Astha Thesis M.tech

OPTIMIZATION AND CHARACTERIZATION OF PECTIN
EXTRACTED FROM CITRUS FRUIT PEELS AND ITS EFFECT ON

QUALITY CHARACTERISTICS OF YOGHURT
by
Astha Adhikari
Central Department of Food Technology, Dharan
Institute of Science and Technology
Tribhuvan University, Nepal
May, 2023
Optimization and Characterization of Pectin Extracted from Citrus Fruit
Peels and Its Effect on Quality Characteristics of Yoghurt
A dissertation submitted to the Central Department of Food Technology, Institute of

Science and Technology, Tribhuvan University, in partial fulfillment of the
requirements for the degree of M. Tech in Food Technology.
by
Astha Adhikari
Central Department of Food Technology, Dharan
Tribhuvan University, Nepal
May, 2023
ii
Tribhuvan University
Central Department of Food Technology
Approval Letter
This dissertation entitled Optimization and Characterization of Pectin Extracted from

Citrus Fruit Peels and Its Effect on Quality Characteristics of Yoghurt presented by Astha
Adhikari has been accepted as the partial fulfillment of the requirements for the M. Tech.
Degree in Food Technology.
Dissertation Committee
1. Head of the Department
(Mr. Basanta Kumar Rai, Prof.)
2. External examiner
(Mr. Birendra Kumar Yadav, Asst. Prof., BPKIHS)
3. Supervisor
(Mrs. Mahalaxmi Pradhananga, Asst. Prof.)
4. Internal examiner
(Mr. Bunty Maskey, Asst. Prof.)
May 16, 2023
iii
Acknowledgements
I would like to express my profound gratitude to all those who gave me immense support to
complete my work. Special thanks to my supervisor Asst. Prof. Mahalaxmi Pradhananga,
for her guidance, supervision and expertise throughout the work. I am really grateful to my
internal Asst. Prof. Bunty Maskey and Prof. Basanta Kumar Rai (Head of Department,
CDFT) for valuable ideas and constructive suggestions.
My sincere thanks to all faculty members of CDFT, staff of library and laboratory who
directly or indirectly have helped in completion of my work. I am very grateful to all my
friends for their encouragement and support.
Finally, I am forever indebted to my parents and my brother for their acceptance, patience,
love and moral support in every step of my life.
………………………………….
Date of submission: May 16, 2023
Astha Adhikari
iv
Abstract
The aim of this study was to optimize and characterize pectin extracted from three citrus
fruit peels namely lemon (Citrus limon), lime (Citrus aurantifolia) and Pomelo (C. maximus)
and study its effect on quality parameters of yoghurt. Pectin was extracted at three different
temperatures (80℃, 90℃ and 100℃), pH (1, 2 and 3) and time (60, 90 and 120 min) using
acid extraction method. Response surface methodology with central composite design at
three-level three-factor general factorial design was applied for the maximum yield and
anhydrouronic acid content. Pectin extracted from optimized condition is further
characterized on equivalent weight, methoxyl content, and anhydrouronic acid content,
degree of esterification, moisture and ash content. Afterwards, 0.2% pectin extracted from
optimized condition was incorporated in yoghurt samples followed by sensory,
physicochemical and microbiological analysis.
For maximize yield and anhydrouronic acid content, the optimum condition of pH,
temperature and time for pectin extraction from lemon peels was 1, 100℃, 118.6 min
respectively whereas from lime peels was 1, 100℃, 120 min respectively and from pomelo
peel was 1, 100℃, 105.4 min respectively. Equivalent weight, methoxyl content,
anhydrouronic acid and degree of esterification of pectin from pomelo peel was significantly
higher than pectin extracted lemon and lime peels. The ash content and moisture content of
extracted pectin from all citrus peels was lower than 10% and 12% respectively which
confirmed the commercial usage of extracted pectin. The yoghurt prepared with 0.2% pectin
had low moisture and high viscosity. During the 7 days storage of yoghurt, titratable acidity
of pectin added yoghurt samples were higher to control sample however, there was decrease
in syneresis content. The sensory parameters aroma, consistency, taste and overall
acceptability of pectin added samples were significantly different (p<0.05) to control
samples. Addition of pectin had no significant effect (p>0.05) on the microbial profile of
yoghurt. Thus, the extracted pectin could be utilized as additives in yoghurt samples.
v
Contents
Approval Letter .................................................................................................................. iii
Acknowledgements ............................................................................................................. iv
Abstract ................................................................................................................................ v
List of tables ........................................................................................................................ xi
List of figures ..................................................................................................................... xii
List of plates ...................................................................................................................... xiv
Abbreviations ..................................................................................................................... xv
1. Introduction .............................................................................................................. 1-4
1.1 Background ......................................................................................................... 1
1.2 Statement of the problem .................................................................................... 2
1.3 Objectives of the study ....................................................................................... 3
1.3.1 General objective .................................................................................. 3
1.3.2 Specific objectives ................................................................................ 3
1.4 Significance of the study .................................................................................... 4
1.5 Limitations of the study ...................................................................................... 4
2. Literature review .................................................................................................... 5-19
2.1 Pectin .................................................................................................................. 5
2.2 Structure of pectin .............................................................................................. 6
2.2.1 Classification of pectin.......................................................................... 7
2.2.1.1 Degree of methylation .......................................................... 7
2.2.1.2 Degree of acetylation and amidation .................................... 8
2.3 Extraction methods ............................................................................................. 8

vi
2.3.1 Acid extraction method ......................................................................... 9
2.3.2 Microwave assisted extraction ............................................................... 9
2.3.3 Ultrasonic assisted extraction.............................................................. 10
2.4 Sources of pectin .............................................................................................. 11
2.4.1 Lemon peels ....................................................................................... 11
2.4.2 Acid lime peels.................................................................................... 12
2.4.3 Pomelo ................................................................................................ 13
2.4.4 Production ........................................................................................... 13
2.5 Techno-functional properties of pectin............................................................. 14
2.5.1 Pectin gelation ..................................................................................... 14
2.5.2 Water/oil holding capacity .................................................................. 15
2.6 Application of pectin ........................................................................................ 15
2.6.1 Pectin in yoghurt ................................................................................. 15
2.7 Yoghurt .............................................................................................................. 16
2.7.1 Types of yoghurt .............................................................................. 16
2.7.2 Health benefits of yoghurt................................................................... 17
2.8 Factors affecting the quality of yoghurt ........................................................... 17
2.8.1 Casein and fat content ......................................................................... 17
2.8.2 Homogenizing ..................................................................................... 17
2.8.3 Acidity ................................................................................................. 18
2.8.4 Heat treatment ..................................................................................... 18
2.8.5 Incubation temperature ....................................................................... 18
vii
2.9 Stabilizers ......................................................................................................... 18
3. Materials and methods ......................................................................................... 20-28
3.1 Material collection ............................................................................................ 20
3.1.1 Chemicals and equipment required ............................................................... 20
3.2 Methods ............................................................................................................ 20
3.2.1 Peel powder preparation...................................................................... 20
3.2.2 Extraction procedure ........................................................................... 21
3.2.3 Experimental Design ........................................................................... 22
3.2.4 Characterization of extracted pectin ................................................... 23
3.3 Preparation of pectin incorporated yoghurt ...................................................... 25
3.4 Physicochemical analysis ................................................................................. 26
3.4.1 Moisture content .................................................................................. 26
3.4.2 Fat........................................................................................................ 26
3.4.3 pH ........................................................................................................ 26
3.4.4 Protein ................................................................................................. 27
3.4.5 Ash ...................................................................................................... 27
3.4.6 Total solids .......................................................................................... 27
3.4.7 Viscosity.............................................................................................. 27
3.4.8 Carbohydrate ....................................................................................... 27
3.4.9 Syneresis ............................................................................................. 27
3.4.10 Titratable acidity ............................................................................... 27
3.5 Microbiological analysis................................................................................... 28
viii
3.6 Sensory evaluation ............................................................................................ 28
3.7 Statistical analysis............................................................................................. 28
4. Results and discussions ........................................................................................ 29-62
4.1 Experimental design for pectin extraction from lemon peels ........................... 29
4.1.1 Model fitting for yield and AUA of pectin from lemon peels ............ 31
4.1.2 Effect of process variables for pectin extraction from lemon peels31
4.2 Experimental design for pectin extraction from lime peels .............................. 37
4.2.1 Model fitting for yield and AUA of pectin from lime peels ............... 38
4.2.2 Effect of process variables for pectin extraction from lime peels ....... 38
4.3 Experimental design for pectin extraction from pomelo peels ......................... 44
4.3.1 Model fitting for yield and AUA of pectin from pomelo peels ..... 45
4.3.2 Effect of process variables for pectin extraction from pomelo peels45
4.4 Validation of optimization condition................................................................. 51
4.5 Physiochemical characterization of pectin ....................................................... 52
4.5.1 Equivalent weight ............................................................................... 53
4.5.2 Methoxyl content ................................................................................ 53
4.5.3 AUA content ....................................................................................... 53
4.5.4 Degree of esterification ....................................................................... 54
4.5.5 Moisture content ................................................................................. 54
4.5.6 Ash content ......................................................................................... 54
4.6 Sensory analysis of pectin added yoghurt ......................................................... 55
4.6.1 Colour.................................................................................................. 55
ix
4.6.2 Aroma.................................................................................................. 56
4.6.3 Taste .................................................................................................... 56
4.6.4 Consistency ......................................................................................... 56
4.6.5 Overall acceptance .............................................................................. 56
4.7 Physiochemical properties of yoghurt .............................................................. 57
4.7.1 Syneresis ............................................................................................. 59
4.7.2 Titratable acidity ................................................................................. 60
4.8 Microbial analysis............................................................................................. 61
5. Conclusions and recommendations ..................................................................... 63-64
5.1 Conclusions ...................................................................................................... 63
5.2 Recommendations ............................................................................................ 63
6. Summary ............................................................................................................... 65-66
References.............................................................................................................. 67-78
Appendices ............................................................................................................ 79-89
x
List of Tables
Table No. Title Page No.
2.1 Sources and different extraction methods of pectin 10
2.2 Production of lemon and limes in different provinces of Nepal 13
2.3 Different stabilizer approved by the FAO and WHO 19
3.1 Range of factors for RSM 22
3.2 Experimental design for extraction of pectin 23
4.1 Experimental design for pectin from lemon peels 30
4.2 ANOVA table for quadratic model of yield from lemon peel 33
4.3 ANOVA table for quadratic model of AUA from lemon peel 33
4.4 Experimental design for pectin from lime peels 37
4.5 ANOVA table for quadratic model of yield from lime peel 39
4.6 ANOVA table for quadratic model of AUA from lime peel 40
4.7 Experimental design for pectin from pomelo peels 44
4.8 ANOVA table for quadratic model of yield from pomelo peel 46
4.9 ANOVA table for quadratic model of AUA from pomelo peel 47
4.10 Different parameters for optimization 51
4.11 Optimized conditions of each peels and their response values 52
4.12 Physiochemical characterization of extracted pectin 52
4.13 Proximate analysis of pectin added yoghurt 57
4.14 Syneresis of yoghurt samples during storage period of 7 days 59
4.15 Titratable acidity of yoghurt samples of 7 days storage 60
4.16 Microbial analysis of pectin added yoghurt (log cfu/ml) 62
xi
List of Figures
Figure No. Title Page No.
2.1 HM pectin 7
2.2 LM pectin 8
3.1 Flow diagram for the preparation of citrus peel powder 20
3.2 Flow diagram for the extraction of pectin 21
3.3 Flow diagram for yoghurt preparation 26
4.1 Response curve 3D plot for effect of pH and temperature on 34

pectin yield from lemon peels
4.2 Response curve 3D plot for effect of pH and time on pectin 34

yield from lemon peels
4.3 Response curve 3D plot for effect of time and temperature on 35

pectin yield from lemon peels

AUA of pectin from lemon peels

AUA of pectin from lemon peels
4.6 Response curve 3D plot for effect of pH and time on AUA of 36

pectin from lemon peels

pectin yield from lime peels
4.8 Response curve 3D plot for effect of pH and time on pectin 41

yield from lime peels

pectin yield from lime peels

AUA of pectin from lime peels
xii
AUA of pectin from lime peels

pectin from lime peels

pectin yield from pomelo peels
4.14 Response curve 3D plot for effect of pH and time on y pectin 48

yield from pomelo peels
4.15 Response curve 3D plot for effect of time and temperature 49

pectin yield from pomelo peels

AUA of pectin from pomelo peels

AUA of pectin from pomelo peels

pectin from pomelo peels
4.19 Sensory characteristics of pectin added yoghurt 55
xiii
List of Plates
Plate No. Title Page No.
P.1 Fresh and dried peels of lemon, lime, pomelo 89
P.2 Extraction of pectin and yoghurt samples 89
xiv
Abbreviations
Abbreviations Full form
ACE Angiotensin converting enzyme
AUA Anhydrouronic Acid
CCD Central Composite Design
CMC Carboxymethyl cellulose
DE Degree of esterification
DFTQC Department of Food Technology and quality control
DM Degree of Methylation
EW Equivalent weight
FAO Food and Agriculture Organization
FDA Food and Drug Administration
GalA Galacturonic acid
HG Homogalacturonan
HM High methoxyl
IPPA International Pectin Producers Association
ITIS Integrated Taxonomic Information System
LAB Lactic Acid Bacteria
LM Low methoxyl
LMA Low methoxyl amidated
MeO Methoxyl Content
MOALD Ministry of Agriculture and Livestock Division
NDDB Nation Dairy Development Board
RG Rhamnogalacturonan
RSM Response Surface Methodology
xv
SMP Skimmed milk powder
TA Titratable acidity
WHC Water holding capacity
xvi
Part I
Introduction
1.1 Background
Pectin is a group of hetero polysaccharides present the primary cell wall and middle lamellae
of the plants. It consists of covalently α-1,4-linked D-galacturonic acid (GalA) units
interrupted by L-rhamnose residues with side chains of neutral sugars (mainly D‐galactose
and L‐arabinose) (Guo et al., 2012). It was first discovered in apple juice by Vauquelin
(1790) and named by Henri Braconnot (1825), derived from the Greek word ‘pektikos’
which means to ‘congeal or solidify’. It is considered a high-value functional food ingredient
because of its excellent emulsifying properties and stability so used as a gelling agent and
stabilizer. (Khamsucharit et al., 2018). The pectin has wide applications in the
pharmaceutical, food industry, medical, dairy, nutritional, health, and cosmetic products
(Saha and Bhattacharya, 2010).
Pectin is extracted from raw materials such as apple pomace or citrus peel in industries
using acid in high temperature. The fruit processing industries produces large quantities of
by-products mainly peels. These are used as cheap sources of feed for cattle, but also peel
contains high sums of pectin. Citrus peel have been utilized in commercial scale pectin
production since quite long (May, 1990). Pectin is the gelatinizing agent naturally found in
fruits and vegetable products. The amount and quality of pectin mainly depend on the type
maturity of fruits and vegetables and method adopted for the extraction of pectin. Citrus
fruits are at the top in the total production and economic value. Citrus fruit peels contains
different layers namely flavedo and albedo. Flavedo is the outer layer, whose colour varies
from green to yellow (Brat et al., 2001) , and have been used in flavor and fragrance industry
(Vekiari et al., 2002). Similarly, albedo is a spongy and cellulosic layer laid under flavedo.
Flavonoids and vitamin C with antioxidant properties are abundantly present in it with
healthier benefits than other dietary fiber sources (Marin et al., 2002). In Nepal, some of the
major citrus plants are mandarin (Citrus reticulate Blanco), sweet orange (Citrus sinensis),
acid lime (Citrus aurantifolia Swing), lemon (Citrus limon), Pomelo (Citrus maxima), citron
(Citrus medica), and rough lemon (Citrus jambhiri Lush.) (Panth and Dhakal, 2019). Pomelo
(Citrus maxima) is one of the popular citrus fruits but extremely underutilized in commercial
level. Acid lime (Citrus aurantifolia) and lemon (Citrus Limon Burm.) are some of the
important citrus fruits with high commercial value (Shrestha et al., 2012).
Yoghurt is a popular fermented milk product made by inoculation of specific bacteria

strains into milk, called Lactic Acid Bacteria (LAB) the most common are Streptococcus
thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. It is asserted with various
therapeutic benefits and refreshing taste. The marketability and consumer acceptance of
yoghurt depends on flavour, texture, consistency and shelf life (Nagaoka, 2019) . Along with
milk, varieties of additives such as fruits, flavours, colours, sweeteners, stabilizers and
preservatives are added to mix in yoghurt. Stabilizers added in yoghurt increases the body
and textural properties improving the firmness, reduction in syneresis and uniform
distribution of ingredients (Gawai et al., 2017). Stabilizers as gelatin, agar, gums, pectin,
carrageenan and pregelatinized starch are generally permitted to use combine and included in
the formulation of the yoghurt (Tamime and Robinson, 2007).
Today, a substantial quantity of pectin is employed to stabilize low pH dairy beverages,

such as fermented beverages and milk-fruit juice mixtures. The drinks may undergo heat
treatment to lengthen their shelf life. The ideal qualities include low viscosity and
homogeneous appearance. However, casein is subject to aggregation at low pH, especially
when heat treated. High viscosity, whey exudation, and sandy mouthfeel are therefore
possible quality flaws in these kinds of drinks in the absence of a stabilizer (Naseri et al.,
2008). Moreover, post-treatment process such as heat treatment and pumping affect the gel
structure of the yoghurt reducing viscosity and causing syneresis of the product. The necessity
for a smooth, stable, and long-lasting yoghurt product without any form of refrigeration
creates additional difficulties for the dairy sector. Stabilizers are added to the yoghurt in
ambient temperatures to prevent syneresis, or the separation of the whey, and to maintain the
desired texture (Henrysson, 2016).
1.2 Statement of the problem
According to the Volza (2020), Nepal is the largest importer of pectin and generally imported
from India. There is no production of pectin within Nepal. The report published by MOALD
(2022) shows the amount of pectin imported is 19,889 kg. Alongside, among 45% of annual
foods wastages from fruits and vegetables 14.8% are during the production and preservation
2
of fruits and vegetables, resulting the largest amounts of food wastes (Maric et al., 2018b).
Also, the wastes collected from citrus fruits amounts for 50% of the total weight of the fruit.
Therefore it could be promising strategy for self-production of commercial pectin finding
the alternative source for commercial pectin. This can be recovered from fruits byproducts
as a plant based pectin (Putra et al., 2022).
Low solid content in yoghurt ensues quality concerns such as weak body, poor texture,
whey separation and variations in consistency. The standard yoghurt is very perishable and
prone quality changes during storage subsequently hampering large-scale production
(Bhattarai et al., 2015). However, the major problem of yoghurt is syneresis. The common
technique practiced in most dairies is the chilling storage to extend the shelf life. But the
frequent shortage of electricity and inefficient transportation system in countries like Nepal
may deteriorates the final quality characteristics of yoghurt.
1.3 Objectives of the study
1.3.1 General objective
The general objective of this study was optimization and characterization of pectin extracted
from citrus fruit peels and its effect on quality characteristics of yoghurt.
1.3.2 Specific objectives
To fulfill the general objectives, the following specific objectives were:
1. To optimize the extraction of pectin from citrus peels of lemon, lime and pomelo (pH,
temperature and time).
2. To carry out characterization of the citrus fruit peels pectin (moisture, ash, equivalent
weight, degree of esterification, methoxyl content, anhydrouronic acid content and
grade).
3. To carry out physical, chemical and microbiological analysis of fresh yoghurt samples
(moisture, protein, fat, total solid, carbohydrate, viscosity, pH, total plate count, coliform,
yeast and mold count, lactic acid bacteria count).
4. To study the changes in syneresis and titratable acidity in yoghurt samples up to 7 days
of storage.
3
1.4 Significance of the study
The aim of this study had undertaken to characterize the pectin extracted from peels of citrus
fruits and its effect in yoghurt. The effective utilization of food wastes protects the
environment and discloses the great potential in the production of functional substances such
as bioactive secondary metabolites, essential oils, dietary fibers, pigments, enzymes, and
non-starch polysaccharides (Di Donna et al., 2020).
Pectin as stabilizer and thickening agent is expected to overcome the problem of syneresis
and to create desired texture and stability of yoghurt. Stabilizer improves the shelf-life of
yoghurt, prevents the product from becoming deteriorated in terms of sensory attributes. It
also enhances properties such as the viscosity, influences texture, creaminess and mouth
feel as well as aids to prevent separation of whey from yoghurt (Alakali et al., 2008). The
viscoelastic properties and water holding capacity of yoghurts is directly proportional to the
total solid level in milk. Stabilizers increases the total solid content of the yoghurt (Sodini et
al., 2004).
This could to minimize the import of pectin and control the losses or damages of fruits
and vegetables, conversing the problem into an asset along with enhancing the quality
characteristics of yoghurt.
1.5 Limitations of the study
The limitations of the work are as follows:
1. Structural analysis of pectin could not be performed.
2. Textural analysis of product could not be performed.
3. Gel strength of pectin and product could not be measured.
4
Part II
Literature review
2.1 Pectin
Pectin is defined as complex mixtures of polysaccharides that make up approximately one third
of the cell-wall dry substance of most types of plants. Pectin consists of blocks of
homogalacturonic acid called ‘smooth regions’ mixed with blocks of homo galacturonic acid
containing many neutral sugars including rhamnose, galactose, arabinose, xylose, and glucose
called ‘hairy regions’ (Brejnholt, 2010). The utilization of gelling ability of pectin with sugar
and acid has been dated back to at least the 18th century in high sugar jams and confectionery
jellies (IPPA, 2001).
Pectin is natural additive for foods considered for a number of applications as thickeners,
water binders, and stabilizers. This valuable functional ingredients has been pursued by
pharmaceutical, plant and food scientists (Saha and Bhattacharya, 2010). In food industry, it
is primarily used for jam, jellies, confectionary products, fruit juices and bakery fillings.
Another major application of pectin is in fermented acidic milk drinks and yoghurts (Willats
et al., 2006). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has
recommended pectin (Codex Alimentarius No. 440) as a safe additive with no limit on
acceptable daily intake. The premature fruits can be a rich source of pectin, and extracted
pectin can be employed in domestic and commercial food (Azad et al., 2014). The
characteristics of pectin vary accordingly to plant origin, extraction conditions and post-
extraction treatment (Munarin et al., 2012).
In recent years, modified pectin (MP) are attracting researchers with higher bioactivities
than the native one as a neutraceutical or pharmaceutical in cancer therapy, mainly due to its
ability to protect the immune system, regulate oncogenes, promote the growth of probiotics
and inhibit the development of tumor (Georgiev et al., 2012). This could induce inhibition
of galectin-3 (Gal-3) interactions with other proteins and peptides so as to bridle cell
adhesion and migration and prevent apoptosis (Maxwell et al., 2012). Moreover, emerging
modified pectin showed various pharmaceutical bioactivities including lipase inhibition
(Kumar and Chauhan, 2010), wound healing (Hokputsa et al., 2004), apoptosis induction of
human cancer cell (Jackson et al., 2007).
2.2 Structure of pectin
Pectins are a group of complex polysaccharides that are found in the primary cell wall and
between the cells of dicotyledons. They give plants flexibility and mechanical strength. The pectin
source, plant developmental phases, and extraction conditions all have a significant impact on the
composition and structure of pectin. The exact chemical structure of pectin is still controversial
depending upon sources and different extraction methods. GalpA-(1,4)-linked D-galacturonic
acids make up the backbone of pectin, which is broken up by (1,2)-linked L-rhamnose (do
Nascimento Oliveira et al., 2018). They include a very complicated set of polysaccharides that
are covalently connected to one another, with homogalacturonan (HG) and rhamnogalacturonan
I (RG-I) being the most prevalent classes. Substituted galacturonans, such as rhamnogalacturonan
II (RG-II), xylogalacturonan (XGA), and apiogalacturonan (AGA) are minor components
(Belkheiri et al., 2021). The major chemical structure presented in pectin are described as:
1. Homogalacturonan (HGA)
HGA is a linear homopolymer and most abundant forms in pectin 57-70% (Jackson et al.,
2007). They are known as smooth region in pectin. The partially C-6 carboxylated and O-2
or O-3 acetylated HGs spears to be synthesized in golgi apparatus transferring to the cell
wall. The methyl esterification of HG regions determines the application and interaction
capacity of pectin industrially (Willats et al., 2006).
2. Rhamnogalacturonan I (RG I)
RG-I consists of alternating GalpA and Rha forming a (1,4)-α-D-galacturonic acid-(1,2)-α-

L-rhamnose repeating unit, the backbone of rhamnogalacturonan I. It makes up 7–14% of
the pectin (Wang et al., 2018). The neutral sugars side chains are attached to C2, C3 OR C4.
Mostly galactose and arabinose, forming galactan, arabinan and arabinogalactans. Other
sugars such as glucose, mannose, fructose, xylose, and glucuronic acid are found covalently
linked to the backbones as side chains. This forms the hairy region of pectin and responsible
for bioactive properties of pectin (Voragen et al., 2009).
3. Substituted galacturonans (GS)
GS consists of rhamnogalacturonan II, xylogalacturonan and apiogalacturonan depending

upon types of pectin. RG-II is included in the hairy region, mostly composed of
6
arabinofuranose, arabinopyranose, glucopyranose, fucopyranose, apiofuranose,
galactopyranose, and other unusual sugars such as 3-deoxy-D-lyxo-2-heptulosaric acid
(Dha), ketodeoxyoctonic acid and aceric acid. Xylogalacturonan and apiogalacturonan are
homogalacturanans substituted with xylose and xylogalacturonan or disaccharide
apiofuranosyl for apiogalacturonan (Ridley et al., 2001).
2.2.1 Classification of pectin
2.2.1.1 Degree of methylation
Pectin can be classified according to the degree of methoxylation (DM). The degree of
methoxylation is expressed as a percentage of esterified galacturonic acid units to total
galacturonic acid units in the molecule of pectin. The methoxyl content shows the dispersibility
of pectin in water and its ability to form hydrogel (Castillo-Israel et al., 2015).
1. High methoxyl pectin
Pectin containing more than 50% of esterified methoxyl groups (Fig. 2.1) is classified as high
methoxyl pectin (HMP). HM pectin are capable of forming gels in aqueous systems with high
contents of soluble solids and low pH values. It is mostly present in nature (Williams, 2011).
COOCH3 COOH
COOH COOCH3 COOCH3
Fig. 2.1 HM pectin
2. Low methoxyl pectin
Low methoxyl pectin (LMP) has less than 50% controlled esterified methoxyl groups (Fig. 2.2).
LM pectin are characterized by their ability to form gels in the presence of bivalent salts, normally
Ca++, in systems with low solids content and a wide pH range It is only obtained after
demethylation of HM pectin (Williams, 2011).
7
COOH COOH
COOH COOCH3 COOCH3
Fig 2.2 LM pectin
2.2.1.2 Degree of acetylation and amidation
The percentage of galacturonosyl residues esterified (on the hydroxyl group) with acetyl is
defined as degree of acetylation (DAC). The gel formation of the pectin is prevented by
acetylation but increase the stabilizing and emulsifying ability. Higher degree of acetylation
(up to 50%) of pectin do not have good gelling properties (Williams, 2011).
When the carboxyl group of pectin reacts with ammonia it forms amidated pectin. Pectin's
amidation makes it more thermo-reversible, soluble and able to resist greater calcium
variation. It specifically involves low methoxyl amidated pectin (LMPA) (Matia-Merino et
al., 2004). Commercially, pectin is classified by International Numbering System as, E440 (i) for
high methoxyl pectin and conventional low methoxyl pectin and E440 (ii) for amidated low
methoxyl pectin (Mortensen et al., 2017).
2.3 Extraction methods
Generally, apple pomace and citrus fruits are used for commercial production of pectin
(Salam et al., 2012). The extraction conditions vary from facility to facility and are
dependent on the pectin source. Pectic substances are extracted by chemical or enzymatic
methods with multiple physical and chemical stages process , in which the hydrolysis,
extraction and solubilization of macromolecules plant tissue are influenced by several factors
such as temperature, pH, acid type and extraction time (Pagan et al., 2001). The most
common extraction methods is acid extraction using a dilute mineral acid, usually
hydrochloric, sulfuric, or nitric acids. Extraction process is significantly impacted by all
processing factors (Fakayode and Abobi, 2018). The right techniques must be used to
transform the problem into an advantage (Girma and Worku, 2016).
The International Pectin Producers Association has described the detailed commercial
8
pectin extraction. A factory receives cleaned and dried apple pomace or citrus peel. The raw
materials are added to hot water and a dilute mineral acid is added for extraction. Sufficient
time intervals to allow extraction and then the solids are separated from the pectin containing
liquid through filtration or centrifugation. The remaining solution is mixed with an alcohol
for pectin precipitation. The precipitated pectin is separated and washed with alcohol to
remove impurities. The pectin is dried, ground to a powder, and blended with other additives,
if necessary (IPPA, 2001).
Recently, others plant sources are reported as excellent yields for pectin extraction. Novel
technologies are being applied for efficient and qualitative pectin extraction. The different
extraction techniques can be used in different sources as in Table 2.1. Other fruits like grapes,
mango, pumpkins, red dragonfruit, carrot and beet are studied for the pectin extraction
(Colodel et al., 2020; Kostalova et al., 2016; Misra and Yadav, 2020; Woo et al., 2010).
2.3.1 Acid extraction method
Strong acid solutions, such as nitric, sulfuric, phosphoric, and hydrochloric acids, are
typically used in industry to extract pectin while they are heated (Freitas et al., 2020). Using
boiling water, conventional pectin extraction requires many h to provide a satisfactory yield.
Low-quality pectin are produced as a result of the lengthy heating process' thermal
degradation of pectin caused by beta-elimination and debranching. As a result, pectin is
extracted in an acidic aqueous medium (pH 1.5–3) at a temperature between 75 and 100℃
for 1-3 h while being continuously stirred. Conventional extraction depends on several
factors, such as temperature, pH, solvent properties, solid to solvent ratio, particle size, and
diffusion rate (Maric et al., 2018a).
2.3.2 Microwave assisted extraction
Microwave-assisted extraction (MAE) is a green method in which consists of two oscillating

perpendicular fields: electric and magnetic fields and polar solvent absorbs microwave
energy (Routray and Orsat, 2012). It is considered as fast extraction method and has higher
efficiency in comparison to the conventional acid extraction method. The heat produced
plays a significant role in the extraction process because higher temperatures increase the
diffusion rate, which improves extraction yields. The rate of extraction and the quality of the
extracted compounds are also influenced by dielectric characteristics of sample and solvent,
9
as well as the solvent solubility of the compounds (Picot-Allain et al., 2022). The extraction
of pectin from apple using microwave assistance and conventional method is performed by
Zheng et al. (2021).
2.3.3 Ultrasonic assisted extraction
The ultrasonic waves of range 20-100 kHz is used for U-assisted extraction method. This
method reduces the extraction time and increases yield in compared to acid extraction
method (Azmir et al., 2013). In this technique, compression and expansion are created as
sound waves travels through a liquid medium resulting cavitation. Consequently, it affects
the plant matrix facilitating more solvent penetration and increasing the efficiency(Cravotto
and Cintas, 2006). Reduced extraction time, equipment size, energy usage, and improved
extraction yield are all significant advantages of U-assisted extraction, which is also said to
be more ecologically friendly than the traditional method. On the other hand, U-extracted
polysaccharides have decreased viscosity, molecular mass, and esterification levels (Freitas
et al., 2020). de Oliveira et al. (2016) and Guandalini et al. (2019) has extracted the pectin
using ultrasound technology.
Table 2.1 Sources and different extraction methods of pectin
Sources Extraction methods References
Lemon peels Acid extraction method Akhtar et al. (2020); Dhushane and
Mahendran (2020)
Citrus peels Acid extraction method Wonago (2016); Kanmani et al.

(2014)
Orange peels Acid extraction method Kamal et al. (2021); Arioui et al.
(2017); Rady et al. (2021)
Pomelo peels Ultrasound, Acid, Nguyen et al. (2020); Liew (2019)

Microwave extraction
Lime peels Microwave extraction Rodsamran and Sothornvit (2019)
Okra Aqueous extraction Kpodo et al. (2017)
Banana peels Acid extraction method Khamsucharit et al. (2018)
10
Mango peels Acid extraction method Sayed et al. (2022)
Dragonfruit peels Acid extraction method Nazaruddin et al. (2011)
Jackfruit; passion fruit Acid extraction method Mbaeyi-Nwaoha et al. (2019)
Apple pomace Microwave extraction Wang et al. (2007)
Peach pomace Acid extraction method Pagan et al. (2001)
Lemon pomace Acid extraction method Azad et al. (2014)
2.4 Sources of pectin
2.4.1 Lemon peels
The lemon (Citrus Limon) is a small evergreen tree native to Asia. The term lemon derived from
ancient French word ‘limon’. It is also known as ‘lamun’ or ‘lmun’ in Arabic and ‘lmun’ in
Persian. Lemon is a flowering plant of the Rutaceae family. Approximately 140 genes and
1300 species are present in the genus citrus (Kamal et al., 2011). The most popular lemons
are Meyer, Eureka and Lisbon. Lemon juice consists of citric acid and most popularly used
in drinks and cooking purposes in Asian households. It is claimed to have various health
benefits. Lemon trees widely grow in tropical and subtropical climates (Duportal et al.,
2013).
Many studies have been done in the functional properties and medicinal values of the
lemon peels. It has shown the great extent of benefits in various areas. The total polyphenols
content, antioxidant activity and pectin present in lemon peels make them promising
resource for food and cosmetic industries (Diankov et al., 2011). The presence of alkaloids,
sterols, terpenoids and steroids exhibits antimicrobial effect on lemon peels extract
(Dhanavade et al., 2011). The lemon peels also have significant effect to cure kidney stone
disease, lower liver and plasma cholesterol and their reversion. The lemon peel has ability
to reduce the nanoparticles of silver following green chemistry methodology (Nisha et al.,
2014).
11
Kingdom Plantae
Sub-division Spermatophytina
Class Magnoliopsida
Sub-class Rosids
Order Sapindales
Family Rutaceae
Genus Citrus
Species C. Limon
Source: ITIS (1998)
2.4.2 Acid lime peels
Acid lime (Citrus aurantifolia) is the popular citrus fruit in Nepal. Trees of acid lime are
small, bushy with small sharp spines. Leaves and flowers are small with narrowly winged
petioles. The fruits are oval shaped and greenish yellow in colour. In Nepal, the area coverage
of production has been reported 16% of total fruit crops. Acid lime has high commercial value
in the market due to better aroma, appropriate size and medicinal value. It is popular for juice,
desert, pickle and other medicinal purpose (Shrestha et al., 2012).
Acid limes are used in various food products such as juices, jellies, jam and pickles.
Essential oils extracted are used in fragrance and perfume industries. Peels of acid limes has
flavedo and albedo layers. Flavedo contains chlorophyll and carotenoids pigments. It has oil
glands from which essential oils are extracted which is used in cosmetics and perfumes.
Some volatile compounds found in oil are isolimonene, terpinene, citronellol and neral.
Similarly. albedo has high cellulose, hemicellulose, lignin and pectin content (Kalatippi and
Hota, 2020).
Kingdom Plantae
Class Magnoliopsida
Order Sapindales
Family Rutaceae
Genus Citrus
Species Citrus aurantifolia
Source: ITIS (1998)
12
2.4.3 Pomelo
Pomelo is the large citrus fruit. It has thick skin or rind and the pulp is sweet or sour. The
size of fruits are up to 25 cm (Bhat and Paliyath, 2016). Peel are light green coloured which
gradually turn yellow when it is ripe. The tree of pummel thrives well in tropical or near
tropical climates. The production of pummel is mostly in the Terai and lower hilly regions
of Nepal. There are three types of pummel viz., white, red and pink pomelo.
The fresh pomelo peels are rich in various nutritional and functional components such as
pectin, essential oils, dietary fibers and phytochemicals. The major components of peels
shows anti-inflammatory, antitumor, anticlotting, antimicrobial and antioxidants activities
(Lan-Phi and Vy, 2015; Wang et al., 2017). Pomelo peels can be used as raw materials for
pickles, jams and teas. The functional ingredients like essential oils, pectin, polyphenols etc.,
can be recovered from peels (Xiao et al., 2021).
Kingdom Plantae
Class Magnoliopsida
Order Sapindales
Family Rutaceae
Genus Citrus
Species Citrus maxima
Source: ITIS (1998)
2.4.4 Production
The report presented in FAO shows the total production of lemons and limes in Nepal is
57912 tonnes. Lime and lemon were widely distributed through the mid hills of Ilam district
in the east to Darchula district in the far west. Table 2.2 shows the amount of production of
lemons and limes in each province. The data for pomelo production has not been published
yet. It has been undervalued in commercial aspect.
13
Table 2.2 Production of lemon and limes in different provinces of Nepal (MT)
Province Lime Lemon
Province 1 21,897 1,679
Madhesh 174 -
Bagmati 7,662 4,751
Gandaki 2,525 1,143
Lumbini 3,787 912
Karnali 1,458 126
Sudurpaschim 2,933 288
Source: MOALD (2022)
2.5 Techno-functional properties of pectin
2.5.1 Pectin gelation
A pectin gel is a three-dimensional network of macromolecules including a solvent. It is

formed by physical or chemical changes that tend to decrease the solubility of pectin,
promoting the formation of local crystallizations (May, 1990). Physicochemical and
functional properties of pectin, such as gelling properties, are highly related to their
structures, including their molecular weight, DE, GalA content, and monosaccharide
composition, which depend on plant sources and extraction methods (Rodsamran and
Sothornvit, 2019). Regardless of DM, pectin can form gels by different mechanisms. In the
case of HM pectin, higher the DM fasten the gel formation. LM pectin is capable of forming
gel by strongly binding divalent ions. The pectin–water interaction is therefore favored by
these two elements. The formation of the three-dimensional network is allowed in part
because of the hydrophobic interactions existing between the methyl groups (Oakenfull and
Scott, 1984). The formation of gel depends on the time, temperature, and the kind of sugar
used (Belkheiri et al., 2021). Pectin quality and purity can vary depending on anhydro
galacturonic acid, degree of esterification, ash content and molecular weight. The low ash
content below 10% content and high anhydrogalacturonic acid above 65% are called good
quality pectin (de Moura et al., 2017).
14
2.5.2 Water/oil holding capacity
Water holding capacity and Oil holding capacity are two functional properties that are related
to texture by the interaction between food product components (Romdhane et al., 2017).
OHC is an important feature of pectin in the food system because pectin with a high OHC
can be used as a stabilizer or emulsifier in high-fat foods, such as some meat products. WHC
depends on the hydration ability of pectin, which could be produced by the OH group in the
structure. The high absorption of water by pectin makes it suitable for reducing the syneresis
rate in some food products (e.g., yoghurt, dairy desserts, etc.) (Khedmat et al., 2020).
2.6 Application of pectin
Pectin are widely popular as textural ingredients in food systems along with cosmetics and
pharmaceutical products. In pharmaceutical industry, pectin is used for release matrix tablets
and encapsulation film. Pectin has vary use in food sectors as stabilizers, thickeners and
emulsifiers. Its major application is in jellies, marmalades, jams, acidified milk products,
yoghurt, spreads, ice cream, bakery glazing, emulsified milk and others (Belkheiri et al.,
2021). Moreover, studied the delivery of volatile system through a layer of emulsion
produced by mixture of whey protein isolate and pectin.
2.6.1 Pectin in yoghurt
Pectin-rich foods like apple pomace, orange fiber, okra and carrot cell wall components have
recently received a lot of attention for their potential to enhance the gel properties, rheology,
texture, and microstructure of set yoghurt (i.e., yoghurt that has been allowed to set in a
container and has a thick texture) by interacting with the casein network (Kieserling et al.,
2019; McCann et al., 2011; Tobil et al., 2020; Wang et al., 2007). It has been noted that the
inclusion of soluble solids in set yoghurts, such as pectin or other hydrocolloids, improves
their physicochemical properties including soluble solids, pH, and acidity (Xu et al., 2019).
The utilization of different hydrocolloids such as flaxseed mucilage, sagu, commercial

pectin has been reported to boost the quality characteristics of yoghurt and storage period
(Basiri et al., 2018; Hasan et al., 2014; Khubber et al., 2021). The changes in composition
and ingredients of yoghurt heightens the sensory appeal of yoghurts. Yekta-Fakhr et al.
(2014) studied the effect of different composition of sugar and pectin in drinking yoghurt.
The low-fat yoghurt, non-fat yoghurt and use of different sweeteners and stabilizers such as
15
CMC, gelatin, carragean, gum has been the recent arising topic for the research to increase
the physiochemical and desirable textural characteristics of yoghurts (Decourcelle et al.,
2004; Kumar and Mishra, 2004; Sobhay et al., 2019).
2.7 Yoghurt
The yoghurt name comes from the Turkish yoğurt and is associated to yoğurmak and yoğun
that means “to knead” and “dense” or “thick” respectively (Yildiz, 2010). The starter culture
start fermentation under controlled temperatures between 42 and 43°C. In the first stage of
the fermentation, the S. thermophilus digest the natural lactose and release lactic acid and
diacetyl as a characteristic flavor component in the yoghurt. The lactic acid lowers the acidity
between pH 4.5 and 3.7 which reduces the activity of S. thermophilus and L. bulgaricus starts
to produce acetaldehyde gives yoghurt typical taste and sour aroma. The low acidity in the
yoghurt does not allow the growing of pathogenic bacteria (Alakali et al., 2008).
2.7.1 Types of yoghurt
Yoghurt can be classified as follows;
1. Set yoghurt
This type of yoghurt is incubated and cooled in the final package and is characterized by a
firm jelly like texture (Aswal et al., 2012).
2. Stirred Yoghurt
This type of yoghurt is incubated in a tank and the final coagulum is "broken" by stirring
before cooling and packing. The texture of stirred yoghurt was less firm than a set yoghurt
somewhat like a very thick (Aswal et al., 2012).
3. Drinking yoghurt
This yoghurt coagulum is broken down to the liquid form before filling (Yekta-Fakhr et al.,
2014).
4. Frozen yoghurt
This type of yoghurt has similar physical state as ice cream, and characterized as sharp, acidic
taste of yoghurt with the coldness of ice-cream. The yoghurt is frozen in batch or continuous
16
freezer after fermentation. It contain high level of sugar and stabilizer (Yildiz, 2010).
5. Concentrated yoghurt
This type of yoghurt is inoculated and fermented in the same manner as stirred yoghurt. The
coagulum the yoghurt is concentrated by boiling off some of the water or filtration (Yildiz,
2010).
6. Flavoured yoghurt
The flavours are added prior to filling or directly into pots. These are in form of either puree
or whole fruit containing as much as 50% sugar in them. Artificial sweetener like aspartame
and saccharine are used in low calorie yoghurt (Aswal et al., 2012).
2.7.2 Health benefits of yoghurt
Yoghurt is suggested to be in daily diet for people of every age group. Yoghurt contains
higher amount of potassium, vitamins, calcium, magnesium, and zinc which improves the
consumer diet. The daily consumer of yoghurt have lower circulating lipids and glucose
levels, as well as reduced systolic blood pressure and insulin resistance. It has positive impact
on osteoporosis, cardiovascular diseases, and diabetes. Also it improves the gut health and
the aid the immune system (Wang et al., 2013).
2.8 Factors affecting the quality of yoghurt
2.8.1 Casein and fat content
Firmness of yoghurt is approximately proportional to the cube of the casein content. Natural
variation in casein content can thus have a marked effect. Evaporating the milk adding skim-
milk powder, or partial ultrafiltration increase firmness. The higher the fat content, the
weaker the gel because the fat globules interrupt the network (Dickinson et al., 1998).
2.8.2 Homogenizing
Homogenization of the milk leads to a much enhanced firmness because the fat globules then
contain fragments of casein micelles in their surface coat by which they can participate in
the network upon acidification. The volume fraction of casein is thus effectively increased.
But homogenization of skim milk makes no difference (Tamime and Robinson, 1985).
17
2.8.3 Acidity
Generally, the preferred pH of yoghurt is between 4.1 and 4.6. (Walstra et al., 2005)
2.8.4 Heat treatment
Heat treatment of the milk considerably enhances firmness. The denatured serum proteins
tend to aggregate, forming large, insoluble complexes that may also increase the viscosity of
the milk. Milk is generally heated for 5 to 10 min at 85 to 90°C (Bhattarai et al., 2015).
2.8.5 Incubation temperature
The lower incubation temperature increases the incubation time as it prolongs the
fermentation process thereby firmness is achieved in the finished product (Walstra et al.,
2005).
2.9 Stabilizers
Stabilizers are hydrocolloids which derived from plants or animals. It modifies the
rheological properties of an aqueous continuous phase in the milk ((Dickinson et al., 1998)).
Stabilizers are added to yoghurt to improve texture, consistency, viscosity and mouth feel,
as well as to lessen whey separation or syneresis in the yoghurt gel (Nauth, 2004). Stabilizers
can trap whey forming a three-dimensional (3D) gel network, limiting the movement of free
water in the system which increases water binding capacity of proteins in yoghurt (Ozer,
2009).
Yoghurt that has less milk solids has a strong propensity for syneresis. However, the total
solids (TS) in the milk determine how much stabilizers are included in the yoghurt mixture.
According to Ozer (2009), "the higher TS level in the milk, the less stabilizer added." The
denaturation of whey proteins increases the firmness and viscosity of the yoghurt. The
addition of milk powder increase total solid in the milk, this result in a variation of proteins
increasing the casein ratio. High protein/TS ratio gives more surface area to make gels more
tight and firmer reducing the syneresis and enhancing the viscosity of the yoghurt (Sodini et
al., 2004).
The stabilizer used in yoghurt depends in their functional properties and usage levels and
the combined interaction effect between stabilizers on the product. FAO and WHO has
18
permitted some stabilizers, a description of the most used stabilizers in yoghurt are shown
in Table 2.3.
Table 2.3 Different stabilizer used in yoghurt approved by the FAO and WHO
Stabilizer Function Recommended

usage level%
Agar Provides viscosity as a gelling agent. 0.25-0.7
CMC Thickener, add viscosity, reduce syneresis. It develops a 0.2-1.5

typical aroma and flavor in the yoghurt.
Carrageenan Stabilizer and gelling agent. Stable at pH 3.5-4.0 0.2-1.5
Gelatin Prevents syneresis. It has gelation properties. 0.3-2.0
Guar gum Thickener. It is stable in higher pH range but lower 0.2-1.5

temperature
Pectin Gelling agent and gives viscosity LM 0.08-0.12
HM 0.08-0.20
Xanthan Thickener, stabilizer. Provides high viscosity, in occasions 0.2-1.5

gum slimy. It does not affect sensory properties in stirred yoghurt.
Maltodextrin Provides a custard-like reversible gel. 1-5
Locust bean Thickener helps to gel properties and reduces syneresis. 0.2-1.5
It contributes in to the formation of aroma and flavor

compounds.
Source: Henrysson (2016)
19
Part III
Materials and methods
3.1 Material collection
Lemon (C. Limon var. Lisbon), lime (C. aurantifolia) and white pomelo (C. maxima) were
collected from local producers of Dharan. The fruits were physically examined for their
wholesomeness. Standard milk was collected from the local market.
3.1.1 Chemicals and equipment required
Chemicals and equipment were used from laboratory of Central Department of Food
Technology and are listed on Appendix A.1 and A.2 respectively.
3.2 Methods
3.2.1 Peel powder preparation
Each fruits were divided and peeled manually. Peels were washed to remove any types of
dirt, dust and residues, then cut in small pieces and dried in hot air oven at 50℃ for 48 h.
The dried peels were grounded using blender. Thus obtained peel powder were packed in
plastic (PP) bags and stored for further extraction. This method was given by Kamal et al.
(2021) as shown in Fig. 3.1.
Fresh Citrus fruits (Lemon, Lime and Pomelo)
Washing with clean water
Splitting fruits in four fractions
Removal of peels manually
Drying in oven at 50℃ for 48 h
Grinding dried peels
Sieving of powder for uniform particle size (Sieve size 80 mm)
Packing the peel powder in PP
Fig. 3.1 Flow diagram for the preparation of citrus peel powder
3.2.2 Extraction procedure
The extraction of pectin was done using the acid extraction method explained by Salam
et al. (2012) and Girma and Worku (2016) with slight modifications shown in Fig. 3.2. The
peel powder of each samples were dissolved in the distilled water at solute-solvent ratio of
1:20. The addition of mineral acid 1N HCl to maintain the pH of solution 1, 2 and 3. The
acidified samples were kept in water bath at temperature of 80, 90 with time to time stirring
till time frame of 60, 90 and 120 min. After heating of solution, supernatant was filtered
using muslin cloth and discarded. The remaining solution was mixed with 96% of alcohol
for precipitation of crude pectin and kept overnight at temperature lower than 10℃. The
gelatinous pectin were skimmed off and washed with 70% ethanol till the clear solution for
four times. Then, it was dried in hot air oven at 50℃ till fully dried. The dried pectin was
grinded to form powder and sealed in Polypropylene plastic for further use.
Peel powder
Addition of 1 N HCl to maintain the pH 2 with solvent ratio 1:20
Heating with stirring for 1.5 h at 80℃ temperature in waterbath
Cooling to room temperature
Filtration with two layer muslin cloth
Addition of 96% ethanol in the ratio 1:1
Precipitate the solution for overnight
Skim off the gelatinous pectin
Washing with 70 % alcohol
Drying in drier at 50℃
Blending in a mortar and pestle
Storage in a plastic bags
Fig. 3.2 Flow diagram for the extraction of pectin
21
3.2.3 Experimental Design
Design expert software (version 13, Stat-Ease Inc., USA) was used for the experimental
design, data analysis and quadratic model construction. The extraction of pectin from peels
was done using different constraints pH, extraction temperature and extraction time. A three-
level; three factor central composite face centered design were employed. The independent
variable selected for the experiments were pH, time, and temperature during extraction and
response variables were yield and anhydrouronic acid content. The design consisting of face
centered one alpha, involved five center points. The variations of the constraints were
determined from Azad et al. (2014) which described pH, time and temperature be 1-3, 80-
100℃ and 60-120 min respectively present in Table 3.1. Table 3.2 represent different range
for the extraction of pectin.
Table 3.1 Range of factors for RSM
Factors Level
-1 0 1
A: pH 1 2 3
B: Temperature (℃) 80 90 100
C: Extraction time (min) 60 90 120
The polynomial equation of yield and AUA for various experimental combinations were
related to coded variables (Xi, i=1, 2 and 3)
Y = β0+ β1X1+ β2X2+ β3X3+β11X12+ β22X22+ β33X32+β12X1.X2+ β13X1.X3+ β23X2.X3+ ε
The polynomial coefficients were expressed by β0 (constant); β1, β2, β3 (linear effects);
β12, β13, β23 (interaction effects); β11, β22, β33 (quadratic effects); and ε (error). Multiple
regression analysis was used for data modelling, while analysis of variance (ANOVA)
for statistical significance of terms.
22
Table 3.2 Experimental design for extraction of pectin
Run pH Extraction time (min) Temperature (℃)
1 1 60 80
2 3 60 80
3 1 120 80
4 3 120 80
5 1 60 100
6 3 60 100
7 1 120 100
8 3 120 100
9 1 90 90
10 3 90 90
11 2 60 90
12 2 120 90
13 2 90 80
14 2 90 100
15 2 90 90
16 2 90 90
17 2 90 90
18 2 90 90
19 2 90 90
20 2 90 90
3.2.4 Characterization of extracted pectin
The obtained dried pectin from optimized conditions of different citrus peels were subjected
to the further characterization and grading.
23
3.2.2.1 Equivalent weight determination
Equivalent weight was determined titration method described by (Ranganna, 1986).

Equivalent wt. was used for calculating the anhydrouronic acid content and the degree of
esterification. It was determined by titration with sodium hydroxide to pH 7.5 using either
phenol red or Hinton’s indicator. Pectin sample weighing 0.5g was taken in a 250 ml conical
flask and 5 ml ethanol was added. Then, 1 g of sodium chloride and 100 ml of distilled water
were added. Finally 6 drops of phenol red was added and titrated against 0.1 N NaOH.
Titration point was indicated by purple colour. This neutralized solution was stored for
determination of methoxyl content.
Weight of sample×1000
Equivalent weight=
ml of alkali ×Normality of alkali
3.2.2.2 Methoxyl content determination (MeO)
The MeO content was determined as described by Ranganna (1986). The neutral solution was
collected from determination of equivalent weight, and 25 ml of sodium hydroxide (0.25 N)
was added. The mixed solution was stirred thoroughly and kept at for 30 min. After 30 min
25 ml of 0.25 N hydrochloric acid was added and titrated against 0.1 N NaOH.
ml of alkali ×Normality of alkali×3.1

MeO=
Weight of sample
3.2.2.3 Determination of degree of esterification (DE)
The DE of pectin was measured on the basis methoxyl and AUA content (Owens et al.,
1952) and calculated by formula given as
176×MeO%
DE%=
31×AUA%
3.2.2.4 Determination of total anhydrouronic acid content (AUA)
Estimation of AUA content was essential to determine the purity and degree of esterification,
and to evaluate the physical properties.
Total AUA of pectin was obtained by the formula described by Mohamed and Hasan
(1995)
24
176 × 0.1z × 100 176 × 0.1y × 100
%AUA = +
W × 1000 W × 1000
Where, z = ml (titer) of NaOH from equivalent weight determination.
y = ml (titer) of NaOH from methoxyl content determination.
W = weight of sample
3.2.2.5 Moisture content
The moisture content was determined by hot air oven method as described by Ranganna
(1986).
3.2.2.5 Ash content
The ash content was determined by dry ashing method at 600℃ as described by Ranganna
(1986).
3.2.2.6 Pectin grades
Pectin grade was determined as per DE value described by Ranganna (1986).
3.2.2.7 Pectin yield
Pectin yield was determined as described by Ranganna (1986).
P
Yield%= ×100%
W
Where, P = amount of the extracted pectin
W= weight of ground peel powder taken
3.3 Preparation of pectin incorporated yoghurt
The pectin extracted from the citrus fruits at optimized conditions were added in production
of yoghurt. The method given by Aswal et al. (2012) was followed for preparation of yoghurt
as given in Fig. 3.3 with addition of 0.2% of pectin suggested by Bhattarai et al. (2015) with
16% TS.
25
Milk (3% fat, 8% SNF)
Preheating
Addition of 4% SMP and 5% sugar
Addition of pectin (0.2%)
Pasteurization (90℃ for 5 min)
Inoculation with 2% starter culture at 40℃
Filling yoghurt mix in 50 ml cups
Incubation of cup (46℃ for 3 h) after covering the top
Cooling
Refrigeration (4℃) for 7 days
Fig. 3.3 Flow diagram for yoghurt preparation
3.4 Physicochemical analysis
The yoghurt samples with addition of pectin (0.2%) from lemon, lime and pomelo peels were
subjected to analysis for the evaluation of physiochemical characteristics along with control
sample.
3.4.1 Moisture content
Moisture content of yoghurt was determined by hot air oven method as described in
Ranganna (1986).
3.4.2 Fat
Fat content of yoghurt was determined by the Gerber method as described in NDDB (2001).
3.4.3 pH
The pH value of yoghurt samples was determined by the direct reading with the digital pH
meter as given by NDDB (2001).
26
3.4.4 Protein
Protein content was determined by formal titration method as described by NDDB (2001).
3.4.5 Ash
Ash content of yoghurt was determined as described by Ranganna (1986).
3.4.6 Total solids
The total solids content of the freshly prepared yoghurt was determined using procedure in
AOAC (1990).
3.4.7 Viscosity
The viscosity of yoghurt was determined as described by Djurdjevic-Denin et al. (2002)

using rotational viscometers (model LCD display, BIOBASE). The temperature of the
system was set and maintained at ambient temperature (25℃) using spindle L3 for 60 s.
3.4.8 Carbohydrate
Carbohydrate (by difference) was determined using method explained in AOAC (1990).
3.4.9 Syneresis
The syneresis in the samples was determined by using the drainage method as Amatayakul
et al. (2006) with slight modifications in the interval of one days up to 7 days.
Yoghurt samples (10 ml) were centrifuged at 3500 rpm for 15 min and supernatants were
weighed to calculate syneresis as follows:
% free whey (g/100 g) = Wt. of initial supernatant×100
Wt. of sample
3.4.10 Titratable acidity
Titratable acidity of yoghurt was determined in every alternate days up to 7 days of storage
by titrimetric method given by AOAC (1990).
27
3.5 Microbiological analysis
Microbiological analysis of fresh yoghurt was done by Total Plate Count Method as
described in Manandhar and Sharma (2013). The count of Yoghurt culture using M 17 and
MRS agar for Steptococccus thermophilus and Lactobacillus bulgaricus (AOAC, 1990) in 1
day interval up to 7 days. The coliform and yeast and mold count was performed for freshly
prepared yoghurt sample as explained by Manandhar and Sharma (2013).
3.6 Sensory evaluation
A panel of ten assessors was selected for the judgment of the samples. The Hedonic scale
(1: dislike very much, 9: like very much) was used for evaluating colour, aroma, taste,
consistency and overall acceptability of the different pectin added yoghurt formulates.
3.7 Statistical analysis
All analysis were performed in triplicates and Excel 2013 measured the means and standard
deviation for the parameters of samples. The data for parameters measures were examined
using SPSS version 20. ANOVA was used to make conclusions about comparison of means
and the post hoc test was used to compare samples and define the significant differences.
28
Part IV
Results and discussions
Citrus fruits lemon, lime and pomelo were chosen as raw materials for the extraction of
pectin. Fruit peels were peeled, dried and grinded to form peel powder. The acid extraction
method was used for the extraction process. The optimization of extraction condition done
from Central Composite Design of Response Surface Method (RSM). The three factor three
level design was followed for the experimental modeling. The independent factors were pH,
extraction temperature and extraction time in response to yield and AUA content of extracted
pectin from different citrus peels. After the optimization of extraction conditions, the
extracted pectin was characterized on the basis of yield, degree of esterification, methoxyl
content, anhydrouronic acid content, moisture, ash and grade.
The pectin was used in preparation of yoghurt. The standard commercial milk was taken
and yoghurt samples with 0.2% of pectin from different peels was produced along with
control sample without pectin. The physiochemical and microbiological analysis of pectin
incorporated yoghurt samples and control sample was performed. For sensory analysis, 10
semi trained panelists judged the sensory parameter on the basis of consistency, colour,
aroma, taste and overall acceptability. The determination of moisture content, fat content,
protein content, total solids, ash, viscosity, sensory analysis were performed as
physiochemical analysis. The change in syneresis and titratable acidity was studied up to 7
days of storage. In case of microbial analysis, TPC, yeast, molds, coliform, and LAB were
analyzed.
4.1 Experimental design for pectin extraction from lemon peels
The effect of independent variables A (pH), B (extraction temperature) and C (extraction

time, min) at three variation levels (in Table 4.1) in the extraction process of lemon peels on
responses yield and AUA.
Table 4.1 Experimental design plan for pectin from lemon peels
Run A: pH B: Temperature Celsius C: Time Min Yield% AUA%
1 2 100 90 16.11 73.22
2 2 90 60 10.6 58.07
3 3 80 120 12.51 69.91
4 2 90 90 12.81 69.90
5 1 100 60 18.45 83.32
6 2 90 90 10.99 72.00
7 3 90 90 8.4 49.23
8 2 90 90 11.51 64.67
9 1 80 60 12.94 83.10
10 2 90 120 15.1 72.29
11 3 100 60 9.5 28.90
12 1 80 120 17.24 75.19
13 2 90 90 12.63 69.24
14 2 80 90 12.03 73.94
15 1 100 120 21.91 86.93
16 3 80 60 6.01 41.39
17 2 90 90 11.71 66.32
18 2 90 90 12.72 70.73
19 1 90 90 17.61 75.32
20 3 100 120 13 71.81
30
4.1.1 Model fitting for yield and AUA of pectin from lemon peels
In this experiment, linear and quadratic model was used for the yield % and AUA% of pectin
extraction from lemon peels respectively. The change in yield % and AUA% of pectin was
represented by the following equation:
Yield= 12.33-3.87A+1.82B+2.23C-0.775AB+0.28AC-0.48BC+0.26A²+1.33 B²+0.1132 C²
AUA= 68.21-14.26A+0.0650B+8.14C-2.82AB+9.47AC+3.24BC-5.02A²+6.28 B²-2.12C²
4.1.2 Effect of process variables for pectin extraction from lemon peels
The analysis of variance (ANOVA) was applied to examine the statistical significance of
model terms A (pH), B (temperature) and C (time) and findings are tabulated in Table 4.2.
The adequacy, fitness, and significance of the linear (A, B, C) quadratic (A2, B2, C2) and
interaction term (AB, BC, AB) effects of variables on the yield and AUA were assessed.
The significant F-value and insignificant lack of fit value showed the regression model
could be fitted well. It was supported by the value of R2 (0.9752) and adjusted R2 (0.9528),
along with C.V. 6.08%. The Table 4.2 showed that temperature (B) and time (C) had the
significant effect whereas pH (A) had negative significant effect (p<0.05). The interaction
terms AB had the positive significant effect (p<0.05) but other terms BC and AB had non-
significant effects (p>0.05). The quadratic term B2 showed positive significant effect
(p<0.05) however other terms did not have any significant effect (p>0.05).
The lower P value (p<0.05) and F-value (76.27) pointed the model was statistically
significant for AUA, together with insignificant lack of fit value as in Table 4.3. For AUA,
pH has negative significant (p <0.05) effect, time had positive significant (p <0.05) effect
and temperature had positive non-significant (p>0.05) effect. The term C2 had no significant
effect (p>0.05) whereas BC, AC, B2 had positive significant (p <0.05) effect. Also quadratic
terms A² and AB had negative significant (p <0.05). The values of R2 (0.9856), adjusted R2
(0.9727) along with C.V. (3.43%) showed model was well correlated. Fig. 4.1, 4.2, 4.3, 4.4,
4.5 and 4.6 showed that increase in both temperature and time along with decreasing pH
increases the yield % and AUA content of extracted pectin from lemon peels.
31
Table 4.2 ANOVA table for model of Yield from lemon peels
Source SS df MS F-value p-value
Model 252.64 9 28.07 43.60 < 0.0001 Significant
A 150.00 1 150.00 233.00 < 0.0001
B 33.27 1 33.27 51.68 < 0.0001
C 49.55 1 49.55 76.97 < 0.0001
AB 4.81 1 4.81 7.46 0.0211
AC 0.6272 1 0.6272 0.9742 0.3469
BC 1.84 1 1.84 2.86 0.1215
A² 0.1978 1 0.1978 0.3072 0.5916
B² 4.89 1 4.89 7.59 0.0203
C² 0.0352 1 0.0352 0.0547 0.8198
Residual 6.44 10 0.6438
Lack of Fit 3.55 5 0.7090 1.23 0.4145 not significant
Pure Error 2.89 5 0.5786
Cor Total 259.08 19
32
Table 4.3 ANOVA table for model of AUA from lemon peels
Model 3700.83 9 411.20 76.27 < 0.0001 significant
A-pH 2034.05 1 2034.05 377.27 < 0.0001
B-Temperature 0.0422 1 0.0422 0.0078 0.9312
C-Time 661.78 1 661.78 122.75 < 0.0001
AB 63.56 1 63.56 11.79 0.0064
AC 716.88 1 716.88 132.97 < 0.0001
BC 83.92 1 83.92 15.56 0.0028
A² 69.43 1 69.43 12.88 0.0049
B² 108.47 1 108.47 20.12 0.0012
C² 12.35 1 12.35 2.29 0.1610
Residual 53.91 10 5.39
Pure Error 38.58 5 7.72
Cor Total 3754.75 19
33
Fig. 4.1 Response curve 3D plot for effect of pH and temperature on pectin yield from
lemon peels
Fig. 4.2 Response curve 3D plot for effect of pH and time on pectin yield from lemon
peels
34
Fig. 4.3 Response surface plot for effect of time and temperature on pectin yield from
lemon peels
Fig. 4.4 Response surface plot for effect of pH and temperature on AUA of pectin from
lemon peels
35
Fig.4.5 Response surface plot for effect of pH and time on AUA of pectin from lemon
peels
Fig. 4.6 Response surface plot for effect of temperature and time on AUA of pectin
from lemon peels
36
4.2 Experimental design for pectin extraction from lime peels
Table 4.4 presented the experimental design for pectin extraction from lime peels.
Table 4.4 Experimental design for pectin extraction from lime peels
Run A: pH B: Temperature℃ C: Time Min Yield% AUA%
1 2 90 120 21.01 38.44
2 1 100 120 39.6 83
3 2 100 90 29.31 62.02
4 1 90 90 26.71 47.74
5 3 100 120 31.93 69.21
6 3 100 60 21.71 42.24
7 2 90 90 19.96 34
8 1 80 60 26.22 51.9
9 2 80 90 21.44 31.21
10 2 90 90 20.11 37.91
11 2 90 90 19.29 33.67
12 1 100 60 36 71.93
13 3 80 120 21.61 35.66
14 3 90 90 17.93 31.18
15 2 90 90 20.01 33.89
16 1 80 120 28.84 46.03
17 2 90 90 19.27 34
18 2 90 60 16.56 30.67
19 3 80 60 13.51 23.91
20 2 90 90 19.32 37.5
37
4.2.1 Model fitting for yield and AUA of pectin from lime peels
In this experiment, quadratic model was suggested for the yield % and AUA% of pectin
extraction from lime peels. The change in yield % and AUA% of pectin was represented by
the following equation:
Yield = 19.62 -5.06A+4.69B +2.89C -0.25AB+1.51AC+0.38BC+2.75A2+5.81B2-0.77C2
AUA=34.77 -9.84A +13.96B+5.16C-0.64AB+4.19AC+4.02BC+5.27A2+12.42B2+0.36C2
4.2.2 Effect of process variables for pectin extraction from lime peels
According to F-value and P values (p <0.05), the model was statistically significant and well
fitted for both yield and AUA of extracted pectin from lime peels. The lack of fit for both
models showed it could be used to determine the optimum parameters of pectin extraction
from lime peels. Table 4.5 and 4.6 showed the pH (A) showed the negative significant (p
<0.05) effect but temperature (B) and time(C) had the positive significant (p <0.05) effect
on the model of yield and AUA at 95% level of confidence. The interaction terms AB and
BC illustrated non-significant (p>0.05) effect whereas AC had positive significant (p <0.05)
effect on yield. Also quadratic terms A² and B² had positive significant (p <0.05) effect but
C² had no significant effect. As for AUA, only AC and BC gave positive significant (p <0.05)
effect along with A² and B² in contrast to terms AB and C2 .The values of adjusted R2 for
yield was 0.9894 and AUA was 0.9849 similarly R2 for yield was 0.9944 and AUA was
0.9921 supporting the fitness of the model as well as lower C.V. for yield (2.93%) and AUA
(4.47%). The surface plots Fig. 4.7, 4.8, 4.9 represents the effect of factors on yield % of
pectin extracted from lime peels. The increase in temperature, time and decrease in pH had
increased the pectin yield. Similarly, the same condition was followed by factors for AUA
content of pectin. The AUA content was increased with increase in temperature, time and
decrease in pH as illustrated in Fig. 4.10, 4.11 and 4.12.
38
Table 4.5 ANOVA table for quadratic model of yield from lime peels
A-pH 256.85 1 256.85 542.49 < 0.0001
B-Temperature 220.24 1 220.24 465.18 < 0.0001
C-Time 84.04 1 84.04 177.51 < 0.0001
AB 0.5100 1 0.5100 1.08 0.3238
AC 18.30 1 18.30 38.65 < 0.0001
BC 1.20 1 1.20 2.54 0.1423
A² 20.91 1 20.91 44.16 < 0.0001
B² 92.90 1 92.90 196.22 < 0.0001
C² 1.66 1 1.66 3.51 0.0904
Residual 4.73 10 0.4735
Pure Error 0.8196 5 0.1639
Cor Total 852.00 19
39
Table 4.6 ANOVA table for quadratic model of AUA from lime peels
Model 4803.75 9 533.75 138.91 < 0.0001 significant
A-pH 968.26 1 968.26 252.00 < 0.0001
B-Temperature 1951.33 1 1951.33 507.85 < 0.0001
C-Time 267.19 1 267.19 69.54 < 0.0001
AB 3.28 1 3.28 0.8528 0.3775
AC 140.45 1 140.45 36.55 0.0001
BC 129.28 1 129.28 33.65 0.0002
A² 76.44 1 76.44 19.89 0.0012
B² 424.70 1 424.70 110.53 < 0.0001
C² 0.3709 1 0.3709 0.0965 0.7624
Residual 38.42 10 3.84
Pure Error 19.56 5 3.91
40
Fig. 4.7 Response surface plot for effect of pH and temperature on pectin yield from
lime peels
Fig. 4.8 Response surface plot for effect of pH and time on pectin yield from lime peels
41
Fig. 4.9 Response surface plot for effect of temperature and time on pectin yield from
lime peels
lime peels
42
Fig. 4.11 Response surface plot for effect of pH and time on AUA of pectin from lime
peels
from lime peels
43
4.3 Experimental design for pectin extraction from pomelo peels
Table 4.7 presented the effect of process variables on pectin extraction from pomelo peels.
Table 4.7 Experimental design for pectin from pomelo peels
Run A: pH B: Temperature C: Time Yield% AUA%
1 1 80 120 20.3 79.00
2 2 90 90 17.9 66.21
3 3 80 120 18.2 55.01
4 2 90 90 18.9 55.90
5 1 80 60 16.4 69.00
6 1 100 60 30.1 73.54
7 3 90 90 12.5 44.92
8 2 90 60 14.6 57.98
9 2 100 90 23.5 73.27
10 2 90 90 17.5 63.01
11 3 100 60 11.8 52.58
12 1 90 90 23.8 68.50
13 2 90 90 19.5 67.50
14 2 90 120 21.5 64.12
15 2 90 90 19.3 66.10
16 3 100 120 19.5 53.48
17 2 90 90 18.3 56.64
18 3 80 60 6.1 54.40
19 2 80 90 17.5 73.50
20 1 100 120 29.2 81.10
44
4.3.1 Model fitting for yield and AUA of pectin from pomelo peels
The quadratic model was suggested for the yield and AUA content of pectin extracted from
pomelo peels. The equation given for change in yield and AUA as following:
Yield = 18.66-5.17A+3.56 B+2.97 C-1.95 AB+2.10 AC-1.15 BC-0.64A²+1.71 B²-0.74C²
AUA= 62.74-11.07A+0.306B+2.52C-1.25AB-2.01AC-0.2688BC-6.31A²+10.37B²-1.97C²
4.3.2 Effect of process variables for pectin extraction from pomelo peels
The ANOVA Table 4.8 and 4.9 explained the significant F-value and insignificant value
for lack of fit confirming the model could be well fitted for yield and AUA of pectin extracted
from pomelo peels. The p-value represented temperature (B) and time(C) had the positive
significant (p<0.05) effect and pH (A) had negative significant (p<0.05) effect on the model
of yield. The model for yield had negative significant (p<0.05) effect from interaction AB
and BC and positive significant (p<0.05) effect from AC and quadratic term B2. However,
A2 and C2 had non-significant (p>0.05) effect on yield. The model for AUA, pH (A) had
negative significant (p<0.05) effect while temperature and time had positive non-significant
(p>0.05) effect on AUA. The quadratic terms A2 and B2 had significant effect though C2 had
no any significant (p>0.05) effect. There interaction terms held non-significant (p>0.05)
effect for model. The R2 value for yield and AUA of pomelo pectin was 0.9907 and 0.9250
along with adjusted R2 value for yield and AUA was 0.9824 and 0.8575 respectively showing
the model were well correlated. This was supported by lower C.V. for yield 3.87% and AUA
5.74%. From Fig 4.13, 4.14 and 4.15, it could be inferred that yield was directly proportional
to the temperature and time but inversely related to the pH used for extraction condition. The
response curve for AUA showed the tortuous surface. Fig. 4.16, 4.17 and 4.18 showed that
the increased pH reduced the AUA content whereas the increasing time upturned the value
of AUA.
45
Table 4.8 ANOVA table for quadratic model of yield of pectin from pomelo peels
A-pH 267.29 1 267.29 504.77 < 0.0001
B-Temperature 126.74 1 126.74 239.34 < 0.0001
C-Time 88.21 1 88.21 166.58 < 0.0001
AB 30.42 1 30.42 57.45 < 0.0001
AC 35.28 1 35.28 66.63 < 0.0001
BC 10.58 1 10.58 19.98 0.0012
A² 1.13 1 1.13 2.13 0.1748
B² 8.03 1 8.03 15.17 0.0030
C² 1.51 1 1.51 2.85 0.1222
Residual 5.30 10 0.5295
Pure Error 3.17 5 0.6347
Cor Total 571.89 19
46
Table 4.9 ANOVA table for quadratic model of AUA of pectin from pomelo peels
Model 1651.66 9 183.52 13.70 0.0002 Significant
A-pH 1226.56 1 1226.56 91.56 < 0.0001
B-Temperature 0.9364 1 0.9364 0.0699 0.7969
C-Time 63.55 1 63.55 4.74 0.0544
AB 12.48 1 12.48 0.9312 0.3573
AC 32.20 1 32.20 2.40 0.1521
BC 0.5778 1 0.5778 0.0431 0.8396
A² 109.45 1 109.45 8.17 0.0170
B² 295.52 1 295.52 22.06 0.0008
C² 10.66 1 10.66 0.7956 0.3934
Residual 133.96 10 13.40
Pure Error 129.86 5 25.97
47
Fig. 4.13 Response surface plot for effect of pH and temperature on pectin yield from
pomelo peels
Fig. 4.14 Response surface plot for effect of pH and time on pectin yield from pomelo
peels
48
Fig. 4.15 Response surface plot for effect of temperature and time on pectin yield from
pomelo peels
pomelo peels
49
Fig. 4.17 Response surface plot for effect of pH and time on AUA of pectin from
pomelo peels
from pomelo peels
50
4.4 Validation of optimization condition
The numerical optimization method was to optimize the variables for yield % of pectin and
AUA % (as responses) with the maximum goal as presented in Table 4.10.
Table 4.10 Different parameters for optimization
Parameters Goal Lower limit Upper limit
pH in the range 1 3
Temperature (℃) in the range 80 100
Time (min) in the range 60 120
Yield% maximize
Lemon 6.01 21.91
Lime 13.51 39.6
Pomelo 6.1 29.2
AUA% maximize
Lemon 28.97 86.93
Lime 23.91 83
Pomelo 44.92 81.10
To verify the stability and accuracy of the experiments; based on higher desirability
values better, three optimum experimental runs were selected as with their corresponding
optimal conditions. Among these three better experimental runs, the best-optimized
condition with highest desirability was observed. The best-optimized condition was verified
through the estimation of yield and AUA of the each citrus peel pectin extract. RSM had
provided the predicted nearly equal to the observed value which confirms it be used
effectually for optimizing the factors in complex processes. The optimized condition of
factors along with their predicted and observed for each citrus peels were explained on the
Table 4.11. The highest desirability of the experimental conditions were taken in account
and performed the conformity experiment. The desirability for the optimized condition of
51
pectin extraction from lemon peels, lime peels and pomelo peels were 0.996, 0.986 and 0.999
respectively. The optimized condition of pH and time was in similar range to the conditions
reported by Rady et al. (2021).
Table 4.11 Optimized conditions of each peels and their response values
pH Temperature Time Predicted Observed
Yield AUA Yield AUA
Lemon 1 100 118.6 21.91 86.49 20.5 82.64
Lime 1 100 120 39.20 82.29 38.97 78.25
Pomelo 1 100 105.4 30.1 82.36 30.2 82.36
4.5 Physiochemical characterization of pectin
The physicochemical analysis of pectin obtained from different citrus peels under the
optimized conditions were evaluated for the purity and fitness in products. The obtained
results of physicochemical properties were presented in Table 4.12.
Table 4.12 Physiochemical characterization of extracted pectin from citrus peels
Parameters Lemon Lime Pomelo
Equivalent Wt. (g/ml) 492.32a±15.83 488.30a±32.3 535.09a±25.91
MeO (%) 8.24b±0.18 6.38a±0.23 8.61b±0.077
AUA (%) 82.6b±1.95 72.45a± 4.2 81.89ab±1.29
D.E. (%) 56.68b±0.67 50.04a±1.28 59.76c±0.58
Moisture (%) 8.18a±0.202 11.36b±0.313 10.79b±0.09
Ash (%) 6.52c±0.028 4.04a±0.09 4.7b±0.17
Pectin grade HM HM HM
Values are the means of triplicate data.
52
4.5.1 Equivalent weight
The mean value of equivalent weight of pomelo peel pectin was higher than lime and lemon.
Statistically, there was no difference among the pectin extracted from different citrus peels
(p>0.05). High equivalent weight would have higher gel forming effect. Azad et al. (2014)
reported the equivalent weight of extracted pectin from lemon pomace ranged from 368 to
1632. In context of lime peel pectin, values slightly low than reported in the study of lime
peels by Rodsamran and Sothornvit (2019) ranged 635.63 to 2219.39 g/ml which could be
due to use of microwave extraction method. The equivalent weight of pectin extracted from
lime and lemon was 326.79- 396.82 g/ml and 980.4-1428.57g/ml respectively suggested by
Wonago (2016). Lower equivalent weight could be higher partial degradation of pectin
(Akhtar et al., 2020).
4.5.2 Methoxyl content
The MeO value of pectin extracted from lime peels was significantly different to the pectin
from lemon and pomelo peels (p<0.05). Also, the study of Rodsamran and Sothornvit (2019)
showed the methoxyl content of lime peel pectin varied from 8.74 to 10.51%. MeO content
of lemon peel pectin ranged 4.24-10.25% as per the maturity stage (Azad et al., 2014).
Different extraction conditions produced pectin with slight differences in the methoxyl
content. The highest methoxyl content was found using citric acid with conventional heating
(Wonago, 2016).
4.5.3 AUA content
The AUA content of pectin from pomelo peels had no significance difference with pectin
from lime and lemon pectin (p>0.05) however there was significance different between
pectins of lemon and lime (p<0.05). The GA content in the extracted lime peel pectin ranged
from 79.29 to 95.93 % Rodsamran and Sothornvit (2019) . The results from study by
Wonago (2016) showed that the AUA content of pectin extracted lemon from 63.712-
77.44% as compared to that of pectin extracted from lime 51.34-67.89%. The anhydrouronic
acid content increased by increasing time of extraction. A minimum value of 65% AUA for
commercial pectin has been specified by FAO (2007) and IPPA (2001). However, the AUA
content obtained of citrus peels pectin were greater than 65%. Result indicated that the pectin
53
was sufficiently pure due to the possible absence of proteins, starch and sugars in the
precipitated pectin (Nazaruddin et al., 2011).
4.5.4 Degree of esterification
The citrus peels pectin designated to be high methoxyl pectin as DE values were higher than
50%. The DE content of 88.6% from lemon peels was reported by Salam et al. (2012) at 80-
90°C. Rodsamran and Sothornvit (2019) stated the values of the DE of pectin extracted from
lime peel were in the range 70.81-91.58%. The difference could be caused by differences in
species, extraction techniques. DE increased significantly with increasing pH (Woo et al.,
2010). The degree of esterification ranged from 49.99 to 63.31%, and when the extraction
time was increased, the degree of esterification decreased significantly (Sayed et al., 2022).
4.5.5 Moisture content
The value of moisture content was similar to the lime (Akhtar et al., 2020). The value of
moisture content of pomelo peels ranged 14.60-18.69% as per different extraction methods
as described by Liew (2019). The study from Wonago (2016) suggested that the pectin
extracted from lime and lemon had moisture content ranged of (9.9-10.2%) and (8.67-8.89%)
respectively. Based on the quality standards of commercial pectin, all of pectin is produced
to meet the standards not far above 12%.
4.5.6 Ash content
The ash content of lemon seemed similar to the study from Dhushane and Mahendran
(2020). The ash content from pomelo peels are higher than the reported Liew (2019) 1.10-
2.96% . Wonago (2016) reported the range of 3.11-3.29% for the Mexican variety of lime at
different pH 1-2.5 and temperature 60 -80. The ash content of pectins extracted from various
banana peels was between 1.38 and 2.87% which was in similar range to that obtained from
the conventional pectin sources, citrus peel (3.46%) and apple pomace (1.96%). The ash
content indicates the purity of the pectin. Lower ash content means higher purity
(Khamsucharit et al., 2018). The ash content in pectin must be less than 10% for commercial
use (Ranganna, 1986).
54
4.6 Sensory analysis of pectin added yoghurt
The yoghurt was prepared using 0.2% extracted pectin with added sugar to maintain the total
solid of yoghurt 16, and control yoghurt (0% pectin) were subjected to sensory evaluation.
The samples were provided to 10 semi trained panelist for judgment of products according
to colour, aroma, taste, consistency and overall acceptability. The average of all the scores
in the evaluation sheet and the statistical method were used to find the relative sensory
characteristics of product summarized in Fig. 4.19.
A B C D
9 ab b ab b
a b b b b ab ab
8 ab b a
7 a a
a
a a a
Sensory score
6
5
4
3
2
1
0
Colour Aroma Taste Consistency OA
Fig. 4.19 Sensory characteristics of pectin added yoghurt
4.6.1 Colour
The mean sensory score of colour for sample A, B, C and D were 5.5±0.5, 5.5±0.8, 7.2±0.97
and 5.5±0.75 respectively. The highest mean score was observed for the sample C. There
was no significance difference among the samples statistically (p>0.05) thus addition of
pectin from different citrus sources at same concentration did not have any significant effect
on appearance or colour of yoghurt. The result was similar to the addition of agbagoma okra
pectin in yoghurt conveyed by Tobil et al. (2020).
55
4.6.2 Aroma
For aroma, the mean sensory score of A, B, C and D were 7.3±0.64, 7.1±1.220, 6.5±1.11and
5.9±0.94 respectively. The sensory score observed for sample A was highest whereas D
obtained lowest score. The sample A and D had no significant (p>0.05) difference. However,
A and B were significantly different to C and D (p<0.05). The presence of polysaccharides
in yoghurts tended to reduce the concentration of aroma compounds in the headspace of the
samples. This effects on aroma release could be due to the interactions between
polysaccharides and aroma compounds (Decourcelle et al., 2004).
4.6.3 Taste
The mean sensory score of taste for A, B, C and D were 6.6±1.35, 7.4±0.48, 6.2±1.07 and
4.9±0.83 respectively. The sample B had the highest score and sample D has the lowest
score. From statistical point of view, sample D was significantly different to sample A, B
and C (p<0.05). It might be resulted from presence of volatile aromatic compounds on lemon
peels due to less purity of pectin (Mao et al., 2014).
4.6.4 Consistency
The average sensory value observed for consistency of yoghurt samples A, B, C and D were
6.2±1.4, 7.6±0.91, 6.9±1.04 and 7.6±0.66 respectively. Statistically, the consistency of
sample B, C and D had no significant different to C (p>0.05) whereas A was significantly
different to B and D (p<0.05). It could be the different gel forming properties of pectin from
different samples that resulted to different consistencies of yoghurt (Gyawali and Ibrahim,
2018).
4.6.5 Overall acceptance
There was no significance difference between the sample C and B to control A (p<0.05).
The mean sensory score of overall acceptance were 7.5±0.5, 7±1.18, 6.9±1.04 and 6.1±0.7
for samples A, B, C and D respectively. So, it could be suggested that yoghurt incorporated
with pomelo pectin was best in comparison to others.
56
4.7 Physiochemical properties of yoghurt
The data of physiochemical analysis of yoghurt samples incorporates with pectin and control
sample were tabulated on Table 4.13. The pH of yoghurt samples were 4.51, 4.49, 4.6 and
4.53 for the sample A, B, C and D respectively.
Table 4.13 Proximate analysis of pectin added yoghurt
A B C D
Moisture 86.13a±0.38 85.73a±0.51 85.33a±0.79 84.94a±0.68
Protein 4.96ab±0.18 5.64b±0.19 5.64b±0.13 5.38a±0.20
Fat 1.062ab±0.004 1.068b±0.008 1.07b±0.006 1.048a±0.003
Ash 1.103a±0.041 1.21a±0.021 1.196a±0.070 1.193a±0.028
TS 14.43a±0.84 15.09a±1.22 15.61a±0.42 15.55a±2.077
Carbohydrate 6.74a±0.84 6.35a±0.8 6.76 a±0.45 7.44 a±0.49
Viscosity(mPa.s) 1713.37a±98.8 2205.97b±63.6 2969.93c±78.1 2963.53c±76.9
Values are the means ± SD of triplicate data.
The proximate analysis of the pectin added yoghurt samples showed sample A had the
highest moisture samples B, C and D. It is evident that the moisture content decreased with
addition of pectin in acidified food products such as yoghurts as it prevents the
agglomeration of the milk proteins, caseins, thus preventing water loss (Mbaeyi-Nwaoha et
al., 2019). Also, the capacity of pectin to form complexes of protein and polysaccharide
stabilizes protein structure through carbohydrate-water interactions. These interactions lead
to the formation of a three dimensional network that traps water within it to form a rigid
structure that is resistant to flow resulting in a higher WHC (Saha and Bhattacharya, 2010).
Statistically, there was no significance difference among samples.
The yoghurt samples B, C and D had the slight increment in protein content with addition
of pectin than the control sample A. Mbaeyi-Nwaoha et al. (2019) had reported the similar
57
result as pectin added samples showed higher value. This might be credited to difference in
moisture content as dilution effect as pectin increase the water binding capacity (Brejnholt,
2010). The significance difference was observed in the protein content of samples with
pectin from different peels.
The effect of fat in pectin added yoghurt and control yoghurt was significant. The results
were compatible to the findings of Sobhay et al. (2019). The yoghurt with 0.2% of pectin
had the fat of 1.46%.
There was no statistical significant difference among the pectin added samples and
control sample regarding about ash content on samples. Sobhay et al. (2019) studied the ash
content of cow milk yoghurt incorporated 0.2 % pectin 0.42% which was lower than value
observed in this study. This difference resulted from variation on ingredients used to prepare
yoghurt.
Moreover, the total solid content of yoghurt samples seemed not to have significant effect
of pectin addition on sample to the control sample with no addition of pectin. The increase
in total solid content had been noticed similar to the Khubber et al. (2021). Although TS
content of control sample is lowest. The readings were slightly more than the study suggested
by Sobhay et al. (2019).
In terms of carbohydrate content, sample A, B, C and D contained 6.76, 6.35, 6.74 and
7.44 respectively. The carbohydrate contents of pectin added yoghurt samples were slightly
higher. That could be attributed to presence of neutral sugars in pectin. The results were
slightly lower than the jackfruit and passion fruit peels pectin added in stirred yoghurt
(Mbaeyi-Nwaoha et al., 2019).
The viscosity of the yoghurt was directly affected by the addition of the pectin. The lowest
viscosity was observed in sample A. Addition of pectin has increased the viscosity of yoghurt
similar to the result shown in study of Arioui et al. (2017) for addition of pectin from orange
peels. The three dimensional network formed by pectin with milk complex could absorb the
maximum amount of water thus resulting the increased viscosity (Dickinson et al., 1998).
58
4.7.1 Syneresis
In this study, the syneresis of yoghurt was calculated on every alternate day up to 7 days
presented in Table 4.14. The addition of pectin had reduced the amount of whey loss in
yoghurt samples A, B, D on comparison to control C. The highest amount of whey was
observes in control samples whereas the least was observed on sample A. Up to the analysis
till day 7, it was clear the stable condition of gel was observed in sample D. There was less
syneresis at the day 7, in sample D followed by sample A, B and finally C with the excess
amount of syneresis. Whey exudation has inverse relation with amount of pectin added on
the yoghurt. Similar result was obtained by Arioui et al. (2017) . Statistically, there was
significant difference among the samples. It could be concluded that pectin addition had the
significant effect on the syneresis of the yoghurt. Yekta-Fakhr et al. (2014) concluded
increasing in syneresis with longer storage time. Meanwhile, Hasan et al. (2014) vouched
against the results with addition of meteroxylon sagu in yoghurt as stabilizer
The post fermentation drainage of whey is prevented through increased protein density
by fat globules yoghurt. Protein-polymer bonding owing to the presence of negatively
charged carboxyl groups in pectin and positively charged casein led to strengthened gel
network (Xu et al., 2019).
Table 4.14 Syneresis of yoghurt samples during storage period of 7 days
Day\Sample A B C D
1 16.40914cA± 13.72266bA± 9.967442aA± 11.2625aA±
0.654593 0.402917 0.611252 0.862761
3 18.17084dB± 15.52909cB± 11.12011aA± 13.46637bB±
0.223932 0.388444 0.605611 0.341055
5 21.20843dC± 18.51975cC± 13.19791aB± 14.87094bC±
0.202873 0.165512 0.368788 0.16101
7 22.5155bC± 20.70194bD± 17.38676aC± 16.17965aC±
0.546978 0.79677 0.419011 0.846308
59
Mean values in each rows with different superscript in lower case are significantly different
at 5% level of significance. Mean values in each column with different superscript in upper
case are significantly different at 5% level of significance.
4.7.2 Titratable acidity
According to Kumar and Mishra (2004), addition of stabilizer slightly increased the rate of
acid development. Titratable acidity was statistically significant among the samples for 7
days. Table 4.15 showed control sample A had the lowest acidity up to 7 days whereas
acidity of pectin added samples had risen significantly. Khubber et al. (2021) had observed
the similar results for addition of pectin in different concentration for yoghurt.
The acid influence on flavor was better predicted by the titratable acidity. The titratable
acidity of sample raised with increase in amount addition of pectin. Sahan et al. (2008)
observed there was an increase in the titratable acid content during the storage period which
could be due to the accumulation of organic acids. The increase in titratable acidity with
increase in pectin concentration was observed by Arioui et al. (2017). The observed
differences are caused by the various structures of pectin recovered from different kinds,
which result in the diverse qualities they give (Kpodo et al., 2017).
Table 4.15 Titratable acidity of yoghurt samples during storage period of 7 days
Day\Sample A B C D
1 0.89059aA± 0.98187aA± 0.962aA± 0.9677aA±
0.03704 0.15454 0.01694 0.17966
3 1.0824aAB± 1.25266aAB± 1.05881aA± 1.01514aA±
0.16437 0.0339 0.16912 0.038062
5 1.19255aBC± 1.32611aB± 1.22534aA± 1.33799aA±
0.04513 0.12985 0.031744 0.14713
7 1.37252aC± 1.57508aB± 1.51185aA± 1.47105aA±
0.04218 0.03978 0.0092 0.07959
60
Values are the means ±SD of triplicate data. Mean values in each rows with different
superscript in lower case are significantly different at 5% level of significance. Mean values
in each column with different superscript in upper case are significantly different at 5% level
of significance
4.8 Microbial analysis
Table 4.16 showed microbiological count of the pectin added set yoghurt samples. There
were no significant differences in among the microbial parameters of sample products
incorporated with pectin and control sample. Therefore, it could be stated that,
concentrations of stabilizers used in making yoghurt do not effect on viability of yoghurt
starter cultures. These results agreed with Basiri et al. (2018) who found that, addition of
mucilage as stabilizer to yoghurt did not significantly effect on the growth of starter culture
in the final product along the storage period. However, samples A and D had significance
difference for the L. bulgaricus and S. thermophiles at 1st day and last day of storage
respectively. The mean count for L. bulgaricus seemed to increase in 7 days storage period
in contrast to meant count for S. thermophilus. This similar condition was observed for
yoghurt incorporated with orange peel pectin in 7th day storage by Arioui et al. (2017).
There was no coliform bacteria in set yoghurt samples because of the efficient heat
treatment of the milk and hygienic conditions during preparation and storage of samples.
Also, the effect of acidity developed during fermentation might prevented the growth of
pathogenic bacteria. All fresh yoghurt samples were free from yeast and mold. The obtained
results were in agreement with Sobhay et al. (2019).
FAO (2003) stated the minimum microbial content including starter culture must be min
1.0 × 107 cfu/ml whichever process was adopted, there was a general understanding that
yoghurt should contain live bacteria unless labelled as heat treated or as pasteurized. Thus,
the results were complaint with the standard just as explained by Mbaeyi-Nwaoha et al.
(2019). The research of Gyawali and Ibrahim (2018) on Greek style yoghurt had shown
slight variation on microbial count of LABs 7.10 ± 0.07 and 8.94 ± 0.06 for L. bulgaricus
and S. thermophlius respectively.
61
Table 4.16 Microbial analysis of pectin added yoghurt (log cfu/ml)
A B C D
TPC Day 1 6.48A±1.05 7.79A±1.39 6.5A±0.53 7.45A±1.167
S. thermophilus Day1 6.93aA±0.66 6.59aA±0.79 6.4aA±1.03 6.04cA±0.34
Day 3 6.38aA±0.53 6.43aA±0.66 6.40aA±1.05 5.63bcA±0.25
Day 5 5.88aA±0.61 5.91a A ±0.7 5.54aA±0.98 4.97abA±0.21
Day 7 5.15aA±0.77 4.98aA±0.61 5.25aA±0.98 4.64aA±0.36
L. bulgaricus Day1 6.49aA±0.37 6.74aA±0.81 6.31aA±1.29 6.31aA±1.41
Day3 6.92abA±0.49 7.01aA±0.79 6.75aA±0.93 6.66aA±0.39
Day5 7.29abA±0.35 7.34aA±1.13 7.24aA±0.77 7.69aA±0.4
Day7 7.66bA±0.28 7.83aA±0.72 7.76aA±0.22 7.84aA±0.45
Values are the means ±SD of triplicate data. Lower case superscript on mean values denotes
samples on storage days are significantly different at 5% level of significance. Upper case
superscript on mean values denotes the samples with different pectin are significantly
different at 5% level of significance
62
Part V
Conclusions and Recommendations
5.1 Conclusions
The study of optimization and characterization of pectin extracted from citrus fruits peels
(Lemon, lime and pomelo) and its effect on quality parameters of yoghurt concluded as:
1. The optimum pH, temperature and time for pectin extraction from lemon peel was 1,
100℃ and 118.6 min, from lime peel was 1, 100℃ and 120 min, and from pomelo peel
was 1, 100℃ and 105.4 min respectively.
2. The characteristics of pectin extracted from lemon, lime and pomelo peels regarding
equivalent weight of E.W. was 492.31, 488.30 and 535.09, methoxyl content was 8.24,
6.38 and 8.61, D.E. was 56.68, 50.04 and 59.76, AUA content was 82.8, 72.45 and 81.89,
ash content was 6.52, 4.04 and 4.7 respectively.
3. The addition of 0.2% of respective citrus peels pectin in yoghurt had significant effect
on sensory characteristics such as aroma, taste, consistency and overall acceptability
except for colour. Sample B has higher mean sensory score in taste, consistency and no
significant difference with control sample A thus could be chosen as best one.
4. The addition of pectin had significant effect on physiochemical parameters of yoghurt

such as moisture, protein, fat and viscosity whereas no effect on ash content and total
solids.
5. There was no significant effect on the microbial profile of yoghurt by addition of pectin.
6. The syneresis content had been significantly affected by the pectin addition in yoghurt
and sample A had lowest syneresis whereas pectin had no any effect on titratable acidity.
5.2 Recommendations
1. Novel methods of pectin extraction such as microwave extraction, ultrasonic extraction,

and supercritical water extraction can be used and characterized.
2. The nature of sugar can be studied for better understanding of structure of pectin.
3. Effect of addition of pectin in quality parameters of yoghurt prepared from the different
milk samples.
4. Extraction of pectin from different fruit pomace and vegetable peels and study of effect
on textural and quality properties of food products
64
Part VI
Summary
Pectin is a polysaccharide, a complex carbohydrate found in the cell walls of many fruits and
vegetables. It is popular as thickeners, emulsifiers and stabilizers and used in various food
products such as jam, acidified milk, yoghurt, confectionaries and others. The by-product
waste in fruit and vegetable processing industries mainly peels has high amount of pectin
content. Thus, peels of the fruits can be used as alternative source of pectin production. The
application of pectin as stabilizer improves the texture, body, consistency and stability of the
yoghurt. The aim of this research is to optimize and characterize the extracted pectin from
citrus peels of lemon, lime and pomelo and to study the effect on the quality parameters of
yoghurt. The peels of citrus fruits were washed, diced and dried then grinded to produce the
powder form. The powdered peels were used to extract the pectin by acid extraction method
with pH 1-3, temperature 80-100℃ and time 60-120 min. RSM with CCD was used to
optimize the extraction condition with response variable yield and AUA. The optimum
condition was used to extract the pectin and characterized for equivalent weight, methoxyl
content, AUA, DE, moisture and ash content. Also, the extracted pectins from each citrus
peels were incorporated in yoghurt sample at 0.2% concentration. The physiochemical
properties and microbial analysis of yoghurt were performed. The syneresis and titratable
acidity of yoghurt were studied up to 7 days storage of yoghurt.
The optimum condition for extraction of pectin from lemon peels was pH 1, 100℃ and
118.6 min, lime peels was pH1, 100℃ and 120 min, and pomelo peels was pH 1, 100℃ and
105.4 min respectively. For characterization of pectin obtained from citrus peels, parameters
such as equivalent weight, methoxyl content, AUA, degree of esterification, moisture
content and ash content were determined. The highest equivalent weight of pectin was of
pomelo as 535.09 followed by lemon with 492.31 then lime as 488.30. The methoxyl content
of lemon, lime and pomelo was 8.24, 6.38 and 8.61 respectively. The AUA content found to
be 82.6, 72.45 and 81.89 for lemon, lime and pomelo respectively. The degree of
esterification was 56.68 for lemon, 50.04 for lime and 59.76 for pomelo. Consequently,
pectin extracted from these citrus peels could be classified as high methoxyl pectin. The ash
content of lemon, lime and pomelo peels pectin were 6.52, 4.04 and 4.7 respectively. These
findings were less than 10% which allowed the commercial usage of pectin in food products.
Yoghurt samples were prepared with incorporation of pectin from citrus peels at 0.2%
concentration and a control sample without pectin. The sensory evaluation suggested that
pectin had significant effect on aroma, taste, consistency, overall acceptability except on
colour. The sample B with pomelo pectin had no significant difference to control in mean
sensory score for overall acceptability therefore it could be suggested as best product. The
physiochemical analysis of yoghurt samples revealed lower moisture content, higher protein
and ash content on samples incorporated with pectin than control samples. In other hand,
statistically the addition of pectin had no significant effect on moisture content, ash, TS and
carbohydrate. Viscosity of pectin added product was higher than control samples and has
significance difference to control samples.
The addition of pectin had minimized the syneresis of the yoghurt samples. The study
conducted till 7 days of storage revealed the whey loss of pectin added samples were lesser
than control sample. Sample C had high mean score than other yoghurt samples however,
D had lowest syneresis value up to 7th day. Statistically, addition of pectin had significant
effect on the syneresis of the yoghurt. In context of titratable acidity, pectin had no significant
effect on yoghurt statistically though, addition of pectin had increased the titratable acidity
of yoghurt samples. There was no significant effect of pectin on starter culture of yoghurt.
Therefore, the optimum conditions and characterization of pectin indicated the use of pectin
on food products. The physiochemical, sensory and microbial data evidenced the application
of pectin as stabilizer in yoghurt commercially.
66
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Appendices
Appendix A
A.1 List of Chemicals
Chemicals Manufacturer
Hydrochloric acid Fischer Scientific
Ethanol Changsung Chemical co.
Sodium chloride Real Chemsys product
Sodium hydroxide Fischer Scientific
Sulphuric acid Fischer Scientific
Formaldehyde Fischer Scientific
Phenol red British Drug House
Phenolpthalein British Drug House
Starter culture CHR Hansen

A.2 List of equipments
Equipments Manufacturer
Weighing machine Prince scale industries
Water bath Mvtex science industries
pH meter Systonic
Hot air oven Y.P. scientific industries
Incubator JSGW
Grinder Crompton
Refrigerator Electrolux
Rotational Viscometer Biobase
Glasswares Borosil
Centrifuge Doctor
80
Appendix B
Sensory Evaluation of Yoghurt
Hedonic Rating Test
Name of Panelist: ……… Date: …………
Name of Product: YOGHURT
Please test the given sample and check how much you prefer each one. Give points for your
degree of preference for each parameter as shown below.
S.N Parameter A B C D
Perceptions Point
1 Colour Like extremely 9
Like very much 8
2 Aroma Like moderately 7
Like slightly 6
3 Taste
Neither like nor dislike 5
4 Consistency Dislike slightly 4
Dislike moderately 3
5 Overall
Dislike very much 2
Acceptability
Dislike extremely 1
Comment……………………............................................................................................
Signature……….………
81
Appendix C
C.1 ANOVA table for characterization of pectin samples
SS Df Mean Square F Sig.
Between Groups 4034.320 2 2017.160 2.049 0.210

E.W. Within Groups 5905.806 6 984.301
Total 9940.127 8
Between Groups 8.580 2 4.290 34.875 0.000
MeO Within Groups 0.738 6 0.123
Total 9.318 8
Between Groups 192.719 2 96.359 6.467 0.032
AUA Within Groups 89.397 6 14.899
Total 282.116 8
Between Groups 148.017 2 74.008 60.838 0.000
DE Within Groups 7.299 6 1.216
Total 155.316 8
Between Groups 4034.320 2 2017.160 2.049 0.210
moisture Within Groups 0.445 6 0.074
Total 17.725 8
Between Groups 9.848 2 4.924 245.775 0.000
Ash Within Groups 0.120 6 0.020
Total 9.969 8
82
C.2 ANOVA table for proximate analysis of yoghurt samples
SS df MS F Sig.
Between Groups 2.372 3 0.791 1.403 0.311
Moisture Within Groups 4.508 8 0.564
Total 6.880 11
Between Groups 0.001 3 0.000 6.331 0.017
Fat Within Groups 0.000 8 0.000
Total 0.001 11
Between Groups 0.909 3 0.303 6.395 0.016
Protein Within Groups 0.379 8 0.047
Total 1.287 11
Between Groups 0.022 3 0.007 2.480 0.135
Ash Within Groups 0.024 8 0.003
Total 0.046 11
Between Groups 1.829 3 0.610 1.321 0.334
Carbohydrate Within Groups 3.694 8 0.462
Total 5.523 11
Between Groups 3878993.3 3 1292997.79 134.199 0.000
Viscosity Within Groups 77079.207 8 9634.901
3956072.6
Total 11
00
83
C.3 ANOVA table for sensory analysis of yoghurt samples
SS Df MS F Sig.
Between
4.875 3 1.625 2.427 0.081
Groups
Colour
Within Groups 24.100 36 0.669
Total 28.975 39
Between
12.000 3 4.000 3.564 0.023
Groups
Aroma
Total 52.400 39
Between
32.675 3 10.892 9.977 0.000
Groups
Taste
Total 71.975 39
Between
13.475 3 4.492 3.734 0.020
Groups
Consistency
Total 56.775 39
Between
10.075 3 3.358 3.743 0.019
Groups
OA
Total 42.375 39
84
C.4 ANOVA table for syneresis
Sample Source Type III Sum of Squares Df MS F Sig.
Corrected
95.959a 3 31.986 81.083 0.00
Model
Intercept 2002.514 1 2002.514 5076.229 0.00
Day 95.959 3 31.986 81.083 0.00
1
Sample .000 0 . . .
Error 3.156 8 .394
Total 2101.629 12
Corrected Total 99.115 11
Corrected
86.588b 3 28.863 78.902 0.00
Model
Intercept 3516.459 1 3516.459 9613.059 0.00
Day 86.588 3 28.863 78.902 0.00
2
Sample 0.000 0 . . .
Error 2.926 8 0.366
Total 3605.973 12
Corrected
69.927c 3 23.309 75.896 0.00
Model
Intercept 4598.629 1 4598.629 14973.506 0.00
Day 69.927 3 23.309 75.896 0.00
3
Sample 0.000 0 . . .
Error 2.457 8 0.307
Total 4671.013 12
Corrected
39.828d 3 13.276 22.087 0.00
Model
Intercept 2333.511 1 2333.511 3882.301 0.00
Day 39.828 3 13.276 22.087 0.00
4
Sample 0.00 0 . . .
Error 4.809 8 6.01
Total 2378.147 12
85
C.5 ANOVA table for titratable acidity of yoghurt
Day Source Type III Sum of Squares df MS F Sig.
Corrected Model 0.015a 3 0.005 0.232 0.872
Intercept 10.847 1 10.847 500.300 0.000
Sample 0.015 3 0.005 0.232 0.872
Day 0.000 0 . . .
1
Sample * Day 0.000 0 . . .
Error 0.173 8 0.022
Total 11.036 12
Corrected Model 0.097b 3 0.032 0.280 0.838
Intercept 14.580 1 14.580 125.705 0.000
Sample 0.097 3 0.032 0.280 0.838
Day 0.000 0 . . .
3
Sample * Day 0.000 0 . . .
Error 0.928 8 0.116
Total 15.605 12
Corrected Model 0.047c 3 0.016 0.175 0.910
Intercept 19.370 1 19.370 215.078 0.000
Sample 0.047 3 0.016 0.175 0.910
Day 0.000 0 . . .
5
Sample * Day 0.000 0 . . .
Error 0.720 8 0.090
Total 20.138 12
Corrected Model 0.065d 3 0.022 0.708 0.574
Intercept 26.378 1 26.378 861.864 0.000
Sample 0.065 3 0.022 0.708 0.574
Day 0.000 0 . . .
7
Sample * Day 0.000 0 . . .
Error 0.245 8 0.031
Total 26.688 12
86
C.6 ANOVA table for L. Bulgaricus count of yoghurt
Day Source Type III Sum of Squares df MS F Sig.
a
Corrected Model 0.387 3 0.129 0.077 0.970
Intercept 501.504 1 501.5 300.875 0.00
Sample 0.387 3 0.129 0.077 0.097
Day 0.000 0 . . .
1
Sample * Day 0.000 0 . . .
Error 13.335 8 1.667
Total 515.226 12
Corrected Model 0.231b 3 0.077 0.121 0.95
Intercept 561.208 1 561.20 884.57 0.00
Sample 0.231 3 0.077 0.121 0.945
Day 0.000 0 . . .
3
Sample * Day 0.000 0 . . .
Error 5.076 8 0.634
Total 566.514 12
Corrected Model 0.370c 3 0.123 0.152 0.926
656.273 1 656.27 807.154 0.000
Intercept
3
Sample 0.370 3 0.123 0.152 0.926
5 Day 0.000 0 . . .
Sample * Day 0.000 0 . . .
Error 6.505 8 0.813
Total 663.147 12
Corrected Model 0.067d 3 0.022 0.045 0.986
725.766 1 725.76 1455.45 0.000
Intercept
6 8
Sample 0.067 3 0.022 0.045 0.986
7 Day 0.000 0 . . .
Sample * Day 0.000 0 . . .
Error 3.989 8 0.499
Total 729.822 12
87
C.7 ANOVA table for L. Bulgaricus count of yoghurt
Day Source Type III Sum df Mean F Sig.

of Squares Square
Corrected
1.232a 3 0.411 0.532 0.673
Model
Intercept 509.334 1 509.334 659.595 0.000
Sample 1.232 3 0.411 0.532 0.673
1 Day .000 0 . . .
Sample * Day .000 0 . . .
Error 6.178 8 0.772
Total 516.743 12
Corrected
1.346b 3 .0449 0.629 0.616
Model
Intercept 463.800 1 463.800 650.756 0.000
Sample 1.346 3 0.449 0.629 0.616
3 Day 0.000 0 . . .
Sample * Day 0.000 0 . . .
Error 5.702 8 0.713
Total 470.847 12
Corrected
1.753c 3 0.584 0.822 0.517
Model
Intercept 373.228 1 373.228 525.081 0.000
Sample 1.753 3 0.584 0.822 0.517
5 Day 0.000 0 . . .
Sample * Day 0.000 0 . . .
Error 5.686 8 0.711
Total 380.668 12
Corrected
0.638d 3 0.213 0.272 0.844
Model
Intercept 300.850 1 300.850 384.445 0.000
Sample 0.638 3 0.213 0.272 0.844
7 Day 0.000 0 . . .
Sample * Day 0.000 0 . . .
Error 6.260 8 0.783
Total 307.749 12
88
Appendix D
P.1 Fresh and dried peels of (a) lemon (b) lime (c) pomelo
P.2 Extraction of pectin and yoghurt samples

89
Name: Astha Adhikari
Email: adk.aastha@gmail.com
TU redg: 5-2-932-4-2014
Mobile no: 9846359910
M. Tech 172/076
90

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OPTIMIZATION AND CHARACTERIZATION OF PECTIN

EXTRACTED FROM CITRUS FRUIT PEELS AND ITS EFFECT ON

Central Department of Food Technology, Dharan

Institute of Science and Technology

Tribhuvan University, Nepal

A dissertation submitted to the Central Department of Food Technology, Institute of

Central Department of Food Technology, Dharan

Institute of Science and Technology

Tribhuvan University, Nepal

Institute of Science and Technology

Central Department of Food Technology

This dissertation entitled Optimization and Characterization of Pectin Extracted from

1. Head of the Department

(Mr. Basanta Kumar Rai, Prof.)

(Mr. Birendra Kumar Yadav, Asst. Prof., BPKIHS)

(Mrs. Mahalaxmi Pradhananga, Asst. Prof.)

(Mr. Bunty Maskey, Asst. Prof.)

May 16, 2023

Approval Letter .................................................................................................................. iii

List of tables ........................................................................................................................ xi

List of figures ..................................................................................................................... xii

List of plates ...................................................................................................................... xiv

1. Introduction .............................................................................................................. 1-4

1.1 Background ......................................................................................................... 1

1.2 Statement of the problem .................................................................................... 2

1.3 Objectives of the study ....................................................................................... 3

1.3.1 General objective .................................................................................. 3

1.3.2 Specific objectives ................................................................................ 3

1.4 Significance of the study .................................................................................... 4

1.5 Limitations of the study ...................................................................................... 4

2. Literature review .................................................................................................... 5-19

2.1 Pectin .................................................................................................................. 5

2.2 Structure of pectin .............................................................................................. 6

2.2.1 Classification of pectin.......................................................................... 7

2.2.1.1 Degree of methylation .......................................................... 7

2.2.1.2 Degree of acetylation and amidation .................................... 8

2.3 Extraction methods ............................................................................................. 8

2.3.2 Microwave assisted extraction ............................................................... 9

2.3.3 Ultrasonic assisted extraction.............................................................. 10

2.4 Sources of pectin .............................................................................................. 11

2.4.1 Lemon peels ....................................................................................... 11

2.4.2 Acid lime peels.................................................................................... 12

2.4.3 Pomelo ................................................................................................ 13

2.4.4 Production ........................................................................................... 13

2.5 Techno-functional properties of pectin............................................................. 14

2.5.1 Pectin gelation ..................................................................................... 14

2.5.2 Water/oil holding capacity .................................................................. 15

2.6 Application of pectin ........................................................................................ 15

2.6.1 Pectin in yoghurt ................................................................................. 15

2.7 Yoghurt .............................................................................................................. 16

2.7.1 Types of yoghurt .............................................................................. 16

2.7.2 Health benefits of yoghurt................................................................... 17

2.8 Factors affecting the quality of yoghurt ........................................................... 17

2.8.1 Casein and fat content ......................................................................... 17

2.8.2 Homogenizing ..................................................................................... 17

2.8.3 Acidity ................................................................................................. 18

2.8.4 Heat treatment ..................................................................................... 18

2.8.5 Incubation temperature ....................................................................... 18