Surfactants in solution, Springer Science & Business Media.
Mnif, I., Ellouze-Chaabouni, S. & Ghribi, D. 2013. Optimization of inocula conditions for
enhanced biosurfactant production by Bacillus subtilis SPB1, in submerged
culture, using Box–Behnken design. Probiotics and antimicrobial proteins, 5, 92-
98.
Moens, K., Tzompa-Sosa, D. A., Van de Walle, D., Van der Meeren, P. & Dewettinck, K.
2019. Influence of cooling rate on partial coalescence in natural dairy cream. Food
research international, 120, 819-828.
Moravkova, T. & Stern, P. 2011. Rheological and textural properties of cosmetic emulsions.
Applied rheology, 21.
Moresi, M., Presti, S. L. & Mancini, M. 2001. Rheology of scleroglucan dispersions. Journal
of food engineering, 50, 235-245.
Morillas, J. R. & de Vicente, J. 2019. Yielding behavior of model magnetorheological fluids.
Soft matter, 15, 3330-3342.
Morita, T., Fukuoka, T., Imura, T. & Kitamoto, D. 2013. Production of mannosylerythritol
lipids and their application in cosmetics. Applied microbiology and biotechnology,
97, 4691-4700.
265
Morita, T., Fukuoka, T., Imura, T. & Kitamoto, D. 2015. Mannosylerythritol lipids:
production and applications. Journal of Oleo Science, 64, 133-141.
Morita, T., Fukuoka, T., Konishi, M., Imura, T., Yamamoto, S., Kitagawa, M., Sogabe, A. &
Kitamoto, D. 2009a. Production of a novel glycolipid biosurfactant,
mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial
properties. Applied microbiology and biotechnology, 83, 1017.
Morita, T., Ishibashi, Y., Fukuoka, T., Imura, T., Sakai, H., Abe, M. & Kitamoto, D. 2009b.
Production of glycolipid biosurfactants, mannosylerythritol lipids, by a smut
fungus, Ustilago scitaminea NBRC 32730. Bioscience, biotechnology, and
biochemistry, 73, 788-792.
Morita, T., Ishibashi, Y., Hirose, N., Wada, K., Takahashi, M., Fukuoka, T., Imura, T., Sakai,
H., Abe, M. & Kitamoto, D. 2011a. Production and characterization of a glycolipid
biosurfactant, mannosylerythritol lipid B, from sugarcane juice by Ustilago
scitaminea NBRC 32730. Bioscience, biotechnology, and biochemistry, 75, 1371-
1376.
Morita, T., Kitagawa, M., Yamamoto, S., Sogabe, A., Imura, T., Fukuoka, T. & Kitamoto, D.
2010. Glycolipid biosurfactants, mannosylerythritol lipids, repair the damaged
hair. Journal of Oleo Science, 59, 267-272.
Morita, T., Ogura, Y., Takashima, M., Hirose, N., Fukuoka, T., Imura, T., Kondo, Y. &
Kitamoto, D. 2011b. Isolation of Pseudozyma churashimaensis sp. nov., a novel
ustilaginomycetous yeast species as a producer of glycolipid biosurfactants,
mannosylerythritol lipids. Journal of bioscience and bioengineering, 112, 137-144.
Morris, S. A., Ananthapadmanabhan, K. P. & Kasting, G. B. 2019a. Anionic Surfactant–
Induced Changes in Skin Permeability. Journal of pharmaceutical sciences, 108,
3640-3648.
Morris, S. A., Thompson, R. T., Glenn, R. W., Ananthapadmanabhan, K. & Kasting, G. B.
2019b. Mechanisms of anionic surfactant penetration into human skin:
Investigating monomer, micelle and submicellar aggregate penetration theories.
International journal of cosmetic science, 41, 55-66.
Mostafa, N. A., Tayeb, A. M., Mohamed, O. A. & Farouq, R. 2019. Biodegradation of
Petroleum Oil Effluents and Production of Biosurfactants: Effect of Initial Oil
Concentration. Journal of Surfactants and Detergents, 22, 385-394.
Mota, T. R., Oliveira, D. M., Morais, G. R., Marchiosi, R., Buckeridge, M. S., Ferrarese-Filho,
O. & dos Santos, W. D. 2019. Hydrogen peroxide-acetic acid pretreatment
increases the saccharification and enzyme adsorption on lignocellulose. Industrial
Crops and Products, 140, 111657.
Moutinho, L. F., Moura, F. R., Silvestre, R. C. & Romão‐Dumaresq, A. S. 2020. Microbial
biosurfactants: A broad analysis of properties, applications, biosynthesis, and
techno ‐ economical assessment of rhamnolipid production. Biotechnology
Progress, e3093.
Mujumdar, S., Bashetti, S., Pardeshi, S. & Thombre, R. S. 2017. Industrial applications of
biosurfactants. Industrial biotechnology: sustainable production and bioresource
utilization. CRC Press.
Mukherjee, S., Das, S. S., Dhar, J., Chakraborty, S. & DasGupta, S. 2017. Electroosmosis of
viscoelastic fluids: Role of wall depletion layer. Langmuir, 33, 12046-12055.
Mukhopadhyay, S., Mukherjee, S., Hashim, M. A. & Sen Gupta, B. 2015. Application of
colloidal gas aphron suspensions produced from Sapindus mukorossi for arsenic
removal from contaminated soil. Chemosphere, 119, 355-362.
Mulligan, C. N. 2005. Environmental applications for biosurfactants. Environmental
pollution, 133, 183-198.
266
Naeeni, S. K. & Pakzad, L. 2019. Droplet size distribution and mixing hydrodynamics in a
liquid–liquid stirred tank by CFD modeling. International Journal of Multiphase
Flow, 120, 103100.
Naghash, A. & Nezamzadeh-Ejhieh, A. 2015. Comparison of the efficiency of modified
clinoptilolite with HDTMA and HDP surfactants for the removal of phosphate in
aqueous solutions. Journal of Industrial & Engineering Chemistry, 31, 185-191.
Najmi, Z., Ebrahimipour, G., Franzetti, A. & Banat, I. M. 2018. In situ downstream
strategies for cost‐effective bio/surfactant recovery. Biotechnology and applied
biochemistry, 65, 523-532.
Nakarapanich, J., Barameesangpet, T., Suksamranchit, S., Sirivat, A. & Jamieson, A. 2001.
Rheological properties and structures of cationic surfactants and fatty alcohol
emulsions: effect of surfactant chain length and concentration. Colloid and
Polymer Science, 279, 671-677.
Nakata, K., Tsuchido, T. & Matsumura, Y. 2011. Antimicrobial cationic surfactant,
cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli
cells. Journal of applied microbiology, 110, 568-579.
Nashida, J., Nishi, N., Takahashi, Y., Hayashi, C., Igarashi, M., Takahashi, D. & Toshima, K.
2018. Systematic and stereoselective total synthesis of mannosylerythritol lipids
and evaluation of their antibacterial activity. The Journal of organic chemistry, 83,
7281-7289.
Nataraj, B. H., Ali, S. A., Behare, P. V. & Yadav, H. 2020. Postbiotics-parabiotics: the new
horizons in microbial biotherapy and functional foods. Microbial cell factories, 19,
1-22.
Nayarisseri, A., Singh, P. & Singh, S. K. 2018. Screening, isolation and characterization of
biosurfactant producing Bacillus subtilis strain ANSKLAB03. Bioinformation, 14,
304.
Ndlovu, T., Khan, S. & Khan, W. 2016. Distribution and diversity of biosurfactant-producing
bacteria in a wastewater treatment plant. Environmental Science and Pollution
Research, 23, 9993-10004.
Ndlovu, T., Rautenbach, M., Vosloo, J. A., Khan, S. & Khan, W. 2017. Characterisation and
antimicrobial activity of biosurfactant extracts produced by Bacillus
amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater
treatment plant. AMB Express, 7, 108.
Nelson, A. Z. & Ewoldt, R. H. 2017. Design of yield-stress fluids: A rheology-to-structure
inverse problem. Soft Matter, 13, 7578-7594.
Neumann, S., van der Schaaf, U. & Karbstein, H. 2018. Investigations on the relationship
between interfacial and single droplet experiments to describe instability
mechanisms in double emulsions. Colloids and Surfaces A: Physicochemical and
Nguyen, Q. T., Di Benedetto, H. & Sauzéat, C. 2015. Linear and nonlinear viscoelastic
behaviour of bituminous mixtures. Materials and Structures, 48, 2339-2351.
Nguyen, T. T., Edelen, A., Neighbors, B. & Sabatini, D. A. 2010. Biocompatible lecithin-
based microemulsions with rhamnolipid and sophorolipid biosurfactants:
formulation and potential applications. Journal of colloid and interface science,
348, 498-504.
Nirosha, M., Badarinath, B. A., Hyndavi, M., Prasad, K., Naveen, N. & Kumar, A. 2016.
Recent advances in self-emulsifying drug delivery system. International Journal of
Research in Phytochemistry and Pharmacology, 6, 9-17.
Nitschke, M. & Pastore, G. M. 2006. Production and properties of a surfactant obtained
from Bacillus subtilis grown on cassava wastewater. Bioresource technology, 97,
336-341.
267
Niu, Y., Fan, L., Gu, D., Wu, J. & Chen, Q. 2017. Characterization, enhancement and
modelling of mannosylerythritol lipid production by fungal endophyte Ceriporia
lacerate CHZJU. Food chemistry, 228, 610-617.
Niu, Y., Wu, J., Wang, W. & Chen, Q. 2019. Production and characterization of a new
glycolipid, mannosylerythritol lipid, from waste cooking oil biotransformation by
Pseudozyma aphidis ZJUDM34. Food science & nutrition, 7, 937-948.
Nwaguma, I., Chikere, C. & Okpokwasili, G. 2019. Isolation and Molecular Characterization
of Biosurfactant-Producing Yeasts from Saps of Elaeis guineensis and Raphia
africana. Microbiology Research Journal International, 1-12.
Ohadi, M., Shahravan, A., Dehghannoudeh, N., Eslaminejad, T., Banat, I. M. &
Dehghannoudeh, G. 2020. Potential use of microbial surfactant in microemulsion
drug delivery system: a systematic review. Drug Design, Development and
Therapy, 14, 541.
Okamoto, T., Tomomasa, S. & Nakajima, H. 2016. Preparation and thermal properties of
fatty alcohol/surfactant/oil/water nanoemulsions and their cosmetic applications.
Journal of oleo science, 65, 27-36.
Olasanmi, I. O. & Thring, R. W. 2018. The Role of Biosurfactants in the Continued Drive for
Environmental Sustainability. Sustainability, 10.
Ong, A. S. Y. 2017. Characterisation of Biosurfactant Produced by Bacteria from
Environmental Isolates. INTI International University.
Onwosi, C. O., Aliyu, G. O., Onu, C. J., Chukwu, K. O., Ndukwe, J. K. & Igbokwe, V. C. 2020.
Microbial-derived glycolipids in the sustainable formulation of biomedical and
personal care products: A consideration of the process economics towards
commercialization. Process Biochemistry.
Oppong, F. K., Rubatat, L., Frisken, B. J., Bailey, A. E. & de Bruyn, J. R. 2006. Microrheology
and structure of a yield-stress polymer gel. Physical Review E, 73, 041405.
Otto, R. T., Daniel, H. J., Pekin, G., Muller-Decker, K., Furstenberger, G., Reuss, M. &
Syldatk, C. 1999. Production of sophorolipids from whey. II. Product composition,
surface active properties, cytotoxicity and stability against hydrolases by
enzymatic treatment. Appl Microbiol Biotechnol, 52, 495-501.
Ozkan, S., Alonso, C. & McMullen, R. L. 2020. Rheological fingerprinting as an effective
tool to guide development of personal care formulations. International Journal of
Cosmetic Science, 42, 536-547.
Pan, L., Zhang, F., Meng, R., Xu, J., Zuo, C. & Ge, B. 2016. Anomalous change of airy disk
with changing size of spherical particles. Journal of Quantitative Spectroscopy and
Radiative Transfer, 170, 83-89.
Pan, Y., Schubert, D. W., Ryu, J. E., Wujick, E., Liu, C., Shen, C. & Liu, X. 2018. Dynamic
oscillatory rheological properties of polystyrene/poly (methyl methacrylate)
blends and their composites in the presence of carbon black. Engineered Science,
1, 86-94.
Pandya, N., Rajput, G., Janni, D. S., Subramanyam, G., Ray, D., Aswal, V. & Varade, D. 2020.
SLES/CMEA mixed surfactant system: Effect of electrolyte on interfacial behavior
and microstructures in aqueous media. Journal of Molecular Liquids, 115096.
Parthipan, P., Preetham, E., Machuca, L. L., Rahman, P. K., Murugan, K. & Rajasekar, A.
2017. Biosurfactant and degradative enzymes mediated crude oil degradation by
bacterium Bacillus subtilis A1. Frontiers in microbiology, 8, 193.
Patel, R. P. & Joshi, J. R. 2012. An overview on nanoemulsion: a novel approach.
International Journal of Pharmaceutical Sciences and Research, 3, 4640.
Patel, S., Homaei, A., Patil, S. & Daverey, A. 2019. Microbial biosurfactants for oil spill
remediation: pitfalls and potentials. Applied microbiology and biotechnology, 103,
27-37.
268
Pati, S. G. & Arnold, W. A. 2020. Comprehensive screening of quaternary ammonium
surfactants and ionic liquids in wastewater effluents and lake sediments.
Environmental Science: Processes & Impacts, 22, 430-441.
Patil, H., Gadhave, A., Mane, S. & Waghmare, J. 2015. Analyzing the stability of the water-
in-diesel fuel emulsion. Journal of Dispersion Science and Technology, 36, 1221-
1227.
Patowary, R., Patowary, K., Kalita, M. C. & Deka, S. 2018. Application of biosurfactant for
enhancement of bioremediation process of crude oil contaminated soil.
International Biodeterioration & Biodegradation, 129, 50-60.
Peffly, M. M., Brown, M. A., Staudigel, J. A. & Zhang, J. J. 2016. Personal care compositions
containing cationic synthetic copolymer and a detersive surfactant. Google
Patents.
Pekin, G., Vardar‐Sukan, F. & Kosaric, N. 2005. Production of sophorolipids from Candida
bombicola ATCC 22214 using Turkish corn oil and honey. Engineering in Life
Sciences, 5, 357-362.
Pele, M. A., Ribeaux, D. R., Vieira, E. R., Souza, A. F., Luna, M. A., Rodríguez, D. M., Andrade,
R. F., Alviano, D. S., Alviano, C. S. & Barreto-Bergter, E. 2019. Conversion of
renewable substrates for biosurfactant production by Rhizopus arrhizus UCP 1607
and enhancing the removal of diesel oil from marine soil. Electronic Journal of
Biotechnology, 38, 40-48.
Pengon, S., Chinatangkul, N., Limmatvapirat, C. & Limmatvapirat, S. 2018. The effect of
surfactant on the physical properties of coconut oil nanoemulsions. Asian Journal
of Pharmaceutical Sciences, 13, 17-22.
Penkina, Y. A., Zolnikov, I. & Perminova, A. Interactions in binary mixtures of nonionic
gemini-surfactants surfynol® 400 series with sodium lauryl sulfate. Micellization
in the aqueous medium. AIP Conference Proceedings, 2020. AIP Publishing LLC,
050039.
Perazzo, A., Preziosi, V. & Guido, S. 2015. Phase inversion emulsification: Current
understanding and applications. Advances in colloid and interface science, 222,
581-599.
Pereira, J., Gudia, E., Dria, M., Domingues, M., Rodrigues, L., Teoxeira, J. & Coutinho, J.
2012. Characterization by electrospray ionization and tandem mass spectrometry
of rhamnolipids produced by two Pseudomonas aeruginosa strains isolated from
Brazilian crude oil. European Journal of Mass Spectrometry, 18, 399.
Perfumo, A., Banat, I. M. & Marchant, R. 2018. Going green and cold: biosurfactants from
low-temperature environments to biotechnology applications. Trends in
Perlekar, P., Biferale, L., Sbragaglia, M., Srivastava, S. & Toschi, F. 2012. Droplet size
distribution in homogeneous isotropic turbulence. Physics of Fluids, 24, 065101.
Petrovic, L. B., Sovilj, V. J., Katona, J. M. & Milanovic, J. L. 2010. Influence of polymer–
surfactant interactions on o/w emulsion properties and microcapsule formation.
Journal of Colloid and Interface Science, 342, 333-339.
Pham, H. T. T., Puig-Gamero, M., Sanchez-Silva, L., Sánchez, P. & Ramirez, A. A. 2018.
Future Market and Policy Initiatives of New High Value Products.
Pivsa-Art, W., Siraworakun, C. & Pivsa-Art, S. 2019. Improvement of Water Cooling System
for Oil in Water Cosmetic Cream Production Process. Journal of Chemical
Engineering of Japan, 52, 789-792.
Podgórska, W. 2006. Modelling of high viscosity oil drop breakage process in intermittent
turbulence. Chemical engineering science, 61, 2986-2993.
269
Poerwadi, B., Kartikowati, C. W., Oktavian, R. & Novareza, O. 2020. Manufacture of a
Hydrophobic Silica Nanoparticle Composite Membrane for Oil-Water Emulsion
Separation. International Journal of Technology, 11, 364-373.
Pooja, Singh, Yogesh, Patil, Vinaykumar & Rale 2018. Biosurfactant Production: Emerging
Trends and Promising Strategies. Journal of applied microbiology.
Porter, M. R. 2013. Handbook of surfactants, Springer.
Posocco, P., Perazzo, A., Preziosi, V., Laurini, E., Pricl, S. & Guido, S. 2016. Interfacial
tension of oil/water emulsions with mixed non-ionic surfactants: comparison
between experiments and molecular simulations. RSC advances, 6, 4723-4729.
Pouget, S., Sauzéat, C., Benedetto, H. D. & Olard, F. 2012. Viscous energy dissipation in
asphalt pavement structures and implication for vehicle fuel consumption.
Journal of Materials in Civil Engineering, 24, 568-576.
Preux, C., Malinouskaya, I., Nguyen, Q. L., Flauraud, E. & Ayache, S. 2020. Reservoir-
Simulation Model with Surfactant Flooding Including Salinity and Thermal Effect,
Using Laboratory Experiments. SPE Journal.
Pulate, V. D., Bhagwat, S. & Prabhune, A. 2013. Microbial oxidation of medium chain fatty
alcohol in the synthesis of sophorolipids by Candida bombicola and its
physicochemical characterization. Journal of Surfactants and Detergents, 16, 173-
181.
Purwasena, I. A., Astuti, D. I., Syukron, M., Amaniyah, M. & Sugai, Y. 2019. Stability test of
biosurfactant produced by Bacillus licheniformis DS1 using experimental design
and its application for MEOR. Journal of Petroleum Science and Engineering, 183,
106383.
Qian, C., Telmadarreie, A., Dong, M. & Bryant, S. 2020. Synergistic Effect between
Surfactant and Nanoparticles on the Stability of Foam in EOR Processes. SPE
Journal, 25.
Racheva, R., Rahlf, A. F., Wenzel, D., Müller, C., Kerner, M., Luinstra, G. A. & Smirnova, I.
2018. Aqueous food-grade and cosmetic-grade surfactant systems for the
continuous countercurrent cloud point extraction. Separation and Purification
Technology, 202, 76-85.
Rahman, K., Rahman, T. J., McClean, S., Marchant, R. & Banat, I. M. 2002. Rhamnolipid
Biosurfactant Production by Strains of Pseudomonas aeruginosa Using Low‐Cost
Raw Materials. Biotechnology progress, 18, 1277-1281.
Rai, A. K., Pandey, A. & Sahoo, D. 2019. Biotechnological potential of yeasts in functional
food industry. Trends in Food Science & Technology, 83, 129-137.
Ramadan, M. S., El-Mallah, N. M., Nabil, G. M. & Abd-Elmenem, S. M. 2018. Hydrophobic
effect for anionic dye-cationic surfactant interaction in aqueous and mixed
solvent. Journal of Dispersion Science and Technology.
Ran, G., Zhang, Y., Song, Q., Wang, Y. & Cao, D. 2009. The adsorption behavior of cationic
surfactant onto human hair fibers. Colloids and Surfaces B: Biointerfaces, 68, 106-
110.
Rau, U., Nguyen, L., Roeper, H., Koch, H. & Lang, S. 2005a. Fed-batch bioreactor
production of mannosylerythritol lipids secreted by Pseudozyma aphidis. Applied
Rau, U., Nguyen, L. A., Roeper, H., Koch, H. & Lang, S. 2005b. Downstream processing of
mannosylerythritol lipids produced by Pseudozyma aphidis. European journal of
lipid science and technology, 107, 373-380.
Recke, V. K., Beyrle, C., Gerlitzki, M., Hausmann, R., Syldatk, C., Wray, V., Tokuda, H.,
Suzuki, N. & Lang, S. 2013. Lipase-catalyzed acylation of microbial
mannosylerythritol lipids (biosurfactants) and their characterization.
270
Reddy, P. V., Karegoudar, T. & Nayak, A. S. 2018. Enhanced utilization of fluorene by
Paenibacillus sp. PRNK-6: Effect of rhamnolipid biosurfactant and synthetic
surfactants. Ecotoxicology and environmental safety, 151, 206-211.
Rehman, N., Ullah, H., Alam, S., Jan, A. K., Khan, S. & Tariq, M. 2017. Surface and
thermodynamic study of micellization of non ionic surfactant/diblock copolymer
system as revealed by surface tension and conductivity. JMES, 8, 1161-1167.
Ren, L., Yu, S., Li, J. & Li, L. 2019. Pilot study on the effects of operating parameters on
membrane fouling during ultrafiltration of alkali/surfactant/polymer flooding
wastewater: optimization and modeling. Rsc Advances, 9.
Ren, Z. H. 2017. A molecular‐thermodynamic approach to predict the micellization of
binary surfactant mixtures containing amino sulfonate amphoteric surfactant and
nonionic surfactant. AIChE Journal, 63, 5076-5082.
Ren, Z. H., Huang, J., Zheng, Y. C., Lai, L. & Hu, L. L. 2017. Interaction and micellar behavior
of binary mixture of amino sulfonate amphoteric surfactant with
octadecyltrimethylammonium bromide in aqueous solutions of NaCl. Journal of
Chemical & Engineering Data, 62, 1782-1787.
Resende, A. H. M., Farias, J. M., Silva, D. D. B., Rufino, R. D., Luna, J. M., Stamford, T. C. M.
& Sarubbo, L. A. 2019. Application of biosurfactants and chitosan in toothpaste
formulation. Colloids and Surfaces B: Biointerfaces, 181, 77-84.
Rhein, L. D. 2017. In vitro interactions: biochemical and biophysical effects of surfactants
on skin. Surfactants in cosmetics. Routledge.
Ribeiro, B. G., Dos Santos, M. M., Pinto, M. I. S., Meira, H. M., Durval, I. B., Guerra, J. M. C.
& Sarubbo, L. 2019. Production and optimization of the extraction conditions of a
biosurfactant of Candida utilis UFPEDA1009 with potential of application in the
food industry. Chemical Engineering Transactions, 74, 1477-1482.
Ribeiro, B. G., Guerra, J. M. C. & Sarubbo, L. A. 2020. Potential Food Application of a
Biosurfactant Produced by Saccharomyces cerevisiae URM 6670. Frontiers in
Bioengineering and Biotechnology, 8.
Ribeiro, H., Morais, J. & Eccleston, G. 2004. Structure and rheology of semisolid o/w
creams containing cetyl alcohol/non ‐ ionic surfactant mixed emulsifier and
different polymers. International journal of cosmetic science, 26, 47-59.
Rincón-Fontán, M., Rodríguez-López, L., Vecino, X., Cruz, J. M. & Moldes, A. B. 2020.
Potential application of a multifunctional biosurfactant extract obtained from
corn as stabilizing agent of vitamin C in cosmetic formulations. Sustainable
Chemistry and Pharmacy, 16, 100248.
Ritter, E., Yordanova, D., Gerlach, T., Smirnova, I. & Jakobtorweihen, S. 2016. Molecular
dynamics simulations of various micelles to predict micelle water partition
equilibria with COSMOmic: influence of micelle size and structure. Fluid Phase
Equilibria, 422, 43-55.
Rodríguez-López, L., Shokry, D. S., Cruz, J. M., Moldes, A. B. & Waters, L. J. 2019. The effect
of the presence of biosurfactant on the permeation of pharmaceutical
compounds through silicone membrane. Colloids and Surfaces B: Biointerfaces,
176, 456-461.
Roelants, S., Solaiman, D. K., Ashby, R. D., Lodens, S., Van Renterghem, L. & Soetaert, W.
2019a. Production and applications of sophorolipids. Biobased Surfactants.
Elsevier.
Roelants, S. L. K. W., Renterghem, L. V., Maes, K., Everaert, B. & Soetaert, W. 2019b.
Microbial Biosurfactants: From Lab to Market.
Rønholt, S., Kirkensgaard, J. J. K., Mortensen, K. & Knudsen, J. C. 2014. Effect of cream
cooling rate and water content on butter microstructure during four weeks of
storage. Food Hydrocolloids, 34, 169-176.
271
Rønholt, S., Kirkensgaard, J. J. K., Pedersen, T. B., Mortensen, K. & Knudsen, J. C. 2012.
Polymorphism, microstructure and rheology of butter. Effects of cream heat
treatment. Food chemistry, 135, 1730-1739.
Rosenberg, E. & Ron, E. 1999. High-and low-molecular-mass microbial surfactants.
Applied Microbiology and Biotechnology, 52, 154-162.
Ruhaak, L. R., Xu, G., Li, Q., Goonatilleke, E. & Lebrilla, C. B. 2018. Mass spectrometry
approaches to glycomic and glycoproteomic analyses. Chemical reviews, 118,
7886-7930.
Saad, E. S., Nasser, A., El-Wahab, A., Hassan, W. A. & Elsayed, A. 2019. Effect of different
surfactant monomers on alkali soluble emulsion polymer as a binder for water
based printing inks. Egyptian Journal of Chemistry, 62, 63-76.
Saarinen, T., Haavisto, S., Sorvari, A., Salmela, J. & Seppälä, J. 2014. The effect of wall
depletion on the rheology of microfibrillated cellulose water suspensions by
optical coherence tomography. Cellulose, 21, 1261-1275.
Sáenz-Marta, C. I., de Lourdes Ballinas-Casarrubias, M., Rivera-Chavira, B. E. & Nevárez-
Moorillón, G. V. 2015. Biosurfactants as useful tools in bioremediation. Advances
in bioremediation of wastewater and polluted soil, 94-109.
Saerens, K. M., Zhang, J., Saey, L., Van Bogaert, I. N. & Soetaert, W. 2011. Cloning and
functional characterization of the UDP ‐glucosyltransferase UgtB1 involved in
sophorolipid production by Candida bombicola and creation of a glucolipid ‐
producing yeast strain. Yeast, 28, 279-292.
Saggese, A., Culurciello, R., Casillo, A., Corsaro, M. M., Ricca, E. & Baccigalupi, L. 2018. A
marine isolate of Bacillus pumilus secretes a pumilacidin active against
Staphylococcus aureus. Marine drugs, 16, 180.
Saika, A., Koike, H., Fukuoka, T. & Morita, T. 2018a. Tailor-made mannosylerythritol lipids:
current state and perspectives. Applied microbiology and biotechnology, 102,
6877-6884.
Saika, A., Koike, H., Yamamoto, S., Kishimoto, T. & Morita, T. 2017. Enhanced production
of a diastereomer type of mannosylerythritol lipid-B by the basidiomycetous
yeast Pseudozyma tsukubaensis expressing lipase genes from Pseudozyma
antarctica. Applied microbiology and biotechnology, 101, 8345-8352.
Saika, A., Utashima, Y., Koike, H., Yamamoto, S., Kishimoto, T., Fukuoka, T. & Morita, T.
2018b. Biosynthesis of mono-acylated mannosylerythritol lipid in an
acyltransferase gene-disrupted mutant of Pseudozyma tsukubaensis. Applied
Saika, A., Utashima, Y., Koike, H., Yamamoto, S., Kishimoto, T., Fukuoka, T. & Morita, T.
2018c. Identification of the gene PtMAT1 encoding acetyltransferase from the
diastereomer type of mannosylerythritol lipid-B producer Pseudozyma
tsukubaensis. Journal of bioscience and bioengineering, 126, 676-681.
Sajna, K. V., Höfer, R., Sukumaran, R. K., Gottumukkala, L. D. & Pandey, A. 2015. White
biotechnology in biosurfactants. Industrial Biorefineries & White Biotechnology.
Elsevier.
Salehiyan, R., Ray, S. S., Stadler, F. J. & Ojijo, V. 2018. Rheology–microstructure
relationships in melt-processed polylactide/poly (vinylidene fluoride) blends.
Materials, 11, 2450.
Salehizadeh, H. & Mohammadizad, S. 2009. Microbial enhanced oil recovery using
biosurfactant produced by Alcaligenes faecalis. Iranian Journal of Biotechnology,
7, 216-223.
Sana, S., Datta, S., Biswas, D. & Sengupta, D. 2018. Assessment of synergistic antibacterial
activity of combined biosurfactants revealed by bacterial cell envelop damage.
Biochimica et Biophysica Acta (BBA)-Biomembranes, 1860, 579-585.
272
Sanchuki, H. B., Gravina, F., Rodrigues, T. E., Gerhardt, E. C., Pedrosa, F. O., Souza, E. M.,
Raittz, R. T., Valdameri, G., de Souza, G. A. & Huergo, L. F. 2017. Dynamics of the
Escherichia coli proteome in response to nitrogen starvation and entry into the
stationary phase. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics,
1865, 344-352.
Sandeep, L. & Rajasree, S. 2017. Biosurfactant: pharmaceutical perspective. J. Anal. Pharm.
Res, 4, 10.15406.
Santos, D. K. F., Meira, H. M., Rufino, R. D., Luna, J. M. & Sarubbo, L. A. 2017. Biosurfactant
production from Candida lipolytica in bioreactor and evaluation of its toxicity for
application as a bioremediation agent. Process Biochemistry, 54, 20-27.
Santos, J., Calero, N. & Muñoz, J. 2016. Optimization of a green emulsion stability by
tuning homogenization rate. RSC advances, 6, 57563-57568.
Santos, W. M., de Caldas Nobre, M. S., Cavalcanti, M. T., dos Santos, K. M. O., Salles, H. O.
& Alonso Buriti, F. C. 2019. Proteolysis of reconstituted goat whey fermented by
Streptococcus thermophilus in co ‐ culture with commercial probiotic
Lactobacillus strains. International Journal of Dairy Technology, 72, 559-568.
Sanz, T., Salvador, A. & Hernández, M. 2017. Creep–Recovery and Oscillatory Rheology of
Flour-Based Systems. Advances in Food Rheology and Its Applications. Elsevier.
Saravanan, A., Kumar, P. S., Vardhan, K. H., Jeevanantham, S., Karishma, S. B., Yaashikaa,
P. R. & Vellaichamy, P. 2020. A review on systematic approach for microbial
enhanced oil recovery technologies: opportunities and challenges. Journal of
Cleaner Production, 120777.
Satpute, S. K., Banpurkar, A. G., Dhakephalkar, P. K., Banat, I. M. & Chopade, B. A. 2010.
Methods for investigating biosurfactants and bioemulsifiers: a review. Critical
reviews in biotechnology, 30, 127-144.
Satpute, S. K., Płaza, G. A. & Banpurkar, A. G. 2017. Biosurfactants’ production from
renewable natural resources: example of innovativeand smart technology in
circular bioeconomy. Management Systems in Production Engineering, 25, 46-54.
Savic, S., Vuleta, G., Daniels, R. & Müller-Goymann, C. C. 2005. Colloidal microstructure of
binary systems and model creams stabilized with an alkylpolyglucoside non-ionic
emulsifier. Colloid and Polymer Science, 283, 439-451.
Schultz, J. & Rosado, A. S. 2020. Extreme environments: a source of biosurfactants for
biotechnological applications. Extremophiles, 24, 189-206.
Sen, S., Borah, S. N., Kandimalla, R., Bora, A. & Deka, S. 2020. Sophorolipid Biosurfactant
can control Cutaneous Dermatophytosis caused by Trichophyton
mentagrophytes. Frontiers in Microbiology, 11, 329.
Seweryn, A. 2018. Interactions between surfactants and the skin – Theory and practice.
Advances in Colloid and Interface Science, 256, 242-255.
Shah, M. U. H., Moniruzzaman, M., Sivapragasam, M., Talukder, M. M. R., Yusup, S. B. &
Goto, M. 2019. A binary mixture of a biosurfactant and an ionic liquid surfactant
as a green dispersant for oil spill remediation. Journal of Molecular Liquids, 280,
111-119.
Shah, M. U. H., Sivapragasam, M., Moniruzzaman, M., Talukder, M. M. R., Yusup, S. B. &
Goto, M. 2017. Production of sophorolipids by Starmerella bombicola yeast using
new hydrophobic substrates. Biochemical Engineering Journal, 127, 60-67.
Shah, N., Nikam, R., Gaikwad, S., Sapre, V. & Kaur, J. 2016. Biosurfactant: types, detection
methods, importance and applications. Indian Journal of Microbiology Research,
3, 5-10.
Sharma, A., Siddiqi, Z. M. & Pathania, D. 2017. Adsorption of polyaromatic pollutants from
water system using carbon/ZnFe2O4 nanocomposite: Equilibrium, kinetic and
thermodynamic mechanism. Journal of Molecular Liquids.
273
Sharma, R. K. 2014. Surfactants: Basics and Versatility in Food Industries. PharmaTutor, 2,
17-29.
Shen, L., Zhu, J., Lu, J., Gong, Q., Jin, M. & Long, X. 2019. Isolation and purification of
biosurfactant mannosylerythritol lipids from fermentation broth with
methanol/water/n-hexane. Separation and Purification Technology, 219, 1-8.
Sherman, P. 1968. Emulsion science, Academic Press.
Shibaev, A. V., Ospennikov, A. S., Kuklin, A. I., Arkharova, N. A. & Philippova, O. E. 2019.
Structure, Rheological and Responsive Properties of a New Mixed Viscoelastic
Surfactant System. Colloids and Surfaces A Physicochemical and Engineering
Aspects, 586, 124284.
Shin, J. D., Lee, J., Kim, Y. B., Han, I.-s. & Kim, E.-K. 2010. Production and characterization
of methyl ester sophorolipids with 22-carbon-fatty acids. Bioresource technology,
101, 3170-3174.
Shinoda, R. & Uchimura, T. 2018. Evaluating the creaming of an emulsion via mass
spectrometry and uv–vis spectrophotometry. ACS omega, 3, 13752-13756.
Shoeb, E., Akhlaq, F., Badar, U., Akhter, J. & Imtiaz, S. 2013. Classification and industrial
applications of biosurfactants. Academic Research International, 4, 243.
Shu, Q., Niu, Y., Zhao, W. & Chen, Q. 2019. Antibacterial activity and mannosylerythritol
lipids against vegetative cells and spores of Bacillus cereus. Food Control, 106,
106711.
Shu, Q., Wei, T., Lu, H., Niu, Y. & Chen, Q. 2020. Mannosylerythritol lipids: dual inhibitory
modes against Staphylococcus aureus through membrane-mediated apoptosis
and biofilm disruption. Applied Microbiology and Biotechnology, 1-12.
Shubair, A., Al-Salih, H., Sabouni, R., Gomaa, H., Hassanin, S., Salem, S., Zeno, T., El Taher,
B. & Zaka, A. 2020. Photocatalytic demulsification of oil/water emulsions
containing nonionic surfactant. Environmental Science and Pollution Research, 1-
9.
Siemes, E., Nevskyi, O., Sysoiev, D., Turnhoff, S. K., Oppermann, A., Huhn, T., Richtering,
W. & Wöll, D. 2018. Nanoscopic visualization of cross‐linking density in polymer
networks with diarylethene photoswitches. Angewandte Chemie International
Edition, 57, 12280-12284.
Silva, E. J., Silva, I. A., Brasileiro, P. P., Correa, P. F., Almeida, D. G., Rufino, R. D., Luna, J.
M., Santos, V. A. & Sarubbo, L. A. 2019a. Treatment of oily effluent using a low-
cost biosurfactant in a flotation system. Biodegradation, 30, 335-350.
Silva, M., Duvoisin Jr, S., Oliveira, R., Banhos, E., Souza, A. & Albuquerque, P. M. 2019b.
Biosurfactant production of Piper hispidum endophytic fungi. Journal of Applied
Microbiology.
Silva, R. d. C. F. S. d., Almeida, D. G. D., Brasileiro, P. P. F., Rufino, R. D., Luna, J. M. D. &
Sarubbo, L. A. 2019c. Production, formulation and cost estimation of a
commercial biosurfactant. Biodegradation.
Silva, S., Farias, C., Rufino, R., Luna, J. & Sarubbo, L. 2010. Glycerol as substrate for the
production of biosurfactant by Pseudomonas aeruginosa UCP0992. Colloids and
Surfaces B: Biointerfaces, 79, 174-183.
Singh, P., Patil, Y. & Rale, V. 2019. Biosurfactant production: emerging trends and
promising strategies. Journal of applied microbiology, 126, 2-13.
Singh, S. 2002. Refractive index measurement and its applications. Physica Scripta, 65,
167.
Sintang, M. D. B., Danthine, S., Patel, A. R., Rimaux, T. & Dewettinck, K. 2017. Mixed
surfactant systems of sucrose esters and lecithin as a synergistic approach for oil
structuring. J Colloid Interface, 504, 387-396.
274
Sobrinho, H., Luna, J. M., Rufino, R. D., Porto, A. & Sarubbo, L. A. 2013. Biosurfactants:
classification, properties and environmental applications. Recent developments in
Solaiman, D. K., Ashby, R. D., Zerkowski, J. A. & Foglia, T. A. 2007. Simplified soy molasses-
based medium for reduced-cost production of sophorolipids by Candida
bombicola. Biotechnology letters, 29, 1341-1347.
Solans, C., Morales, D. & Homs, M. 2016. Spontaneous emulsification. Current Opinion in
Colloid & Interface Science, 22, 88-93.
Song, Xiaoyong, Zuo, Guanjie, Chen & Fusheng 2018. Effect of essential oil and surfactant
on the physical and antimicrobial properties of corn and wheat starch films.
International Journal of Biological Macromolecules: Structure, Function and
Interactions.
Song, H. Y., Salehiyan, R., Li, X., Lee, S. H. & Hyun, K. 2017. A comparative study of the
effects of cone-plate and parallel-plate geometries on rheological properties
under oscillatory shear flow. Korea-Australia Rheology Journal, 29, 281-294.
Soukoulis, C., Behboudi-Jobbehdar, S., Macnaughtan, W., Parmenter, C. & Fisk, I. D. 2017.
Stability of Lactobacillus rhamnosus GG incorporated in edible films: Impact of
anionic biopolymers and whey protein concentrate. Food hydrocolloids, 70, 345-
355.
Souza, A. F., Rodriguez, D. M., Ribeaux, D. R., Luna, M. A., Lima e Silva, T. A., Andrade, R.
F. S., Gusmão, N. B. & Campos-Takaki, G. M. 2016. Waste soybean oil and corn
steep liquor as economic substrates for bioemulsifier and biodiesel production by
Candida lipolytica UCP 0998. International journal of molecular sciences, 17, 1608.
Souza, K. S. T., Gudiña, E. J., Schwan, R. F., Rodrigues, L. R., Dias, D. R. & Teixeira, J. A. 2018.
Improvement of biosurfactant production by Wickerhamomyces anomalus CCMA
0358 and its potential application in bioremediation. Journal of Hazardous
Materials, 346, 152-158.
Spina, F., Giulia, S., POLI, A., ROMAGNOLO, A., ZANELLATI, A., Bentivegna, N. G., Najoi, E.-
A., Tiffanie, R., Anne-Laure, B. & Abdelwahad, E. 2018. Screening of Anionic
Biosurfactants Production among Fungi and Bacteria.
Stan, F., Stanciu, N. V. & Fetecau, C. 2017. Melt rheological properties of ethylene-vinyl
acetate/multi-walled carbon nanotube composites. Composites Part B:
Engineering, 110, 20-31.
Strathclyde, G. G. 1990. Multiple-phase oil-in-water emulsions. J. Soc. Cosmet. Chem, 41,
1-22.
Sun, W., Zhu, B., Yang, F., Dai, M., Sehar, S., Peng, C., Ali, I. & Naz, I. 2020. Optimization of
biosurfactant production from Pseudomonas sp. CQ2 and its application for
remediation of heavy metal contaminated soil. Chemosphere, 129090.
Sun, Y., Mei, L., Han, N., Ding, X., Yu, C., Yang, W. & Ruan, G. 2017. Examining the roles of
emulsion droplet size and surfactant in the interfacial instability-based fabrication
process of micellar nanocrystals. Nanoscale research letters, 12, 434.
Tadros, T. F. 2009. Emulsion science and technology: a general introduction. Emulsion
science and technology, 1-56.
Tadros, T. F. 2013. Emulsion Formation, Stability, and Rheology. Emulsion Formation and
Stability. Wiley-VCH Verlag GmbH & Co. KGaA.
Tai, X.-m., Song, J.-y., Du, Z.-p., Liu, X., Wang, T. & Wang, G. 2018. The performance test
of fatty acid methyl ester sulfonates and application in the dishwashing liquid
detergent. Journal of Dispersion Science and Technology, 39, 1422-1426.
Tang, J., He, J., Liu, T., Xin, X. & Hu, H. 2017. Removal of heavy metal from sludge by the
combined application of a biodegradable biosurfactant and complexing agent in
enhanced electrokinetic treatment. Chemosphere, 189, 599-608.
275
Tang, Y., Ma, Q., Du, Y., Ren, L., Van Zyl, L. J. & Long, X. 2020. Efficient purification of
sophorolipids via chemical modifications coupled with extractions and their
potential applications as antibacterial agents. Separation and Purification
Technology, 116897.
Tao, Geng, Chunqiao, Zhang, Yajie, Jiang, Hongbin, Ju, Yakui & Wang 2017. Synergistic
effect of binary mixtures contained newly cationic surfactant: Interaction,
aggregation behaviors and application properties. Journal of Molecular Liquids.
Tatar, B. C., Sumnu, G. & Sahin, S. 2017. Rheology of Emulsions.
Terescenco, D., Picard, C., Clemenceau, F., Grisel, M. & Savary, G. 2018a. Influence of the
emollient structure on the properties of cosmetic emulsion containing lamellar
liquid crystals. Colloids and Surfaces A: Physicochemical and Engineering Aspects,
536, 10-19.
Terescenco, D., Savary, G., Clemenceau, F., Merat, E., Duchemin, B., Grisel, M. & Picard,
C. 2018b. The alkyl polyglucoside/fatty alcohol ratio effect on the formation of
liquid crystal phases in binary systems. Journal of Molecular Liquids, 253, 45-52.
Thavasi, R. & Banat, I. M. 2019a. Downstream Processing of Microbial Biosurfactants.
Thavasi, R. & Banat, I. M. 2019b. Introduction to Microbial Biosurfactants. Microbial
Biosurfactants and their Environmental and Industrial Applications, 1-15.
Theron, F., Le Sauze, N. & Ricard, A. 2010. Turbulent Liquid− Liquid Dispersion in Sulzer
SMX Mixer. Industrial & engineering chemistry research, 49, 623-632.
Tiwari, S., Mall, C. & Solanki, P. P. 2018. Surfactant and its applications: A review. Int. J.
Eng. Res. Afr., 8, 61-66.
Torres, A. d. C. L., de Lima, L. N., Tardioli, P. W. & de Sousa Júnior, R. 2020. Mathematical
modeling of enzymatic syntheses of biosurfactants catalyzed by immobilized
lipases. Reaction Kinetics, Mechanisms and Catalysis, 130, 699-712.
Trujillo-Cayado, L., Alfaro, M., Muñoz, J., Raymundo, A. & Sousa, I. 2016. Development
and rheological properties of ecological emulsions formulated with a biosolvent
and two microbial polysaccharides. Colloids and Surfaces B: Biointerfaces, 141,
53-58.
Tschoegl, N. W. 2012. The phenomenological theory of linear viscoelastic behavior: an
introduction, Springer Science & Business Media.
Tummino, A., Toscano, J., Sebastiani, F., Noskov, B. A., Varga, I. & Campbell, R. A. 2018.
Effects of Aggregate Charge and Subphase Ionic Strength on the Properties of
Spread Polyelectrolyte/Surfactant Films at the Air/Water Interface under Static
and Dynamic Conditions. Langmuir, acs.langmuir.7b03960.
Tzocheva, S. S., Danov, K. D., Kralchevsky, P. A., Georgieva, G. S., Post, A. J. &
Ananthapadmanabhan, K. P. 2015. Solubility limits and phase diagrams for fatty
alcohols in anionic (SLES) and zwitterionic (CAPB) micellar surfactant solutions.
Journal of colloid and interface science, 449, 46-61.
Udomrati, S., Ikeda, S. & Gohtani, S. 2013. Rheological properties and stability of oil-in-
water emulsions containing tapioca maltodextrin in the aqueous phase. Journal
of food engineering, 116, 170-175.
Uhl, V. 2012. Mixing V1: Theory And Practice, Elsevier.
Van Bogaert, I., Ciesielska, K., Devreese, B. & Soetaert, W. 2014. Microbial Synthesis and
Application. Biosurfactants: Production and Utilization-Processes, Technologies,
and Economics, 159, 19.
Van Bogaert, I., Develter, D., Soetaert, W. & Vandamme, E. 2008. Cerulenin inhibits de
novo sophorolipid synthesis of Candida bombicola. Biotechnology letters, 30,
1829-1832.
Van Bogaert, I. N., Zhang, J. & Soetaert, W. 2011. Microbial synthesis of sophorolipids.
Process Biochemistry, 46, 821-833.
276
van Hoogevest, P. & Fahr, A. 2019. Phospholipids in cosmetic carriers. Nanocosmetics.
Springer.
van Os, N. M., Haak, J. R. & Rupert, L. A. M. 2012. Physico-chemical properties of selected
anionic, cationic and nonionic surfactants, Elsevier.
Varjani, S. J. & Upasani, V. N. 2017. Critical review on biosurfactant analysis, purification
and characterization using rhamnolipid as a model biosurfactant. Bioresource
Technology, 232, 389.
Vecino, X., Ferreira, A., Cruz, J., Moldes, A. & Rodrigues, L. 2016. Essential oil-water
emulsions containing a biosurfactant from Lactobacillus paracasei.
Vecino, X., Rodríguez-López, L., Ferreira, D., Cruz, J. M., Moldes, A. B. & Rodrigues, L. R.
2017. Bioactivity of glycolipopeptide cell-bound biosurfactants against skin
pathogens. International Journal of Biological Macromolecules,
S0141813017337017.
Verboom, G. M. & Bauer, K. L. 2003. Hair conditioning composition. Google Patents.
Verma, R., Sharma, S., Kundu, L. M. & Pandey, L. M. 2020. Experimental investigation of
molasses as a sole nutrient for the production of an alternative metabolite
biosurfactant. Journal of Water Process Engineering, 38, 101632.
Vikhansky, A. 2020. CFD modelling of turbulent liquid–liquid dispersion in a static mixer.
Chemical Engineering and Processing-Process Intensification, 149, 107840.
Vilasau, J., Solans, C., Gómez, M., Dabrio, J., Mújika-Garai, R. & Esquena, J. 2011a. Phase
behaviour of a mixed ionic/nonionic surfactant system used to prepare stable oil-
in-water paraffin emulsions. Colloids and Surfaces A: Physicochemical and
Vilasau, J., Solans, C., Gómez, M. J., Dabrio, J., Mújika-Garai, R. & Esquena, J. 2011b.
Stability of oil-in-water paraffin emulsions prepared in a mixed ionic/nonionic
surfactant system. Colloids and Surfaces A: Physicochemical and Engineering
Aspects, 389, 222-229.
Wada, K., Koike, H., Fujii, T. & Morita, T. 2020. Targeted transcriptomic study of the
implication of central metabolic pathways in mannosylerythritol lipids
biosynthesis in Pseudozyma antarctica T-34. PloS one, 15, e0227295.
Wan, J., Tokunaga, T. K., Dong, W. & Kim, Y. 2017. Extracting natural biosurfactants from
humus deposits for subsurface engineering applications. Energy & Fuels, 31,
11902-11910.
Wang & Peng 2015. Application of Green Surfactants Developing Environment Friendly
Foam Extinguishing Agent. Fire Technology, 51, 503-511.
Wang, B., Wang, Q., Liu, W., Liu, X., Hou, J., Teng, Y., Luo, Y. & Christie, P. 2017.
Biosurfactant-producing microorganism Pseudomonas sp. SB assists the
phytoremediation of DDT-contaminated soil by two grass species. Chemosphere,
182, 137-142.
Wang, C., Guo, H., Meng, J., Wang, S., Kong, Y., Jiang, J., Hu, R. & Zhou, G. 2020a. Improved
lignocellulose saccharification of a Miscanthus reddish stem mutant induced by
heavy‐ion irradiation. Global Change Biology. Bioenergy, 12, 1066-1077.
Wang, F. C. & Marangoni, A. G. 2016. Advances in the application of food emulsifier α-gel
phases: Saturated monoglycerides, polyglycerol fatty acid esters, and their
derivatives. J Colloid Interface Sci, 394-403.
Wang, H., Kaur, G., To, M. H., Roelants, S., Tsang, C.-W., Dharma, R., Soetaert, W. & Lin, C.
S. K. Sophorolipid Production from Food Waste. Food Innovation and Engineering
(FOODIE) Asia Conference, 2020b. Food Innovation and Engineering (FOODIE)
Asia Conference.
277
Wang, K., Li, G. & Zhang, B. 2018a. Opposite results of emulsion stability evaluated by the
TSI and the phase separation proportion. Colloids and Surfaces A: Physicochemical
and Engineering Aspects, 558, 402-409.
Wang, M., Kong, C., Liang, Q., Zhao, J., Wen, M., Xu, Z. & Ruan, X. 2018b. Numerical
simulations of wall contact angle effects on droplet size during step emulsification.
RSC advances, 8, 33042-33047.
Wang, R., Wu, B., Zheng, J., Chen, H., Rao, P., Yan, L. & Chai, F. 2020c. Biodegradation of
total petroleum hydrocarbons in soil: isolation and characterization of bacterial
strains from oil contaminated soil. Applied Sciences, 10, 4173.
Wang, X., Sun, L., Wang, H., Wu, H., Chen, S. & Zheng, X. 2018c. Surfactant-enhanced
bioremediation of DDTs and PAHs in contaminated farmland soil. Environmental
technology, 39, 1733-1744.
Wang, Y., Liu, H., Wang, J., Dong, X. & Chen, F. 2018d. Formulation development and
visualized investigation of temperature-resistant and salt-tolerant surfactant-
polymer flooding to enhance oil recovery. Journal of Petroleum ence and
Engineering, 174.
Wenzel, M., Rautenbach, M., Vosloo, J. A., Siersma, T., Aisenbrey, C. H., Zaitseva, E.,
Laubscher, W. E., Van Rensburg, W., Behrends, J. C. & Bechinger, B. 2018. The
multifaceted antibacterial mechanisms of the pioneering peptide antibiotics
tyrocidine and gramicidin S. MBio, 9.
Whang, L.-M., Liu, P.-W. G., Ma, C.-C. & Cheng, S.-S. 2008. Application of biosurfactants,
rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated
water and soil. Journal of hazardous materials, 151, 155-163.
Worakitkanchanakul, W., Imura, T., Fukuoka, T., Morita, T., Sakai, H., Abe, M., Rujiravanit,
R., Chavadej, S., Minamikawa, H. & Kitamoto, D. 2008. Aqueous-phase behavior
and vesicle formation of natural glycolipid biosurfactant, mannosylerythritol lipid-
B. Colloids and Surfaces B: Biointerfaces, 65, 106-112.
Worakitkanchanakul, W., Imura, T., Fukuoka, T., Morita, T., Sakai, H., Abe, M., Rujiravanit,
R., Chavadej, S., Minamikawa, H. & Kitamoto, D. 2009. Phase behavior of ternary
mannosylerythritol lipid/water/oil systems. Colloids and Surfaces B: Biointerfaces,
68, 207-212.
Wriedt, T. 2012. Mie theory: a review. The Mie Theory. Springer.
Xia, Y., Zhou, J. J., Gong, Y. Y., Li, Z. J. & Zeng, E. Y. 2020. Strong influence of surfactants on
virgin hydrophobic microplastics adsorbing ionic organic pollutants.
Environmental Pollution, 265, 115061.
Xie, K., de Loubens, C., Dubreuil, F., Gunes, D. Z., Jaeger, M. & Leonetti, M. 2017.
Interfacial rheological properties of self-assembling biopolymer microcapsules.
Soft matter, 13, 6208-6217.
Xu, J., Zhou, H., Yu, Q., Manners, I. & Winnik, M. A. 2018. Competitive self-assembly
kinetics as a route to control the morphology of core-crystalline cylindrical
micelles. Journal of the American Chemical Society, 140, 2619-2628.
Xu, L. & Amin, S. 2019. Microrheological study of ternary surfactant ‐ biosurfactant
mixtures. International Journal of Cosmetic Science, 41, 364-370.
Xu, M., Fu, X., Gao, Y., Duan, L., Xu, C., Sun, W., Li, Y., Meng, X. & Xiao, X. 2020.
Characterization of a biosurfactant-producing bacteria isolated from Marine
environment: Surface activity, chemical characterization and biodegradation.
Journal of Environmental Chemical Engineering, 8, 104277.
Xu, Q., Liu, X., Wang, D., Liu, Y., Wang, Q., Ni, B.-J., Li, X., Yang, Q. & Li, H. 2019. Enhanced
short-chain fatty acids production from waste activated sludge by sophorolipid:
performance, mechanism, and implication. Bioresource technology, 284, 456-465.
278
Yamamoto, S., Morita, T., Fukuoka, T., Imura, T., Yanagidani, S., Sogabe, A., Kitamoto, D.
& Kitagawa, M. 2012. The moisturizing effects of glycolipid biosurfactants,
mannosylerythritol lipids, on human skin. Journal of Oleo Science, 61, 407-412.
Yang, L., Li, Y., Zhang, X., Liu, T., Chen, J., Wei, L. & Hua, Q. 2019. Metabolic profiling and
flux distributions reveal a key role of acetyl-CoA in sophorolipid synthesis by
Candida bombicola. Biochemical Engineering Journal, 145, 74-82.
Yang, X., Zhu, L., Xue, C., Chen, Y., Qu, L. & Lu, W. 2012. Recovery of purified lactonic
sophorolipids by spontaneous crystallization during the fermentation of
sugarcane molasses with Candida albicans O-13-1. Enzyme and microbial
technology, 51, 348-353.
Yang, Z., Zu, Y., Zhu, J., Jin, M., Cui, T. & Long, X. 2020. Application of biosurfactant
surfactin as a pH-switchable biodemulsifier for efficient oil recovery from waste
crude oil. Chemosphere, 240, 124946.
Yarveicy, H. & Haghtalab, A. 2018. Effect of amphoteric surfactant on phase behavior of
hydrocarbon-electrolyte-water system-an application in enhanced oil recovery.
Journal of Dispersion Science and Technology, 39, 522-530.
Yavrukova, V. I., Radulova, G. M., Danov, K. D., Kralchevsky, P. A., Xu, H., Ung, Y. W. &
Petkov, J. T. 2020. Rheology of mixed solutions of sulfonated methyl esters and
betaine in relation to the growth of giant micelles and shampoo applications.
Advances in Colloid and Interface Science, 275, 102062.
Ying, G. G. 2010. Personal Care Products, Springer Berlin Heidelberg.
Yoo, B.-H., Zhu, K., Zuo, C. & Lee, S. B. 2020. Preparation of Coconut Oil in Water Emulsions
Using Tween-Span Type Mixed Surfactant: Optimization of CCD-RSM. Applied
Chemistry for Engineering, 31, 19-24.
Yu, M., Liu, Z., Zeng, G., Zhong, H., Liu, Y., Jiang, Y., Li, M., He, X. & He, Y. 2015.
Characteristics of mannosylerythritol lipids and their environmental potential.
Zembyla, M., Murray, B. S. & Sarkar, A. 2020. Water-in-oil emulsions stabilized by
surfactants, biopolymers and/or particles: a review. Trends in Food Science &
Technology.
Zhang, D., Atkinson, H. V., Dong, H. & Zhu, Q. 2017a. Differential scanning calorimetry
(DSC) and thermodynamic prediction of liquid fraction vs temperature for two
high-performance alloys for semi-solid processing (Al-Si-Cu-Mg (319s) and Al-Cu-
Ag (201)). Metallurgical and Materials Transactions A, 48, 4701-4712.
Zhang, G., Wang, H., Wang, J., Zheng, J. & Ouyang, Q. 2019a. Dynamic rheological
properties of polyurethane-based magnetorheological gels studied using
oscillation shear tests. RSC advances, 9, 10124-10134.
Zhang, L., Somasundaran, P., Singh, S. K., Felse, A. P. & Gross, R. 2004. Synthesis and
interfacial properties of sophorolipid derivatives. Colloids and Surfaces A:
Zhang, L., Zhang, G., Ge, J., Jiang, P. & Pei, H. CO2/N2 responsive Pickering emulsion
stabilized by silica nanoparticles and a common nonionic surfactant. IOP
Conference Series: Earth and Environmental Science, 2018a. 012012.
Zhang, M., Feng, Y., Zhang, D., Dong, L. & Pan, X. 2019b. Ozone-encapsulated colloidal gas
aphrons for in situ and targeting remediation of phenanthrene-contaminated
sediment-aquifer. Water Research, 160, 29-38.
Zhang, R., Cai, Y., Wang, J., Zhao, Z., Mao, X. & Peng, Z. 2017b. A Star-Shaped Anionic
Surfactant: Synthesis, Alkalinity Resistance, Interfacial Tension and Emulsification
Properties at the Crude Oil–Water Interface. Journal of Surfactants and
Detergents, 20, 1027-1035.
279
Zhang, W. & Liu, L. 2013. Study on the formation and properties of liquid crystal emulsion
in cosmetic.
Zhang, Y., Jia, D., Sun, W., Yang, X., Zhang, C., Zhao, F. & Lu, W. 2018b. Semicontinuous
sophorolipid fermentation using a novel bioreactor with dual ventilation pipes
and dual sieve ‐ plates coupled with a novel separation system. Microbial
Zhao, F., Zhang, J., Shi, R., Han, S., Ma, F. & Zhang, Y. 2015. Production of biosurfactant by
a Pseudomonas aeruginosa isolate and its applicability to in situ microbial
enhanced oil recovery under anoxic conditions. RSC Advances, 5, 36044-36050.
Zhao, F., Zhou, J.-D., Ma, F., Shi, R.-J., Han, S.-Q., Zhang, J. & Zhang, Y. 2016. Simultaneous
inhibition of sulfate-reducing bacteria, removal of H2S and production of
rhamnolipid by recombinant Pseudomonas stutzeri Rhl: Applications for microbial
enhanced oil recovery. Bioresource Technology, 207, 24-30.
Zhao, Y., Paso, K., Kumar, L., Safieva, J., Sariman, M. Z. B. & Sjöblom, J. 2013. Controlled
shear stress and controlled shear rate nonoscillatory rheological methodologies
for gelation point determination. Energy & fuels, 27, 2025-2032.
Zhou, J., Hu, M. & Jing, D. 2019. The synergistic effect between surfactant and
nanoparticle on the viscosity of water-based fluids. Chemical Physics Letters, 727,
1-5.
Zhu, H., Kim, Y. & De Kee, D. 2005. Non-Newtonian fluids with a yield stress. Journal of
Non-Newtonian Fluid Mechanics, 129, 177-181.
Zhu, L. L. 2011. Solubilization properties of polycyclic aromatic hydrocarbons by saponin,
a plant-derived biosurfactant. Environmental Pollution.
Zhu, Q., Qiu, S., Zhang, H., Cheng, Y. & Yin, L. 2018. Physical stability, microstructure and
micro-rheological properties of water-in-oil-in-water (W/O/W) emulsions
stabilized by porcine gelatin. Food chemistry, 253, 63-70.
Zígolo, M. A., Irazusta, V. P. & Rajal, V. B. 2020. Correlation between initial
biodegradability determined by docking studies and structure of alkylbenzene
sulfonates: A new tool for intelligent design of environmentally friendly anionic
surfactants. Science of the Total Environment, 728, 138731.
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FORMULATION OF PERSONAL CARE PRODUCTS WITH BIO-

A thesis submitted to The University of Manchester

Chapter 1. Introduction ........................................................................................ 17

iv. Further information on the conditions under which disclosure, publication

spectrometry measurement. Thanks to University of Manchester for providing the

top educational resources for me.

material and emotional support that are essential to rely on

1.1 Research System

a. Crystalline/Hydrophilic gel phase, consisting of bilayer of the mixed

The microstructure of multicomponent emulsion system is macro-reflected by its

Table 1.1 Classification of ingredients formulated in E45 cream based on function

1.2 Research Motivation

In the field of pollution abatement, surfactants are capable of dealing with

The study of application of surfactants in food, pharmaceutical and cosmetic

The commercialisation of microbial-derived biosurfactants is promising but also

As for downstream processes, complex mixtures of biosurfactant after

Properties characterisations of biosurfactants along with their potential application

The application of biosurfactants in pharmaceutical industry mainly focused on

Through the mechanisms including increasing substrate bioavailability for

1.4 Research Objectives and Aims

The objectives of the project are:

1. to produce biousurfactants using fermentation technology and characterise

1.5 Overview of Thesis

Sodium lauryl ether sulfate SLES

Mannosylerythritol lipids MELs

2.1.1 Structure of Surfactants

The surfactant molecule consists of a water-favouring hydrophilic head group

Hydrophobic tail (Non polar)

2.1.2 Classification of Surfactants

The hydrophilic head group of cationic surfactant molecules dissociates cations in

Table 2.1 Examples of cationic surfactants and corresponding chemical structures

Name and Structure

In slightly acidic, neutral or alkaline aqueous solutions, the hydrophilic ‘‘head’’

Table 2.2 Examples of anionic surfactants and corresponding chemical structures

Type Name and Structure

Sulfonates Sodium Dodecyl Benzene Sulfonate (SDBS)

Sulfate Sodium Cetostearyl Sulphate

By ionization, anionic surfactants increase the negative potential of the interface

Carboxylated salts are a subgroup of carboxylates, generally applied as cleansing

c) Non-Ionic Surfactants Surfactants

Non-ionic surfactants do not dissociate into ions in an aqueous solution. Their

Table 2.3 Example of non-ionic surfactants and corresponding chemical structures

Name and Structure

The hydrophilic group of amphoteric (zwitterionic) surfactants carry both of positive

When dissolving in water, surfactant molecules align at the surfaces or interfaces

Surfactants exhibits various surface or interfacial activities, where surface tension

Figure 2.3 schematic diagram of different types of surfactant molecules alignment at

Further increasing surfactant concentration in the solution results in the self-

2.1.3.3 Liquid Crystals

Micelle Solution Liquid Crystal Formation

BSs possess advantages over chemically synthesized surfactants in terms of low

2.2.1 Classification of Biosurfactants (BSs)

Biosurfactants (BSs) are classified according to their microbial sources, chemical

In addition, according to molecular weight, Rosenberg and Ron (Rosenberg and

Table 2.4 Classification of bio-surfactants, adapted from Shoeb et al., 2013

Type of BSs Examples

2.2.2 The Production and Extraction of Biosurfactants (BSs)

2.2.3 Characterization of Biosurfactants (BSs)