Food Hydrocolloids 106 (2020) 105878

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Food Hydrocolloids
journal homepage: http://www.elsevier.com/locate/foodhyd

Nanochitin-stabilized pickering emulsions: Influence of nanochitin on lipid

digestibility and vitamin bioaccessibility
Hualu Zhou a, Yunbing Tan a, Shanshan Lv b, Jinning Liu a, Jorge L. Muriel Mundo a, Long Bai c,
Orlando J. Rojas c, David Julian McClements a, *
a
Biopolymers and Colloids Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA, 01003, USA
b
College of Forestry, Northwest A&F University, Shanxi, 712100, China
c
Department of Chemical Engineering, Department of Biological Engineering, Department of Chemistry, Department of Wood Science, 2360 East Mall, The University of
British Columbia, Vancouver, BC V6T 1Z3, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Nature-derived nanoparticles are increasingly being explored for their potential to regulate the digestion of
Vitamins ingested lipids. In this study, we examined the impact of emulsifier format (molecular versus particle) on the
Nanoparticles gastrointestinal fate of vitamin D3-fortified emulsions by measuring their physicochemical properties, micro­
Nanochitin
structure, digestibility, and bioaccessibility using an in vitro human gastrointestinal tract (GIT) model. Nanochitin
Lipid digestion
Bioavailability
(NCh) was used as an example of a particle-based (Pickering) emulsifier, whereas Tween 80 (T80) was used as an
example of a small molecule surfactant. A series of 2 wt% oil-in-water emulsions containing different initial
levels and locations of nanochitin and Tween 80 were prepared: (i) nanochitin-emulsions (0.02 wt% NCh); (ii)
Tween 80-emulsions (0.02 wt% T80); (iii) nanochitin-emulsions (0.02 wt% NCh) plus Tween 80 (0.02 wt% T80);
and (iv) Tween 80-emulsions (0.02 wt% T80) plus nanochitin (0.02 wt% NCh). The nanochitin-emulsions were
much more prone to droplet aggregation within the simulated GIT than the T80-stabilized ones. Lipid digestion
was 30% less and vitamin bioaccessibility was 45% less for the nanochitin-emulsions than for the T80-stabilized
ones. Adding T80 to the nanochitin-emulsions, increased the rate of lipid digestion, whereas adding nanochitin to
the Tween 80-stabilized ones, decreased the rate. The reduction in lipid digestion and bioaccessibility in the
emulsions containing nanochitin may have been for a number of reasons: the adsorbed nanochitin layer hindered
the ability of lipase to reach the lipid phase; the nanochitin-coated droplets were highly aggregated in the GIT,
which reduced the area of lipids accessible to the lipase; and, the cationic nanochitin bound to anionic bile acids,
fatty acids, or lipase. Our results suggest that nanochitin slows down lipid digestion, which may be advantageous
for developing high-satiety foods, but that it also reduces vitamin bioaccessibility, which would be undesirable
from a nutritional perspective.

1. Introduction
Obesity is a pressing global health concern that has been linked to the
rise of various chronic diseases, including cancer, depression, diabetes,
and heart disease (Bray & Popkin, 1998; Hariri & Thibault, 2010;
Madadlou, Rakhshi, & Abbaspourrad, 2016). Reducing dietary fat intake
may help prevent obesity, but it is often difficult for individuals to
control their fat intake due to its important role in sensory attributes and
hormonal responses (Bellissimo & Akhavan, 2015; Guo, Ye, Bellissimo,
Singh, & Rousseau, 2017). One promising approach is to use structural
design principles to create reduced-fat foods that promote satiety,
thereby reducing the tendency to overeat (Guo et al., 2017; Helgason
et al., 2009; McClements, Decker, & Park, 2009). These functional foods
often rely on controlling the lipid digestion profile within the gastroin­
testinal tract (GIT): with slower digestion promoting higher satiety
(Poppitt et al., 2018).
The fats in many high-calorie foods are present in an emulsified form
and so their digestion can be controlled using the principles of colloid
and interface science (Guo et al., 2017). Indeed, many researchers have
already demonstrated that lipid hydrolysis and absorption can be
modulated by controlling the surface properties of emulsified fats e.g.,
composition, area, thickness, structural organization, and charge

* Corresponding author.
E-mail address: mcclements@foodsci.umass.edu (D.J. McClements).

https://doi.org/10.1016/j.foodhyd.2020.105878
Received 20 January 2020; Received in revised form 18 March 2020; Accepted 20 March 2020
Available online 29 March 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
H. Zhou et al.
(Armand et al., 1999; Golding & Wooster, 2010; Mun, Decker, &
McClements, 2007; Mun, Decker, Park, Weiss, & McClements, 2006;
Sarkar, Li, Cray, & Boxall, 2018; Sarkar, Zhang, Murray, Russell, &
Boxal, 2017; Tzoumaki, Moschakis, Scholten, & Biliaderis, 2013; Xiao
et al., 2018). A number of interfacial engineering approaches have been
developed to inhibit the digestion of emulsified fats, including nano­
lamination (Corstens et al., 2017b; Sarkar et al., 2018; Sarkar et al.,
2017), particle adsorption (Borreani et al., 2017; DeLoid et al., 2018;
Liang et al., 2016; Zhou, Yan, Yin, Tang, & Yang, 2018), and interfacial
gelation (Corstens et al., 2017a; Sarkar et al., 2015; Sarkar et al., 2016).
These approaches typically rely on the formation of thick cohesive
coatings around the emulsified fats that restrict the ability of the lipase
molecules to contact the underlying triacylglycerol molecules, as well as
on the high desorption energies of particles that limit their displacement
by bile acids (Sarkar et al., 2018; Sarkar, Zhang, Holmes, & Ettelaie,
2019; Sarkar et al., 2017; Tzoumaki et al., 2013; Xiao et al., 2018).
Chitin nanoparticles (“nanochitin”) are polysaccharide-based nano­
materials isolated from sustainable natural sources that have the po­
tential to inhibit lipid digestion (Tzoumaki et al., 2013; Xiao et al.,
2018). Nanochitin may therefore be suitable for creating functional
foods that can improve human health by modulating the hydrolysis of
emulsified fats inside the GIT. Indeed, anti-obesity effects have been
reported for chitin-based polymers included in a high-fat diet fed to
mice, which was attributed to their tendency to inhibit fat digestion and
absorption (Han, Kimura, & Okuda, 1999). Several in vitro and in vivo
studies have indicated that chitin-based polymers can strongly bind to
bile acids, which may partly account for their ability to inhibit lipid
digestion or absorption (Ebihara & Schneeman, 1989; Gallaher, Munion,
Hesslink, Wise, & Gallaher, 2000; Lee, Kim, & Kim, 1999; Sugano et al.,
1980). For instance, the cationic polymers may promote the precipita­
tion of anionic mixed micelles, thereby reducing the ability of the free
fatty acids to be transported to the epithelium cells.
Nanochitin has previously been shown to be a highly effective
particle-based emulsifier, with the ability to strongly adsorb to fat
droplet surfaces during homogenization, and then stabilize them during
storage (Bai et al., 2019). Nevertheless, there is still a rather limited
understanding of the gastrointestinal fate of nanochitin-stabilized
Pickering emulsions, particularly the effects of a nanochitin coating on
the digestion of emulsified fats. In particular, there have been few
studies directly comparing the gastrointestinal fate of Pickering emul­
sions with conventional emulsions, as well as on the behavior of emul­
sions containing a mixture of molecular and particulate emulsifiers. In
addition, there have been few studies on the impact of Pickering
emulsifiers on the bioavailability of any encapsulated ingredients
(Araiza-Calahorra, Akhtar, & Sarkar, 2018; Shah, Zhang, Li, & Li, 2016).
One recent study showed that nanocellulose reduces the rate of lipid
digestion and decreases the bioaccessibility of vitamin D when present
at sufficiently high levels (Winuprasith et al., 2018).
In the current research, we aimed to address some of these issues.
First, we compared the gastrointestinal fate of nanochitin-stabilized
Pickering emulsions with that of surfactant-stabilized conventional
emulsions. In these studies, a non-ionic surfactant (Tween 80, T80) was
used to prepare the conventional emulsions. Second, we examined the
impact of adding a second emulsifier to the system on their gastroin­
testinal fate, either T80 to nanochitin-stabilized emulsions or nanochitin
to T80-stabilized emulsions. Third, we investigated the impact of
nanochitin coatings on lipid digestion and vitamin bioaccessibility. Our
working hypothesis was that the nanochitin coating would suppress the
digestion of the fat droplets, which would then reduce the bio­
accessibility of the vitamin. Our research therefore builds on earlier
studies on the gastrointestinal fate of nanochitin-stabilized emulsions
(Bai et al., 2019; Tzoumaki et al., 2013; Xiao et al., 2018; Zhou et al.,
2020). These results may prove useful for the creation of functional food
products that can inhibit lipid digestion, thereby enhancing the satiety
response.
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Food Hydrocolloids 106 (2020) 105878
2. Materials and methods
2.1. Materials
Deacetylated chitin (degree of deacetylation ¼ 27.3%) from crab
shells was provided by Aalto University (Finland) (Bai et al., 2019).
Tween 80 and Nile red were obtained from Sigma-Aldrich (Sigma
Chemical Co., St. Louis, Mo, USA). Corn oil was purchased from a su­
permarket (Mazola, ACH Food Company, Memphis, TN). Vitamin D3 (1,
0 Mill. I.U./g) was kindly provided by BASF (Ludwigshafen, Germany).
The in vitro digestion chemicals, such as mucin, pepsin, lipase, bile
extract, and inorganic salts, were obtained from Sigma-Aldrich. Double
distilled water was utilized to prepare all solutions and emulsions.
2.2. Emulsion fabrication
Nanochitin was prepared by alkaline-mechanical treatment of
deacetylated crab shell chitin as described previously (Bai et al., 2019).
Small pieces of deacetylated-chitin were dissolved in an acetic acid so­
lution. The mixture was then adjusted to pH 3.0 and stirred for 2 h to
ensure complete dissolution, followed by sonication for 40 min (50%
power, on/off: 3s/2s) using a sonicator (FB505, Fisher Scientific, USA).
A nanochitin stock solution was prepared containing 0.5 wt% nano­
chitin, which was then stored at 4 � C. The nanochitin particles used in
the study have previously been reported to have a mean particle
diameter of 0.220 μm, which was determined by laser diffraction (Bai
et al., 2019; Zhou et al., 2020). It should be noted, however, that
nanochitin particles are non-spherical and so this value should be
treated only as an effective particle size. The same source of nanochitin
has previously been reported to contain particles with a mean length of
166 � 25 nm and a mean width of 11 � 2 nm, which was determined by
electron microscopy (Bai et al., 2019).
A Pickering emulsion was creating by homogenization of a 90 wt%
aqueous nanochitin (0.11 wt%) suspension with a 10 wt% oil phase (2
wt% vitamin D3 in corn oil). A hand-blender was used to homogenize the
oil and aqueous phases for 2 min, and then they were sonicated for 4 min
(50% power, on/off: 3s/2s) in an ice-water bath. The final stock Pick­
ering emulsions contained 0.1 wt% nanochitin and 10.0 wt% oil.
To obtain more insight into the impact of nanochitin on the digestion
of the Pickering emulsions, three other types of emulsions were prepared
as controls. A Tween 80-stabilized emulsion was formed by homogeni­
zation of 10 wt% corn oil with 90 wt% aqueous surfactant solution (0.1
wt% Tween 80, pH 3). Mixed nanochitin/surfactant emulsions were
formed by adding Tween 80 solution to a nanochitin-stabilized emulsion
or by adding nanochitin to a Tween 80-stabilized emulsion (1:1 v/v) and
then stirring for 30 min.
All the emulsions were then diluted so that they had the same final
oil content (2% corn oil). This led to systems with the following final
compositions: (i) nanochitin-emulsions (0.02 wt% NCh); (ii) Tween 80-
emulsions (0.02 wt% T80); (iii) nanochitin-emulsions (0.02 wt% NCh)
plus Tween 80 (0.02 wt% T80); and (iv) Tween 80-emulsions (0.02 wt%
T80) plus nanochitin (0.02 wt% NCh). It should be noted that the
interfacial composition of the emulsions containing both Tween 80 and
nanochitin may change after preparation due to competitive and/or co-
adsorption effects.
2.3. In vitro digestion
A three-stage simulated GIT (oral, gastric, small intestine) was used
to study the physicochemical and structural changes, lipid digestion
rate, and vitamin bioaccessibility of the various emulsions, which has
been described in detail elsewhere (Schoener, Zhang, Lv, Weiss, &
McClements, 2019; Winuprasith et al., 2018). In brief, the emulsions
were first diluted to the same final oil concentration (2 wt%) as
described in the previous section, and then preheated to 37 � C. For the
oral phase, emulsions (7.5 mL) were added to simulated saliva (7.5 mL)
H. Zhou et al.
that contained 3 mg/mL mucin, and then the system was adjusted to pH
6.8 and incubated in a shaker at 37 � C for 2 min (Innova Incubator
Shaker, Model 4080, New Brunswick Scientific, NJ, USA). For the gastric
phase, the mixture from the oral phase (15 mL) was added to simulated
gastric fluids (15 mL) containing pepsin (3.2 mg/mL, 800 U/mL in the
final stomach phase), adjusted to pH 2.5, and then incubated in a shaker
at 37 � C for 2 h. For the small intestine phase, the mixture from the
gastric phase (30 mL) was placed in the vessel of an automatic titration
unit and then 1.5 mL of salt solution (3.75 M NaCl and 0.25 M CaCl2),
3.5 mL of bile extract solution (5 mg/mL), and 2.5 mL of fresh lipase
solution (24 mg/mL, 100 U/mL in the final small intestine phase) were
added sequentially. The mixture was then changed to pH 7.0 and a pH
stat (Metrohm, USA Inc, Riverview, FL, USA) was used to maintain the
pH at 7.0 by titration of NaOH solution throughout lipid digestion. The
free fatty acids (FFA) liberated from the sample at a particular time was
determined as follows (Li & McClements, 2010):
VNaOH ​ CNaOH Mlipid
FFA ​ ð%Þ ¼
2Wlipid
� 100
Here, VNaOH is the volume of NaOH solution consumed, CNaOH is the
concentration of the NaOH solution (0.25 M), Mlipid is the molecular
weight of the carrier lipid (824 g/mol), and Wlipid is the weight of the
carrier lipid.
2.4. Emulsion characterization
2.4.1. Particle dimensions and surface charge
Laser diffraction was used to measure the mean particle diameter
(d32) and particle size distribution (PSD) of the particles in the samples
(Mastersizer 2000, Malvern Instruments, Worcestershire, United
Kingdom), while particle electrophoresis was used to measure their
surface potentials (Nano-ZS, Malvern Instruments). Samples were
diluted using pH-adjusted distilled water before analysis (same pH as
sample) to reach an optimal particle concentration for light scattering
analysis. It should be stressed that in the mixed emulsions, which con­
tained both solid particles (nanochitin) and liquid droplets (oil), the
results obtained from laser diffraction should be treated with caution
because both kinds of particles contribute to the overall light scattering
signal used to determine the particle dimensions and charge.
2.4.2. Microstructure
Emulsion microstructures were observed using confocal microscopy
with a 40�objective lens (Nikon D-Eclipse C1 80i, Nikon, NY, United
States). The lipids were dyed using a fat-soluble stain (1 mg/mL Nile Red
in ethanol). A small volume (5 μL) of dyed sample was put onto a mi­
croscope slide and then a cover slip was used to prevent evaporation.
2.5. Vitamin bioaccessibility
After passing through the entire GIT model, the samples were
centrifuged at 18,000 rpm for 50 min at 4 � C (Thermo Fisher Scientific,
MA, USA). The upper layer (supernatant) was considered to contain the
bioaccessible vitamin-loaded “mixed micelles”. The mixed micelles are
small colloidal particles formed from lipid digestion products (free fatty
acids and monoacylglycerols) and bile salts. The extraction and quan­
tification methods for vitamin D3 have been described in detail in earlier
studies (Schoener et al., 2019; Tan, Liu, Zhou, Mundo, & McClements,
2019). Briefly, a solvent-extraction procedure was used to isolate the
oil-soluble vitamins from the samples, which consisted of a three-step
process: (i) adding 3 mL of sample to 3 mL of organic solvent (hex­
ane/ethanol: v/v ¼ 1/1); (ii) vortexing the sample to ensure it was ho­
mogeneous; and (iii) centrifuging at 4000 rpm for 2 min to promote
separation of the phases (Sorvall ST8, Thermo Fisher Scientific, MA,
USA). This procedure was repeated three times to ensure good extrac­
tion efficiency. The supernatant layers were then collected, combined,
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Food Hydrocolloids 106 (2020) 105878
and dehydrated with saturated sodium chloride before they were dried
using nitrogen. Afterward, the dehydrated samples were dispersed
within HPLC grade methanol, passed through a 0.45 μm membrane
filter, and then analyzed by HPLC (Agilent 1100 series, Agilent Tech­
nologies, CA, USA) using a UV detector at 265 nm (column: C18 (4.6 �
250 nm, 5 μm, Agilent Technologies). The mobile phase was comprised
of a mixture of methanol and double distilled water (v/v ¼ 95/5).
Finally, the vitamin bioaccessibility was determined as follows:
CM
Bioaccessibilityð%Þ ¼ � 100
CD
Here, CM and CD represent the vitamin concentrations within the mixed
micelle phase and the total digested sample, respectively.
3. Statistical analysis
All measurements were carried out at least in triplicate, with the
exception of the emulsion preparation and digestion processes, which
were carried out in duplicate. The means and standard deviations of the
data were calculated, and then the Duncan test was used in the analysis
of variance (ANOVA, p < 0.05) to establish statistical differences among
treatments (SPSS, IBM Corp., Armonk, NY, USA).
4. Results and discussion
4.1. Impact of pH on nanochitin-stabilized emulsions
Initially, the impact of pH on the size and charge of the particles in
the nanochitin-stabilized emulsions was determined (Fig. 1), as this in­
formation was useful in interpreting the gastrointestinal studies dis­
cussed later. The ζ-potential on the nanochitin was strongly positive at
low pH but strongly negative at high pH (Fig. 1a). The positive charge at
low pH values is a result of amino group protonation on the nanochitin
surfaces, whereas the negative charge at high pH values is due to
carboxyl group de-protonation (Liu et al., 2014). The strength of the
positive charge decreased as the system was reduced from pH 3 to 2
because of the increase in ionic strength under highly acidic conditions.
The size of the particles in the nanochitin-stabilized emulsions was
relatively low from pH 2 to 4, but relatively high from pH 5 to 8, which is
indicative of extensive aggregation of nanochitin-coated fat droplets at
higher pH values (Fig. 1b). The particle size results were supported by
the confocal microscopy analysis of the samples (Fig. 2).
The nanochitin-stabilized emulsions were initially prepared under
acidic conditions (pH 3) and then adjusted to other pH values. At low pH
values, there was a strong positive charge on the particle-coated drop­
lets, which inhibited their aggregation because of the strong electro­
static repulsion between them. As the pH was increased, the surface
potential became less strongly positive, then zero, and then increasingly
negative. Interestingly, the emulsions were highly aggregated at pH 8,
even though they had a high negative charge. Presumably, the oil
droplets in the emulsions became irreversibly aggregated when the pH
was raised and passed through their isoelectric point. Under these
conditions, there would only be a weak electrostatic repulsion between
the nanochitin-coated oil droplets, which may have promoted their
irreversible aggregation. Indeed, extensive coalescence of the droplets
was observed in the confocal microscopy images around pH 5 and higher
(Fig. 2), which would not be broken down when the pH was raised
further.
The confocal fluorescence microscopy images also indicate that the
nanochitin-stabilized emulsions were fairly resistant to aggregation at
pH 3, exhibiting a uniform distribution of relatively small lipid droplets
(Fig. 2). At all other pH values, however, some aggregation of the lipid
droplets occurred, with the nature of the aggregates formed being highly
pH-dependent. At pH 2 and 4 there appeared to be fairly loose “cloud-
like” flocs formed, which presumably broke down when these samples
H. Zhou et al. Food Hydrocolloids 106 (2020) 105878

Fig. 1. Impact of pH on charge and size of the particles in nanochitin-stabilized oil-in-water emulsions.

Fig. 2. Impact of pH on the microstructure of nanochitin-stabilized emulsions assessed using confocal fluorescence microscopy. The oil phase was stained red and the
scale bar is 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

were diluted for laser diffraction analysis (accounting for the small
measured particle size). At pH 5 and higher, the lipid droplets were
highly aggregated with clear evidence of droplet coalescence having
occurred (i.e., the presence of large individual fat droplets). The pH-
dependence of the particle size and charge would be expected to influ­
ence the GIT fate of the Pickering emulsions because the pH changes as
the samples move from mouth to stomach to small intestine.
Tween 80 (“NCh(þTween)”); and, (iv) Tween 80-stabilized emulsions þ
nanochitin (“Tween(þNCh)”). These emulsions were prepared to
distinguish the effects of adsorbed and non-adsorbed nanochitin on their
potential gastrointestinal behavior. Previous studies have shown that
Tween 80 does not strongly inhibit lipid digestion at the concentrations
used here, which means that Tween 80-stabilized emulsions can be
utilized as a reference system (Li & McClements, 2011).

4.2. Physicochemical properties of the emulsions in the gastrointestinal 4.2.1. Appearance

tract
Initially, the influence of gastrointestinal conditions on the structural
and physicochemical characteristics of the various emulsions was
determined.
The four emulsions used in this study are shown schematically in
Fig. 3a: (i) nanochitin-stabilized emulsions (“NCh”); (ii) Tween 80-stabi­
lized emulsions (“Tween”); (iii) nanochitin-stabilized emulsions þ
Emulsion appearance was measured after 0, 1, and 7 days storage
(Fig. 3b). Immediately after production, all emulsions appeared white
and homogeneous, showing they were resistant to rapid gravitational
separation, presumably due to their fairly small initial droplet size. After
prolonged storage, creaming was observed in a number of the emulsions.
The nanochitin-emulsions were the most stable to creaming, with no
evidence of phase separation after 7 days storage, which is consistent
with our previous study on similar systems (Zhou et al., 2020). This

4
H. Zhou et al. Food Hydrocolloids 106 (2020) 105878

Fig. 3. (a) Schematic diagram of the initial structure of the four emulsions used in this study. (b) Appearances of the different emulsions after storage for different
times. The initial emulsions contained 10 wt% oil phase (98% corn oil and 2% vitamin D3) and 90 wt% aqueous phase (0.1 wt% Tween 80 or nanochitin, pH 3).

effect was probably partly because the nanochitin coating increased the
effective density of the fat droplets, thereby decreasing their density
contrast with the surrounding aqueous phase. Moreover, there may have
been some loose flocculation of the nanochitin-coated droplets, leading
to the formation of a 3D-network that inhibited their movement. Some
creaming was also observed in the Tween 80-emulsions at the end of
storage, with a fairly thick white creamed layer forming at the top of the
samples, which was probably because the initial droplet size was too
large to fully resist gravitational forces (see next section). Interestingly,
for the NCh (þTween) emulsions there was also evidence of a cream
layer in the samples after 7 days storage, which may have been because
the surfactant caused desorption of some of the nanochitin or inhibited
droplet flocculation. Normally, the nanoparticles used to stabilize
Pickering emulsions are assumed to be so strongly adsorbed that they
cannot be displaced from the droplet surfaces. However, it may still be
possible for a small molecule surfactant, like Tween 80, to promote the
displacement of nanochitin by adsorbing to the surfaces of the nano­
particles and changing their wetting characteristics.

4.2.2. Particle size

The particle size characteristics of the four emulsions were also
measured after each GIT phase (Figs. 4 and 5). All the emulsions initially
had fairly similar mean particle diameters (~1.5 μm), but pronounced
differences were observed after passage through the mouth, stomach,
and small intestine.
For the nanochitin-emulsions, the particle size was very large in the
mouth, stomach, and small intestine phases, due to the formation of a
monomodal population of large particles in the samples. Within the
mouth, the nanochitin-coated lipid droplets probably aggregated
because the pH was around neutral, which was seen to induce aggre­
gation in the preliminary experiments (Fig. 1). Within the stomach,
aggregation may have been a result of bridging flocculation of the
Fig. 4. Influence of gastrointestinal region on mean particle diameters of
different emulsions. The significant difference between different emulsion
samples was labeled by capital letters (A, B, C, D), while the significant dif­
ference between different GIT phases was labeled by low-case letters (a, b, c, d).
cationic nanochitin-coated lipid droplets by anionic mucin molecules
(Sarkar, Goh, & Singh, 2010). Within the small intestine, aggregation
may have again been due to the neutral pH, as well as the action of the
lipase. In general, the pH and solution composition change as the par­
ticles mover from the mouth to the stomach, which alters the electro­
static and other colloidal interactions acting between them. In addition,

5
H. Zhou et al. Food Hydrocolloids 106 (2020) 105878

Fig. 5. Influence of gastrointestinal region on PSDs of different emulsions.

Fig. 6. Impact of gastrointestinal region on the microstructure of the emulsions monitored by confocal fluorescence microscopy (the lipid phase is stained red). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

6
H. Zhou et al.
there is some dilution and mixing of the samples that could disrupt any
large aggregates.
For the Tween 80-emulsions, the particles remained relatively small
throughout the entire GIT, although there was some increase after
exposure to the stomach phase (Fig. 4). Moreover, the particle size
distribution (PSD) was monomodal in the initial, mouth, and stomach
samples, but multimodal in the small intestine samples. The surfactant-
coated droplets therefore appeared to be fairly resistant to gastrointes­
tinal conditions until they reached the small intestine, where the lipid
phase was digested by lipase.
The Tween 80-emulsions containing added nanochitin had a rela­
tively small particle size in the mouth and stomach stages, but a large
one after the small intestine stage. The PSD measurements show that
these Tween(þNCh) emulsions were bimodal in the mouth, stomach,
and small intestine (Fig. 5b). By comparison with the emulsions pre­
pared using a single type of emulsifier (Fig. 5a and c), the smaller par­
ticles can be assumed to be surfactant-coated lipid droplets, whereas the
larger ones can be assumed to be aggregated nanochitin particles or
nanochitin-coated oil droplets. The light scattering data is supported by
the confocal microscopy measurements (Fig. 6), which show that some
extensive aggregation occurred in all the emulsions, except the Tween
stabilized ones. The nanochitin-emulsions with added Tween 80
behaved fairly similarly to the nanochitin-emulsions without Tween 80.
This is probably because their behavior was dominated by the aggre­
gation of the chitin-coated lipid droplets. This result also suggests that
the Tween 80 did not completely displace the nanochitin from the lipid
droplet surfaces, which was supported by ζ-potential measurements (see
later).
4.2.3. Microstructure
Confocal fluorescence microscopy analysis showed that the fat
droplets in the nanochitin-emulsions aggregated within the mouth and
stomach but were largely digested in the small intestine (Fig. 6). Mi­
croscopy analysis also showed that the fat droplets in the Tween 80-
emulsions remained relatively small in the mouth and stomach, but
were almost completely digested in the small intestine. Addition of the
surfactant to the nanochitin-emulsions (NCh(þTween)) appeared to
reduce the size of the aggregates formed, but there was still evidence of
extensive droplet flocculation within the mouth and stomach.
Conversely, the addition of nanochitin to the surfactant-stabilized
emulsions (Tween(þNCh)) appeared to promote some aggregation of
the lipid droplets in the mouth and stomach.
4.2.4. Surface potential
Surface potential analysis gives useful insights into alterations in the
interfacial properties of emulsions under GIT conditions. The ζ-poten­
tials of the four initial emulsions (pH 3) depended on their composition
and structure (Fig. 7). The nanochitin-emulsions contained strongly
positively charged droplets due to the adsorbed nanochitin particles.
The Tween 80-emulsions had a low negative charge, which was prob­
ably due to some anionic impurities in the surfactant, as has been re­
ported previously (Uluata, McClements, & Decker, 2015). The NCh
(þTween) emulsions had a high positive charge but the magnitude was
less than for the NCh emulsions, suggesting that the small-molecule
surfactant may have displaced some of the nanoparticles or
co-adsorbed with them. As mentioned earlier, it is possible that the
surfactant molecules could have adsorbed to the surfaces of the nano­
chitin particles and altered their wetting properties, thereby altering
their affinity for the droplet surface. Conversely, the Tween (þNCh)
emulsions had a small negative charge, which was somewhat smaller in
magnitude than for the Tween emulsions, which suggests that the
nanochitin may have displaced the Tween or co-adsorbed with it.
Further studies are required to elucidate the precise mechanisms
involved.
In the mouth, the four emulsions had fairly similar moderately
negative surface potentials (around 15 mV), probably because of the
7
Food Hydrocolloids 106 (2020) 105878
Fig. 7. Impact of gastrointestinal region on the surface potential of the particles
in the emulsions. The significant difference between different emulsion samples
was labeled by capital letters (A, B, C, D), while the significant difference be­
tween different GIT phases was labeled by low-case letters (a, b, c, d).
neutral pH and adsorption of mucin to the droplet surfaces. In the
stomach phase, all four emulsions also had fairly similar surface po­
tentials but this time they were close to zero. The low net surface po­
tential in the stomach is partly due to the highly acidic gastric fluids
(which cause protonation of carboxyl groups), as well as the presence of
negatively-charged mucin molecules that adsorb to the surfaces of any
positively-charged particles thereby neutralizing their charge, probably
resulting in droplet flocculation (Sarkar et al., 2010). Within the small
intestine, the four emulsions all had relatively strong negative charges
because they contained colloidal particles assembled from
negatively-charged species such as bile acids, fatty acids, and peptides.
4.3. Changes in interfacial composition
We hypothesized that the different behaviors of the emulsions con­
taining mixed emulsifiers under simulated gastrointestinal conditions
may have been due to differences in their interfacial compositions. In­
sights into the ability of added emulsifiers to displace the existing
emulsifiers from the fat droplet surfaces was therefore obtained by
measuring alterations in particle surface potential and size (Fig. 8). In
one set of experiments, increasing levels of nanochitin were added to a
Tween 80-emulsion (Fig. 8a and b), whereas in the other set, increasing
levels of Tween 80 were added to a nanochitin-emulsion (Fig. 8c and d).
The incorporation of nanochitin into the Tween 80-emulsions caused
the ζ-potential to change from slightly negative to highly positive
(Fig. 8a). This effect may be a result of various phenomenon: the
nanochitin particles displaced some of the adsorbed surfactant mole­
cules from the lipid droplet surfaces; some of the nanochitin particles co-
adsorbed to the droplet surfaces; or, non-adsorbed nanochitin particles
contributed to the light scattering signal utilized in the ζ-potential
calculation. Conversely, the addition of Tween 80 to the nanochitin-
emulsions caused the ζ-potential to change from highly positive to
moderately positive (Fig. 8b). This phenomenon may have occurred
because some of the nanochitin particles displaced the Tween 80 mol­
ecules from the droplet surfaces. Alternatively, some of the anionic
species associated with the surfactant may have co-adsorbed on to the
surfaces of the fat droplets. Under these conditions, any non-adsorbed
surfactant micelles would be expected to be too small to scatter light
strongly and contribute to the ζ-potential measurements. Interestingly,
the incorporation of additional emulsifiers to both emulsions did not
cause a change in their mean particle diameter (Fig. 8a and c), which
suggests that they did not promote strong droplet aggregation. More
detailed studies are required in future to elucidate whether the observed
H. Zhou et al.
Fig. 8. Studies of the competition between nanochitin and Tween 80 in emulsions: (a) and (b) nanochitin was added to Tween 80-emulsions; (c) and (d) Tween 80
was added to nanochitin-emulsions.
changes in surface potential are due to displacement or co-adsorption.
4.4. Lipid digestion
The impact of nanochitin on lipid hydrolysis was assessed by
measuring the percentage of free fatty acids (FFAs) generated within the
simulated small intestine over time (Fig. 9). The fastest and most
extensive lipid digestion occurred in the reference Tween 80-emulsions,
which contain fat droplets coated by a thin layer of surfactant molecules.
The majority of the triacylglycerol molecules were hydrolyzed during
the first 10 min, with the rest being digested slowly throughout the
Fig. 9. Impact of emulsion type on FFA release under simulated small intesti­
nal conditions.
8
Food Hydrocolloids 106 (2020) 105878
remainder of the small intestine period. The initial rate of lipid digestion
was calculated from the slope of the FFA-time profiles during the first 10
min of digestion: 4.1, 8.5, 5.1, and 6.0 FFA/min for NCh, Tween, NChþ
(Tween), and Tweenþ(NCh) systems, respectively. In addition, the final
extent of lipid digestion in the same four systems after 2 h of digestion
was around 75%, 103%, 84%, and 69%, respectively. This, for the
nanochitin-stabilized emulsions, the initial lipid digestion rate was
considerably slower than for the reference emulsion. Indeed, the total
amount of FFAs released by the end of digestion was about 30% less. The
nanochitin layer coating the lipid droplets therefore appeared to retard
the hydrolysis of the triacylglycerols. This effect may have been due to
the nanocrystals forming a physical barrier around the lipid droplets,
which reduced the ability of the lipase molecules to assess the tri­
acylglycerol molecules. Even so, most of the lipid phase was still
digested, suggesting that the nanochitin coating could not completely
inhibit lipid digestion. This may have been because the small lipase
molecules could penetrate through the gaps separating the nanochitin
particles at the droplet surfaces. Alternatively, the bile acids may have
displaced some or all of the nanochitin from the surfaces of the fat
droplets.
The mixed-emulsifier emulsions exhibited behavior somewhere be­
tween that of the two single-emulsifier emulsions. For the NCh
(þTween) emulsions, the initial digestion rate was slower than the
reference emulsion, while the final lipid digestion extent was lower. This
phenomenon may have again been because the layer on nanochitin
particles retarded the ability of the lipase molecules to access the tri­
acylglycerol molecules inside the lipid droplets. Having said this, the
digestion rate and extent were somewhat higher in this system than in
the pure nanochitin-emulsions, suggesting that the surfactant may have
facilitated lipid digestion. The enhanced lipid digestion may have
H. Zhou et al. Food Hydrocolloids 106 (2020) 105878

occurred because the surfactant molecules partially displaced the

nanochitin particles from the surfaces of the lipid droplets, thereby
making it easier for the bile salts and lipase to adsorb. The Tween
(þNCh) emulsions had a relatively rapid initial digestion rate but still
slightly less than for the reference emulsions. There was, however, a
pronounced reduction in the total amount of FFAs generated during the
digestion period, which suggests non-adsorbed nanochitin may have
interfered with lipid digestion. The non-adsorbed nanochitin could bind
to bile salts or free fatty acids, thereby altering lipid digestion.

4.5. Vitamin bioaccessibility

We also examined the impact of emulsion type on the bioaccessibility

of vitamin D3 dissolved in their lipid phase (Fig. 10). The vitamin bio­
accessibility decreased in the following order: T80-stabilized emulsion
(73%) > NCh-stabilized emulsion þ T80 (41%) > NCh-stabilized
emulsion (28%) > T80-stabilized emulsion þ NCh (23%). The highest
bioaccessibility was therefore observed for the reference T80-emulsion,
which may be attributed to the rapid and complete digestion of the lipid
phase leading to the full release of the fat-soluble vitamin. Moreover, the
free fatty acids and monoacylglycerols released, as well as the surfactant
molecules themselves, could participate in mixed micelle formation,
thereby increasing the solubilization capacity of system. A much lower
bioaccessibility was observed in the emulsions containing nanochitin,
which suggests that these cationic nanoparticles interfered with the
release or solubilization of the vitamin molecules.
The bioaccessibility of the vitamin in the nanochitin-emulsion (28%)
was much lower than in the Tween 80-emulsion (73%), which suggests
that the nanochitin particles that coated the fat droplets had an impor­
tant influence. The final percentage of FFAs released was considerably
lower for the nanochitin-emulsion (76%) than for the Tween 80-emul­
sion (104%), which may at least partly account for this effect. Specif­
ically, a fraction of the vitamin may have stayed inside the non-digested
lipid phase and there would have been fewer mixed micelles present to
incorporate any vitamin that was released. In addition, some of the
vitamin-loaded mixed micelles may have precipitated in the presence of
the nanochitin and therefore not have contributed to the bio­
accessibility. The addition of the surfactant to the nanochitin-emulsions
(NCh(þTween)) led to a slight increase in bioaccessibility, possibly
because the surfactant displaced a fraction of the nanochitin particles
from the surfaces of the fat droplets, thus facilitating lipid digestion,
vitamin release, mixed micelle formation, and vitamin solubilization.
Interestingly, adding nanochitin to the surfactant-stabilized emulsions
(Tween(þNCh)) caused a large decrease in vitamin bioaccessibility,
which suggests that the non-adsorbed nanochitin can also impact the
release and solubilization of the vitamin. As mentioned previously, this
may have been a result of nanochitin promoting precipitation of the
vitamin-loaded mixed micelles, so they were not part of the supernatant
collected after centrifugation of the digested samples. Other studies have
also shown a correlation between the bioaccessibility of hydrophobic
substances and lipid digestion (Silva et al., 2019; Zhang et al., 2015;
Zhang et al., 2019).
5. Conclusion
In this study, we prepared nanochitin-stabilized Pickering emulsions
and then characterized their properties as they traveled through a
human GIT model. The behavior of these emulsions was compared to
those of a surfactant-stabilized emulsion, as well as to emulsions con­
taining a combination of nanochitin and surfactant. Our results showed
that both adsorbed and non-adsorbed nanochitin may interfere with
lipid digestion and vitamin bioaccessibility. The presence of the nano­
chitin reduces the initial digestion rate, as well as the total extent of lipid
digestion, which may be linked to at least three mechanisms: (i) the
nanochitin surrounding the fat droplets hindered the ability of lipase to
reach the lipid phase; (ii) nanochitin promoted flocculation of the fat
Fig. 10. Impact of emulsion type on the bioaccessibility of vitamin D. The
significant difference between different emulsion samples was labeled by cap­
ital letters (A, B, C, D).
droplets, thereby reducing the surface area of lipids accessible to the
lipase; and, (iii) cationic nanochitin bound to anionic bile acids, fatty
acids, and/or lipase, as well as promoting precipitation of the mixed
micelles. Our results may be useful for the formulation of functional
foods containing nanochitin that slow down lipid digestion inside the
human GIT. However, it is crucial to consider the impact that these
functional ingredients may have on the bioaccessibility of any fat-
soluble vitamins in the system.
Declaration of competing interest
The authors declared that they have no conflicts of interest to this
work.
Acknowledgements
This material was partly based upon work supported by the National
Institute of Food and Agriculture, USDA, Massachusetts Agricultural
Experiment Station (MAS00491). Hualu Zhou sincerely thanks the
Chinese Scholarship Council for financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodhyd.2020.105878.
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