D.R.A. Muhammad, et al.

Food Hydrocolloids 100 (2020) 105377

polyphenol-rich cinnamon extract due to light, heat, and oxygen (Joye Table 1
& McClements, 2014; Teixeira, Ozdemir, Hill, & Gomes, 2013). As far as Antioxidant properties of cinnamon extract.
is known, studies into the synthesis and stability of nanocapsules con- Parameter Value Unit (per gram of dry weight)
taining polyphenol-rich cinnamon extract are still scarce up till now.
Among the existing methods, anti-solvent precipitation is con- Total phenolic compound 273 ± 8 mg epicatechin equivalent
Total flavonoid 25 ± 1 mg quercetin equivalent
sidered as an efficient technique for synthesizing nanocapsules due to
Total antioxidant activity 208 ± 4 mg tannic acid equivalent
its low cost and easy operation (Esfanjani & Jafari, 2016; Sedaghat Ferric reducing antioxidant power 208 ± 3 mmol L−1 ascorbic acid
Doost et al., 2019a). In this method, a solution of a hydrophobic activity equivalent
compound dissolved in an organic solvent is added to an anti-solvent
(aqueous phase). The nanoparticles are formed by three main steps: (a)
generation of supersaturation; (b) nucleation; and (c) growth of nuclei 2. Materials and methods
(Joye & McClements, 2013; Sedaghat Doost, Muhammad, Stevens,
Dewettinck, & Van der Meeren, 2018b). 2.1. Materials
In the last several years, special emphasis has been given to the
functionality of shellac as a base material for nanoparticles. Shellac is a Cinnamon bark (Cinnamomum burmannii Blume) was collected from
hard, tough and amorphous resin consisting of polyhydroxy poly- Mount Kerinci, Indonesia. Fine shellac powder (SSB 55 Astra FP) and
carboxylic esters, lactones and anhydrides secreted by lac insects xanthan gum (Satiaxane CX 931) were provided by SSB Stroever GmbH
(Kerria lacca) (Patel et al., 2013). It has interesting properties as a na- & Co. KG (Bremen, Germany) and Cargill France SAS (France), re-
nocapsule wall material given its tremendous film-forming properties spectively.
such as excellent gloss, low gas permeability and the ability to prevent
moisture transfer. Moreover, shellac is of natural origin, biodegradable, 2.2. Preparation of polyphenol-rich cinnamon extract
odourless in cold conditions, non-toxic, physiologically harmless and
generally recognised as safe (so-called “GRAS’ status in the USA). For Briefly, 10 g of cinnamon powder was subjected to extraction in
these reasons, shellac is an acceptable material for the food and phar- 100 ml of ethanol before being stirred for 48 h at 20 °C. Next, the
maceutical industries, particularly for the coating of phytochemicals mixture was filtered by vacuum (Laboport, KNF Neuberger, Inc., USA)
and controlled drug delivery purposes (Byun, Ward, & Whiteside, 2012; (Muhammad et al., 2017). The antioxidant properties of the cinnamon
Farag & Leopold, 2011; Poovarodom & Permyanwattana, 2015). Re- extract after the removal of the solvent are given in Table 1.
cently, a study performed by Sedaghat Doost et al. (2018b) successfully
encapsulated quercetin as a bioactive compound within core-shell na- 2.3. Preparation of colloidal nanoparticles
noparticles containing shellac and almond gum. In similar works, Sun
et al. (2017) and Chen et al. (2018) studied the fabrication and char- Colloidal nanoparticles were prepared by anti-solvent precipitation,
acterization of binary composite particles based on zein and shellac. according to our previous work (Sedaghat Doost et al., 2018b). In brief,
Nevertheless, shellac is restricted in its application as a delivery 2% (w/w) of shellac powder was dissolved in ethanol using a magnetic
system for bioactive compounds because it tends to aggregate at an stirrer. Xanthan gum was prepared separately in distilled water at dif-
acidic pH (1.2), which might be solved by the incorporation of a sta- ferent concentration levels (0–0.5% (w/w)) to examine the minimum
biliser (Patel, Heussen, Hazekamp, & Velikov, 2011). A nano-particu- concentration of xanthan gum required to stabilise the shellac at pH
late carrier of bioactive compounds targeting the colon has to survive 1.2. The shellac solution was injected into the xanthan gum solution
the harsh acidic conditions of the stomach; otherwise, the distribution, using a syringe at a shellac-to-xanthan gum ratio of 1:3 (w/w), and then
release, and bioavailability of the compound can be uncontrolled mixed using a magnetic stirrer to obtain a homogeneous mixture. Next,
(Jafari & McClements, 2017; Park, Saravanakumar, Kim, & Kwon, colloidal nanoparticles were formed by removing the ethanol through
2010). Natural polymers have been widely acknowledged to have many rotary evaporation (Laborota 4000 Heidolph, Germany). To prepare the
functions in foods, including as a stabilising agent (Manuhara, nanoparticles containing cinnamon, the solvent (ethanol) was partially
Praseptiangga, Muhammad, & Maimuni, 2016; Muhammad et al., 2019; replaced by cinnamon extract in various proportions (12.5%–50% (w/
Praseptiangga, Giovani, Manuhara, & Muhammad, 2017; Sedaghat w)).
Doost et al., 2019b). Xanthan gum, a non-toxic biopolymer produced by
Xanthomonas campestris through fermentation, has been widely used as 2.4. Characterization of colloidal nanoparticles
a thickening and stabilising agent in various products, including
emulsion systems and micro particles. The main chain of xanthan gum 2.4.1. Determination of particle size and surface charge
is based on a linear backbone of 1,4-linked β-D-glucose (Garcıa-Ochoa, Photon correlation spectroscopy (PCS100M, Malvern Instrument
Santos, Casas, & Gomez, 2000; Palaniraj & Jayaraman, 2011). Ltd, UK) was used to determine the z-average particle size, while a
Thus, the purpose of this study was to investigate the functionality Zetasizer IIC (Malvern Instrument Ltd., UK) was utilised to measure the
of xanthan gum in stabilising shellac nanoparticles containing cin- ζ-potential of the colloidal nanoparticles. A few drops of the colloidal
namon extract. The novelty of this work lay in the functionality of dispersion was appropriately diluted prior to the analysis of the particle
xanthan gum in shellac nanoparticles and the use of a spice extract size and ζ-potential. The z-average particle diameter was obtained by a
instead of a single compound as the core material for the nanocapsules. cumulant analysis of the light that was scattered at an angle of 150°.
The use of an herb or spice extract with high antioxidant properties is The measurements were reported as the averages of 3 separate injec-
preferable for the manufacture of polyphenol- and antioxidant-rich tions, with three readings made per injection. The measurements were
foods compared to using a large amount of a single antioxidant as it carried out at 25 °C.
may have an adverse effect on health (Shahidi & Ambigaipalan, 2015).
In this study, therefore, the synthesis of nanocapsules containing cin- 2.4.2. Morphological characterization
namon extract was conducted. The physicochemical and antioxidant A JEOL JSM 7100F scanning electron microscope equipped with a
properties as well as the release behaviour and thermal stability of the PP3010T Cryo-SEM preparation system (Oxford Instruments, UK) was
nanoparticles were also evaluated. used to investigate the shape of the synthesised particles. A few milli-
grams of lyophilised colloidal nanoparticles were rehydrated in a few
drops of water. The samples were placed on the cryo-specimen holder,
and then cryo-fixed in slush nitrogen (−210 °C). Subsequently, the

2
D.R.A. Muhammad, et al. Food Hydrocolloids 100 (2020) 105377

sample was transferred to the cryo-unit in a vitrified state. After the UV–visible spectrophotometer (Udayaprakash et al., 2015). A standard
sample was fractured, it was sublimated (20 min, −70 °C) and sputter- plot of ascorbic acid was used to measure the FRAP activity of the
coated with platinum (4 min, 0.5 mbar). Finally, the sample was samples. The FRAP activity was expressed as mmol L−1 of ascorbic acid
transferred to the microscope, where it was observed at −140 °C. equivalent per gram dry weight (mmol L−1 AAE/g DW) of the nano-
particles.
2.4.3. Determination of encapsulation efficiency and loading capacity
The encapsulation efficiency was determined by measuring the 2.4.7. pH-responsive study
difference between the phenolic content on the surface of the nano- The protocol of Patel et al. (2011) was used to test the release of
particles and the total phenolic yield in the dispersion, according to phenolic compounds from the shellac colloidal nanoparticles at dif-
Zheng, Ding, Zhang, and Sun (2011) with minor modifications. To ferent pH conditions. An aqueous medium (25 ml at pH 1.2) was pre-
dissolve and determine the phenolic content on the surface of the na- pared using 0.1 M HCl. Cinnamon extract and the nanoparticles were
noparticles, 20 mg of the sample were added to distilled water (10 ml). separately introduced into the release medium, which was slowly
Next, the mixture was centrifuged (Sigma 4K15 Sartorius AG, Germany) stirred for 2 h at 37 °C in the absence of light. Immediately afterwards, a
at 13102 g for 30 min. The supernatant was collected and the total few drops of NaOH were added to change the pH to 7.4, and the in-
phenolic content was analysed using Folin-Ciocalteu reagent (see Sec- cubation was continued for another 2 h. Periodically, a sample was
tion 2.4.4). To determine the total phenolic yield, 20 mg of particles taken, and the total phenolic content was analysed by means of the
were dissolved in 10 ml of buffer solution at pH 7.4. The encapsulation Folin–Ciocalteu method (see Section 2.4.4).
efficiency was calculated using Eq. (1).
2.4.8. Stability test
Encapsulation efficiency The test for the thermal stability of the cinnamon extract (free and
total phenolic yield − phenolic content on the surface nanoencapsulated forms) was conducted in a water bath at a tem-
= x100%
total phenolic yield perature of 90 °C, according to the protocol of Sedaghat Doost et al.
(Eq. 1) (2018b). The samples were collected after 20 min of heat treatment.
The total phenolic content and antioxidant activity of the samples were
The loading capacity was determined according to Teixeira et al.
analysed (see Section 2.4.4 and Section 2.4.5, respectively) before and
(2013) (Eq. (2)).
after the heat treatment.
Loading capacity
total phenolic yield − phenolic content on the surface 2.5. Experimental design and statistical analysis
= x100%
amount of particles produced
The parameters that were considered to be affecting the character-
(Eq. 2)
istics of the nanoparticles were the xanthan gum concentration, the
cinnamon loading and the storage temperature. The effect of each
2.4.4. Analysis of total phenolic content compositional and operational parameter on the characteristics of the
The total phenolic content was determined using the nanoparticles was studied based on a single factor experiment with 5
Folin–Ciocalteu method, as described by Udayaprakash et al. (2015), (five) levels, except for the study on the effect of the xanthan gum
whereby 0.2 ml of extract solution was mixed with 0.2 ml of Fo- concentration on the particle size, the ζ-potential, the encapsulation
lin–Ciocalteu reagent and 1 ml of distilled water. After incubation for efficiency and the loading capacity of the nanoparticles. The experi-
6 min, 2.5 ml of 7% Na2CO3 were added. Afterwards, the mixture was ment was performed by means of a completely randomized design
maintained at ambient temperature ( ± 20 °C) for 90 min, and then, the (CRD), and the results represented the means of three replicates. A data
absorbance was measured at 760 nm using a UV–visible spectro- analysis was performed by SPSS Statistics 23 using an analysis of var-
photometer (Varian Cary 50 Bio, Agilent Technology). The total phe- iance (one-way ANOVA). A DMRT (Duncan's multiple range test) and T-
nolic content was expressed as milligrams of epicatechin equivalent test were performed to analyse the differences between the means. A
(ECE) per gram of dry weight of nanoparticles. significance level of 5% was applied.

2.4.5. Total antioxidant content assay 3. Results and discussion

The total antioxidant content was assayed by using the phospho-
molybdenum method by Udayaprakash et al. (2015). The analysis was 3.1. Preparation of stable shellac colloidal particles
conducted after the wall material had been solubilised (see Section
2.4.3). The reagent solution for performing the assay was created by As shellac tends to aggregate at an acidic pH, xanthan gum was
mixing 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM am- incorporated into the anti-solvent phase to stabilise the system. It was
monium molybdate at a ratio of 1:1:1. Next, 0.5 ml of the sample was even found that an instant aggregation of some parts of shellac occurred
added to 4.5 ml of the reagent solution. The solution was incubated at during the injection of the solvent phase into the anti-solvent phase
95 °C for 90 min using a water bath. The absorbance was measured at when xanthan gum was absent. The instant aggregation could be pre-
695 nm using a UV–visible spectrophotometer after the sample reached vented by the incorporation of 0.1% of xanthan gum. A minimum
room temperature. The total antioxidant content was stated as milli- concentration of 0.3% of xanthan gum was required to prevent ag-
grams of tannic acid equivalent per gram of dry weight (mg TAE/g DW) gregation at a pH of 1.2. As the pH decreased, the negative charges
of the nanoparticles. present within the chemical structure of the xanthan gum were
screened and thus, xanthan gum at a concentration below 0.3% was
2.4.6. FRAP (Ferric Reducing Antioxidant Power) assay inadequate to induce repulsion against aggregation. While the un-
Phosphate buffer (0.2 M, pH 7, 2.5 ml) was added to the colloidal controlled aggregation was occurring, particles of irregular shape and
nanoparticles (1 ml), and then mixed with potassium ferricyanide (1%, size were formed (Fig. 1). At an acidic pH, H+ ions will tend to accu-
2.5 ml). The solution was incubated at 50 °C for 30 min before the ad- mulate on negatively-charged nanoparticles and the ζ-potential value
dition of 2.5 ml of 10% trichloroacetic acid. In order to separate the may become less negative. In terms of the oral delivery system for
larger aggregates, the solution was centrifuged for 10 min. The super- bioactive compounds, the aggregation of nanoparticles is undesired
natant was mixed well with distilled water and 0.1% FeCl3 at a ratio of since the release of the bioactive compounds can be uncontrolled. To
5:5:1, and then, the absorbance was measured at 700 nm using a get a better insight into the stabilisation effect of xanthan gum on

3
D.R.A. Muhammad, et al. Food Hydrocolloids 100 (2020) 105377

Fig. 1. Image of the colloidal particles at neutral pH (small container) and pH 1.2 (large container): (A) without the use of xanthan gum and (B) with the presence of
0.3% xanthan gum. Insets: Microscopic image of the shellac colloidal dispersion obtained by Cryo-SEM.

0 Xanthan gum concentra on 250

A B
200
0.0% 0.1% 0.2% 0.3% 0.4% 0.5%
ζ-poten al (mV)

-20

Par cle size (nm)

150

100

-40
50

0
0.0% 0.1% 0.2% 0.3% 0.4% 0.5%
-60
Xanthan gum concentra on

Fig. 2. The effect of xanthan gum incorporation on the zeta-potential (A) and (B) particle size of the shellac colloidal nanoparticles at neutral pH.

shellac nanoparticles, the ζ-potential of the nanoparticles was eval- xanthan gum, which might hamper the diffusion between the solvent
uated. and anti-solvent (Joye & McClements, 2013; Kakran, Sahoo, Li, &
As shown in Fig. 2, the incorporation of a higher concentration of Judeh, 2012). Rheological experiments confirmed that a higher con-
xanthan gum had a substantial impact on the ζ-potential of the nano- centration of xanthan gum caused a higher apparent viscosity at all the
particles. As xanthan gum is a negatively-charged biopolymer with a tested shear rate levels (not shown).
high surface charge density, the higher percentage of xanthan gum led
to more negative ζ-potential values. The highly significant correlation 3.2. Impact of cinnamon extract loading and gum concentration on the
between the incorporation of xanthan gum and the ζ-potential was characteristics of the nanoparticles
verified by a Pearson's correlation analysis (−0.983), which showed
that the correlation was significant at p < 0.01, which was in agree- Cinnamon extract was loaded into the shellac-xanthan gum com-
ment with a previous study by Patel et al. (2011). plex, and the effect of this on the characteristics of the nanoparticles is
It should be noted that the concentration of xanthan gum that was shown in Table 2. The nanoparticles with a higher level of cinnamon
required to stabilise the shellac nanoparticles at pH 1.2 was lower than extract loading tended to induce a bigger particle size and less negative
that of almond gum, which was previously reported to be at a con- charges. The latter indicated that the cinnamon extract was at least
centration of 0.7% (Sedaghat Doost et al., 2018b). According to Joye partly present at the surface of the shellac-xanthan gum system. To
and McClements (2013), the stabilisation effect of different types of challenge this hypothesis, a study investigating the effect of the pro-
gums is determined by their composition, molecular weight and chain portion of cinnamon extract on the encapsulation efficiency was carried
length. The difference in the charge density of xanthan and almond gum out.
seems to play a significant role in the stabilisation effect. For instance, The phenolic content was used as the parameter for the en-
in this study the ζ-potential of xanthan gum at a concentration of 0.5% capsulation efficiency since polyphenols are acknowledged to be the
was found to be −59 mV, while, at a similar concentration, almond main constituent offering health benefits (Gruenwald, Freder, &
gum resulted in a ζ-potential of −32 mV (Sedaghat Doost et al., 2018b). Armbruester, 2010; Li et al., 2013; Lu et al., 2011; Muthenna, Raghu,
Increasing the xanthan gum concentration resulted in a significant Kumar, Surekha, & Reddy, 2014; Ribeiro-Santos et al., 2017). It was
growth in the size of the particles (with a correlation coefficient of shown that about 30% of cinnamon polyphenols were encapsulated
0.975) (Fig. 2). The growth in particle size indicated that more xanthan within the nanoparticles and hence, the rest might have been associated
gum molecules were adsorbed onto the colloidal shellac particles (Patel with the surface of the nanoparticles. Cinnamon extract does not consist
et al., 2011). In a previous study, it was shown by transmission electron of only one compound but is rich in structurally diverse phytochemi-
microscopy that the shellac was surrounded by almond gum, which led cally active constituents, such as cinnamic acid, sinapic acid, p-cou-
to the stabilisation of the formed nanoparticles (Sedaghat Doost et al., maric acid, cafeic acid, protocatechuic acid, quercetin, kaempferol,
2019a). The ability of the xanthan gum to cover the surface of the gallic acid, vanillic acid, syringic acid, ferulic acid, quercetin-3-rham-
shellac particles was due to the hydrogen bonding between the shellac noside, tannic acid, caffeic acid, chlorogenic acid, rosmarinic acid and
and xanthan gum, as reported by Patel et al. (2013). The adsorption of salicylic acid (Muhammad & Dewettinck, 2017). Some of these cin-
an amphiphilic stabiliser at the surface of the hydrophobic colloidal namon extract constituents are highly soluble in water. During the
particles is the basis for a steric stabilisation effect (Cerrutti, de Souza, nanoparticle formation (precipitation of shellac in the evaporation
Castellan, Ruggiero, & Frollini, 2012). process), some of these compounds might have been in a solute state in
A rise in the size dispersity, which is a measure of the distribution of the water, and thus, could not be entrapped in the colloidal shellac
width in the samples, from 0.14 to 0.46 was observed when the xanthan nanoparticles. When the colloid was lyophylised, the compounds were
gum concentration was increased from 0 to 0.5%. An increase in size deposited onto the surface of the nanoparticles. This fact implies that
dispersity indicated the formation of agglomerates of nanoparticles, the constituents of a spice extract can have an important influence on
possibly due to a higher viscosity upon the introduction of more the encapsulation efficiency.

4
D.R.A. Muhammad, et al. Food Hydrocolloids 100 (2020) 105377

Table 2
Characteristics of nanoparticles loaded with different concentrations of cinnamon extract and xanthan gum.
Cinnamon extract (%) Xanthan gum (%) Z-average particle size (nm) ζ-potential (mV) Encapsulation Efficiency (%) Loading Capacity (%)

0 0.3 149 ± 3a
−53.6 ± 0.4a

12.5 159 ± 5ab −45.3 ± 0.5bc 33.6 ± 0.5a 2.4 ± 0.1a

25 157 ± 5ab −43.8 ± 1.4c 31.7 ± 1.3ab 2.9 ± 0.1b
37.5 154 ± 6ab −45.1 ± 0.7bc 32.0 ± 1.0ab 3.6 ± 0.1c
50 162 ± 6b −46.1 ± 0.7b 29.7 ± 1.8b 3.5 ± 0.1c

12.5 0.3 159 ± 5a −45.3 ± 0.5a 33.6 ± 0.5a 2.4 ± 0.1a

0.4 225 ± 8b −48.5 ± 1.0b 27.1 ± 2.9b 2.0 ± 0.3a
0.5 241 ± 5c −53.6 ± 1.3c 27.9 ± 2.7b 2.1 ± 0.3a

Mean values with the same letter do not differ significantly (p > 0.05) in the same column. Statistical analysis was performed separately for each combination for
each experimental set up (different concentration of cinnamon loading and different level of xanthan gum).

The encapsulation efficiency was also significantly affected by the

initial amount of cinnamon extract, where the encapsulation efficiency
was lower at higher concentrations of the cinnamon extract. This result
was in accordance with the finding of Xu and Du (2003), who worked
with bovine serum albumin (BSA) and chitosan. They discovered that a
low BSA loading led to high encapsulation efficiency, while a high BSA
loading resulted in low encapsulation efficiency. When a smaller ratio
of cinnamon extract to shellac was employed, the shellac had a higher
chance of entrapping the cinnamon extract. As expected, at a higher
initial cinnamon extract concentration, the actual amount of phenolic
compounds entrapped in the nanoparticles was higher. Therefore, the
loading capacity increased (Table 2).
Confirming the results from the previous experiment (Section 3.1), Fig. 3. Typical morphology of shellac nanoparticles incorporated with 0.3%
Table 2 also shows that the incorporation of a higher concentration of xanthan gum loaded with 50% cinnamon extract.
xanthan gum led to larger particles and more negative surface charges.
The drawback in increasing the xanthan gum concentration was that it
used to investigate the antioxidant potency of the nanoparticles. In the
led to a significant decrease in the encapsulation efficiency. This could
phosphomolybdenum method, the formation of a phosphomolybdenum
have been due to an increase in the viscosity. This result was in
complex is based on the reduction of Mo (VI) to Mo (V) by the sample
agreement with the negative relationship between the viscosity and
analyte, and the subsequent formation of a green complex at an acidic
encapsulation efficiency in the fabrication of nanoparticles, as reported
pH. The FRAP assay measures the ability for the reduction of Fe3+ to
by Gan and Wang (2007) as well as Kraisit, Limmatvapirat, Nunthanid,
Fe2+. Fig. 4 shows that without the cinnamon extract loading, the na-
Sriamornsak, and Luangtana-anan (2013). The reduction in the en-
noparticles did not exhibit antioxidant activity when evaluated in both
capsulation efficiency was directly proportional to the loading capacity.
assays, thereby strongly indicating that cinnamon extract plays a sig-
nificant role on the antioxidant activity of the nanoparticle system. The
3.3. Microstructural characterization antioxidant activity of the nanoparticles was highly correlated with the
cinnamon loading. As shown in both assays, a higher level of cinnamon
A microstructural visualisation was performed to study the surface loading led to a higher antioxidant activity. A previous study showed
morphology of the shellac-xanthan gum nanoparticles containing cin- that the antioxidant activity of cinnamon extract is highly correlated
namon extract. To study the morphology of the nanoparticles, they with its phenolic content (Muhammad et al., 2017). This result in-
were initially freeze-dried and then, a small amount of water was added dicates that the nanoparticles have a great potential as a natural
to rehydrate the particles. The freeze-drying step was conducted to
make the colloidal nanoparticles denser and, therefore, easier to ob- 250
Total antioxidant activity (mg TAE/g DW)
serve. The rehydration step was carried out to reverse the form of the
FRAP Activity (mM AAE/g DW) d
colloidal nanoparticles. It was found that the nanoparticles were 200
spherical with a smooth surface. There was no significant difference in
the morphology between the blank nanoparticles (Fig. 1B) and the 150 c
d
Value

nanoparticles loaded with cinnamon extract (Fig. 3). This result was in d
accordance with the previously published literature explaining that b c
100
nanoparticles, produced using anti-solvent precipitation, are often b
b
spherical (Joye & McClements, 2013; Sedaghat Doost et al., 2018b).
50
Fig. 3 shows that the particles containing cinnamon extract in shellac-
xanthan gum were of nanoscale size. The size corresponded well with a a
0
the information on the particle size obtained by photon correlation 0% 12.5% 25% 37.5% 50%
spectroscopy, which indicated an intensity-weighted particle size dis-
Cinnamon loading
tribution ranging from 73 to 366 nm.
Fig. 4. Total antioxidant activity assayed using phospomolybdenum method
3.4. Antioxidant activity and Ferric Reducing Antioxidant Power (FRAP) of nanoparticles stabilised with
xanthan gum (0.3%) loaded with various levels of cinnamon. Mean values
As there is no single method that can provide unequivocal results, within each antioxidant assay with different lowercase letter differ significantly
two different assays (phospomolybdenum and FRAP methods) were (p < 0.05).

5
D.R.A. Muhammad, et al. Food Hydrocolloids 100 (2020) 105377

3.6. Thermal stability test

100 pH 1.2 pH 7.4
As polyphenols are prone to degradation upon heat treatment
Phenolic compound release (%)

during processing, the thermal resistance of cinnamon extract in its free

80 form and in its nanoencapsulated form was investigated. It is useful to
draw a conclusion on the effectiveness of nanoencapsulation to prevent
60
polyphenol degradation. As shown in Fig. 6, after the heat treatment,
Dried particle in the polyphenol retention of the free cinnamon extract was about 84%,
the release medium (0 min)
while that of the nanoencapsulated cinnamon extract was 94%. This
40 indicated that nanoencapsulation can really have an impact on the
Clear solution
(240 min) thermal stability of polyphenols. However, in this study, na-
noencapsulation could not perfectly prevent the polyphenols of cin-
20
namon from degrading upon heat treatment. This was probably because
Colloidal formation
some of the polyphenols of cinnamon were not well-encapsulated, as
(120 min)
0 shown in the encapsulation efficiency test. The nanoencapsulated cin-
0 40 80 120 160 200 240 namon extract also had better antioxidant activity retention as com-
Time (min) pared to the free cinnamon extract, at the level of 88% and 85%, re-
spectively. This was reasonable since the antioxidant activity of
Fig. 5. Release profile of cinnamon phenolic compounds from freeze-dried
cinnamon extract is directly proportional to its polyphenol content
colloidal nanoparticles in a release medium (2 h at pH 1.2 followed by in-
cubation for 2 h at pH 7.4). Insets: physical appearance of freeze-dried nano- (Muhammad et al., 2017). Thus, based on the result of the thermal
particles during the treatment. stability test, it was concluded that nanoencapsulation can significantly
improve the stability of polyphenol and antioxidant compounds upon
heat treatment.
antioxidant to replace synthetic antioxidants.

4. Conclusions
3.5. pH-responsive release
This work confirmed the potential use of xanthan gum to stabilise
Fig. 5 illustrates the release profile of the phenolic compounds of shellac colloidal nanoparticles at a low pH. Upon the addition of cin-
cinnamon extract from the freeze-dried nanoparticles. There was a namon polyphenols, about a third of the initial load was encapsulated
burst release of around 40% in the first 15 min, which was associated within the core of the shellac-xanthan gum nanoparticles, while the
with the loosely attached polyphenols on the surface of the nano- remaining part was attached to the surface of the nanoparticles. The
particles. Afterwards, the release level remained constant. The dried encapsulated cinnamon polyphenols were completely released in al-
nanoparticles at the low pH changed to a turbid colloidal form. When kaline pH conditions. The nanoparticles containing cinnamon extract
the pH was increased to 7.4, a sharp rise in the release of the phenolic exhibited high antioxidant activity, indicating that the shellac-xanthan
content from the particles was observed. It was then followed by a gum nanoparticle system may be used as a carrier of polyphenols for
gradual increase to more than 90% at the end of the study. This in- improving the health-promoting properties of food. Nanoencapsulation
dicated that at pH 7.4, the shellac had more solubility, as could be also effectively improved the thermal stability of the polyphenol-rich cin-
visibly observed from the decreased turbidity. As shown, in the first namon extract. Further studies are required to improve the encapsula-
120 min, the dried nanoparticles changed to a turbid colloidal form in tion efficiency of engineered cinnamon nanoparticles.
the release medium. In the next 120 min at pH 7.4, the hazy colloidal
appearance changed into a clear, brown-coloured solution (Fig. 5, in- Conflicts of interest
sets). This pH-responsive behaviour indicated that the shellac-xanthan
gum system can play an important role in preventing the discharge of The authors declare that they have no conflict of interest.
the content and subsequently, controlling the release of polyphenol in
the digestion system. Once the bioactive compounds are perfectly en-
Acknowledgements
capsulated in a shellac gum system, it can be expected that an un-
controlled burst release can be prohibited, and then, the bioactive
The first and second authors would like to thank Dr. Ashok R. Patel
compounds can be released in the targeted site triggered by the in-
for his valuable input in the laboratory work. Hercules Foundation is
testinal alkaline pH.
acknowledged for its financial support in the acquisition of the
Scanning Electron Microscope JEOL JSM-7100F equipped with cryo-
transfer system Quorum PP3000T (grant number AUGE-09-029).

A Control Heat treatment B Control Heat treatment

100 100
% an oxidant reten on
% polyphenol reten on

80 80

60 60

40 40

20 20

0 0
Free form nanoencapsulated Free form nanoencapsulated
form form

Fig. 6. The retention of polyphenols (A) and antioxidant activity (B) cinnamon extract upon heat treatment (90 °C, 20 min).

6
D.R.A. Muhammad, et al. Food Hydrocolloids 100 (2020) 105377

Appendix A. Supplementary data proceedings, Vol. 1710, AIP Publishing030043.

Muhammad, D. R. A., & Dewettinck, K. (2017). Cinnamon and its derivatives as potential
ingredients in functional foods - a review. International Journal of Food Properties, 20,
Supplementary data to this article can be found online at https:// 2237–2263.
doi.org/10.1016/j.foodhyd.2019.105377. Muhammad, D. R. A., Gonzalez, C. G., Sedaghat Doost, A., Van de Walle, D., Van der
Meeren, P., & Dewettinck, K. (2019). Improvement of antioxidant activity and phy-
sical stability of chocolate beverage using colloidal cinnamon nanoparticles. Food and
References Bioprocess Technology, 12, 976–989.
Muhammad, D. R. A., Praseptiangga, D., Van de Walle, D., & Dewettinck, K. (2017).
Assadpour, E., & Jafari, S. M. (2018). A systematic review on nanoencapsulation of food Interaction between natural antioxidants derived from cinnamon and cocoa in binary
bioactive ingredients and nutraceuticals by various nanocriiers. Critical Reviews in and complex mixtures. Food Chemistry, 231, 356–364.
Food Science and Nutrition, 1–23. Muhammad, D. R. A., Saputro, A. D., Rottiers, H., Van de Walle, D., & Dewettinck, K.
Byun, Y., Ward, A., & Whiteside, S. (2012). Formation and characterization of shellac- (2018). Physicochemical properties and antioxidant activities of chocolates enriched
hydroxypropyl methylcellulose composite films. Food Hydrocolloids, 27(2), 364–370. with engineered cinnamon nanoparticles. European Food Research and Technology,
Cerrutti, B. M., de Souza, C. S., Castellan, A., Ruggiero, R., & Frollini, E. (2012). 244, 1185–1202.
Carboxymethyl lignin as stabilizing agent in aqueous ceramic suspensions. Industrial Muthenna, P., Raghu, G., Kumar, P. A., Surekha, M. V., & Reddy, G. B. (2014). Effect of
Crops and Products, 36(1), 108–115. cinnamon and its procyanidin-B2 enriched fraction on diabetic nephropathy in rats.
Chen, S., Xu, C., Mao, L., Liu, F., Sun, C., Dai, L., et al. (2018). Fabrication and char- Chemico-Biological Interactions, 222, 68–76.
acterization of binary composite nanoparticles between zein and shellac by anti- Palaniraj, A., & Jayaraman, V. (2011). Production, recovery and applications of xanthan
solvent co-precipitation. Food and Bioproducts Processing, 107, 88–96. gum by Xanthomonas campestris. Journal of Food Engineering, 106(1), 1–12.
Durak, A., Gawlik-Dziki, U., & Pecio, Ł. (2014). Coffee with cinnamon–Impact of phy- Park, J. H., Saravanakumar, G., Kim, K., & Kwon, I. C. (2010). Targeted delivery of low
tochemicals interactions on antioxidant and anti-inflammatory in vitro activity. Food molecular drugs using chitosan and its derivatives. Advanced Drug Delivery Reviews,
Chemistry, 162, 81–88. 62(1), 28–41.
Esfahani, R., Jafari, S. M., Jafarpour, A., & Dehnad, D. (2019). Loading of fish oil into Patel, A., Heussen, P., Hazekamp, J., & Velikov, K. P. (2011). Stabilisation and controlled
nanocarriers prepared through gelatin-gum Arabic complexation. Food Hydrocolloids, release of silibinin from pH responsive shellac colloidal particles. Soft Matter, 7(18),
90, 291–298. 8549–8555.
Esfanjani, A. F., & Jafari, S. M. (2016). Biopolymer nano-particles and natural nano- Patel, A. R., Remijn, C., Cabero, A.i. M., Heussen, P., ten Hoorn, J. W. M., & Velikov, K. P.
carriers for nano-encapsulation of phenolic compounds. Colloids and Surfaces B: (2013). Novel all‐natural microcapsules from gelatin and shellac for biorelated ap-
Biointerfaces, 146, 532–543. plications. Advanced Functional Materials, 23(37), 4710–4718.
Farag, Y., & Leopold, C. S. (2011). Development of shellac-coated sustained release pellet Poovarodom, N., & Permyanwattana, W. (2015). Development of starch/shellac-based
formulations. European Journal of Pharmaceutical Sciences, 42(4), 400–405. composites for food contact applications. Journal of Thermoplastic Composite Materials,
Gan, Q., & Wang, T. (2007). Chitosan nanoparticle as protein delivery carrier—systematic 28(5), 597–609.
examination of fabrication conditions for efficient loading and release. Colloids and Praseptiangga, D., Giovani, S., Manuhara, G. J., & Muhammad, D. R. A. (2017).
Surfaces B: Biointerfaces, 59(1), 24–34. Formulation and characterization of novel composite semi-refined iota carrageenan-
Garcıa-Ochoa, F., Santos, V. E., Casas, J. A., & Gomez, E. (2000). Xanthan gum: based edible film incorporating palmitic acid. AIP conference proceedings: Vol.
Production, recovery, and properties. Biotechnology Advances, 18(7), 549–579. 1884AIP Publishing030006.
Ghasemi, S., Jafari, S. M., Assadpour, E., & Khomeiri, M. (2017). Production of pectin- Ribeiro-Santos, R., Andrade, M., Madella, D., Martinazzo, A. P., Moura, L. D. A. G., de
whey protein nano-complexes as carriers of orange peel oil. Carbohydrate Polymers, Melo, N. R., et al. (2017). Revisiting an ancient spice with medicinal purposes:
177, 369–377. Cinnamon. Trends in Food Science & Technology, 62, 154–169.
Ghasemi, S., Jafari, S. M., Assadpour, E., & Khomeiri, M. (2018). Nanoencapsulation of d- Sedaghat Doost, A., Dewettinck, K., Devlieghere, F., & Van der Meeren, P. (2018a).
limonene within nanocarriers produced by pectin-whey protein complexes. Food Influence of non-ionic emulsifier type on the stability of cinnamaldehyde nanoe-
Hydrocolloids, 77, 152–162. mulsions: A comparison of polysorbate 80 and hydrophobically modified inulin. Food
Gruenwald, J., Freder, J., & Armbruester, N. (2010). Cinnamon and health. Critical Chemistry, 258, 237–244.
Reviews in Food Science and Nutrition, 50(9), 822–834. Sedaghat Doost, A., Kassozi, V., Grootaert, C., Claeys, M., Dewettinck, K., Van Camp, J.,
Helal, A., Tagliazucchi, D., Verzelloni, E., & Conte, A. (2014). Bioaccessibility of poly- et al. (2019a). Self-assembly, functionality, and in-vitro properties of quercetin
phenols and cinnamaldehyde in cinnamon beverages subjected to in vitro gastro- loaded nanoparticles based on shellac-almond gum biological macromolecules.
pancreatic digestion. Journal of Functional Foods, 7, 506–516. International Journal of Biological Macromolecules, 129, 1024–1033.
Ilmi, A., Praseptiangga, D., & Muhammad, D. R. A. (2017). Sensory attributes and pre- Sedaghat Doost, A., Muhammad, D. R. A., Stevens, C. V., Dewettinck, K., & Van der
liminary characterization of milk chocolate bar enriched with cinnamon essential oil. IOP Meeren, P. (2018b). Fabrication and characterization of quercetin loaded almond
conference series: Materials science and engineering, Vol. 193IOP Publishing012031. gum-shellac nanoparticles prepared by antisolvent precipitation. Food Hydrocolloids,
Jafari, S. M., & McClements, D. J. (2017). Nanotechnology approaches for increasing 83, 190–201.
nutrient bioavailability. In F. Toldra (Vol. Ed.), Advances in food and nutrition research: Sedaghat Doost, A., Nikbakht Nasrabadi, M., Kassozi, V., Dewettinck, K., Stevens, C. V., &
Vol. 81, (pp. 1–30). Academic Press. Van der Meeren, P. (2019b). Pickering stabilization of thymol through green emul-
Joye, I. J., Davidov-Pardo, G., & McClements, D. J. (2014). Nanotechnology for increased sification using soluble fraction of almond gum-whey protein isolate nano-complexes.
micronutrient bioavailability. Trends in Food Science & Technology, 40(2), 168–182. Food Hydrocolloids, 88, 2018–2027.
Joye, I. J., & McClements, D. J. (2013). Production of nanoparticles by anti-solvent Shahidi, F., & Ambigaipalan, P. (2015). Phenolics and polyphenolics in foods, beverages
precipitation for use in food systems. Trends in Food Science & Technology, 34(2), and spices: Antioxidant activity and health effects–A review. Journal of Functional
109–123. Foods, 18, 820–897.
Joye, I. J., & McClements, D. J. (2014). Biopolymer-based nanoparticles and micro- Shori, A. B., & Baba, A. S. (2011). Cinnamomum verum improved the functional prop-
particles: Fabrication, characterization, and application. Current Opinion in Colloid & erties of bioyogurts made from camel and cow milks. Journal of the Saudi Society of
Interface Science, (5), 417–427. Agricultural Sciences, 10(2), 101–107.
Kakran, M., Sahoo, N. G., Li, L., & Judeh, Z. (2012). Fabrication of quercetin nanoparticles Sun, C., Xu, C., Mao, L., Wang, D., Yang, J., & Gao, Y. (2017). Preparation, character-
by anti-solvent precipitation method for enhanced dissolution. Powder Technology, ization and stability of curcumin-loaded zein-shellac composite colloidal particles.
223, 59–64. Food Chemistry, 228, 656–667.
Katouzian, I., & Jafari, S. M. (2016). Nano-encapsulation as a promising approach for Teixeira, B. N., Ozdemir, N., Hill, L. E., & Gomes, C. L. (2013). Synthesis and char-
targeted delivery and controlled release of vitamins. Trends in Food Science & acterization of nano‐encapsulated black pepper oleoresin using hydroxypropyl be-
Technology, 53, 34–48. ta‐cyclodextrin for antioxidant and antimicrobial applications. Journal of Food
Kraisit, P., Limmatvapirat, S., Nunthanid, J., Sriamornsak, P., & Luangtana-anan, M. Science, 78(12), 1913–1920.
(2013). Nanoparticle formation by using shellac and chitosan for a protein delivery Udayaprakash, N. K., Ranjithkumar, M., Deepa, S., Sripriya, N., Al-Arfaj, A. A., &
system. Pharmaceutical Development and Technology, 18(3), 686–693. Bhuvaneswari, S. (2015). Antioxidant, free radical scavenging and GC–MS compo-
Li, R., Liang, T., Xu, L., Li, Y., Zhang, S., & Duan, X. (2013). Protective effect of cinnamon sition of Cinnamomum iners Reinw. ex Blume. Industrial Crops and Products, 69,
polyphenols against STZ-diabetic mice fed high-sugar, high-fat diet and its underlying 175–179.
mechanism. Food and Chemical Toxicology, 51, 419–425. Xu, Y., & Du, Y. (2003). Effect of molecular structure of chitosan on protein delivery
Lu, Z., Jia, Q., Wang, R., Wu, X., Wu, Y., Huang, C., et al. (2011). Hypoglycemic activities properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250(1),
of A-and B-type procyanidin oligomer-rich extracts from different Cinnamon barks. 215–226.
Phytomedicine, 18(4), 298–302. Zheng, L., Ding, Z., Zhang, M., & Sun, J. (2011). Microencapsulation of bayberry poly-
Manuhara, G. J., Praseptiangga, D., Muhammad, D. R. A., & Maimuni, B. H. (2016, phenols by ethyl cellulose: Preparation and characterization. Journal of Food
February). Preparation and characterization of semi-refined kappa carrageenan-based Engineering, 104(1), 89–95.
edible film for nano coating application on minimally processed food. AIP conference