Food Hydrocolloids 109 (2020) 106136

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

Mixed plant-based emulsifiers inhibit the oxidation of proteins and lipids in

walnut oil-in-water emulsions: Almond protein isolate-camellia saponin
Shulin Zhang a, Li Tian a, Jianhua Yi b, **, Zhenbao Zhu b, Eric Andrew Decker c,
David Julian McClements c, *
a
College of Biology and Food Engineering, Anyang Institute of Technology, Huanghe Road, An yang, Henan, 455000, PR China
b
School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xuefu Road, Xi’an, Shaanxi, 710021, China
c
Department of Food Science, University of Massachusetts, Amherst, MA, 01003, United States

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

Keywords: The oxidation of lipids and proteins often occurs at the oil-water interface in emulsions, and so emulsifiers may
Emulsions influence the oxidative stability of these systems. The objective of this work was to assess the influence of mixed
Protein oxidation plant-based emulsifiers, almond protein isolate (API) and camellia saponin (CS), on the physical and oxidative
Lipid oxidation
stability of walnut oil-in-water emulsions. Initially, a 5 wt% walnut oil emulsion was formulated using 0.5 wt%
Almond protein
API as an emulsifier, then different levels of CS (1.0–2.0 wt%) were added. Experiments were carried out under
Camellia saponins
conditions where the API and CS had different charges: pH 3 (protein positive, saponin negative) and pH 7 (both
negative). Analysis of the surface-potential and interfacial protein concentration indicated that mixed API-CS
interfacial layers were present at the oil droplet surfaces, with the main driving forces for adsorption being
electrostatic and/or hydrophobic interactions. Emulsions prepared with the mixed emulsifier system had greater
resistance to droplet flocculation than those stabilized by API alone when incubated at 45 � C for 0, 3, and 6 days.
Markers of lipid oxidation (hydroperoxides and TBARS) and protein oxidation (carbonyl formation, sulfhydryl
loss, intrinsic fluorescence loss, and electrophoresis) were recorded during storage. These measurements showed
that API-CS-coated droplets were more resistant to oxidation than API-coated ones. Oxidation was faster at pH 3
than pH 7, which was mainly linked to the higher water-solubility and chemical reactivity of the transition
metals under acidic conditions. Our results indicate that a combination of almond protein and camellia saponin is
suitable for forming plant-based emulsions with high physical and chemical stability.

1. Introduction
Almonds are widely consumed globally because they have good
flavor, nutritional and health attributes (Sze-Tao and Sathe (2000).
Indeed, almonds were the most highly produced tree nut in 2016 (1.06
million tons), with the United States being responsible for around half of
global production (Zhang, Zhang, Sheng, Wang, & Fu, 2016). They
provide relatively high levels of dietary proteins (16–22%) and lipids
(50–55%), with most of these lipids being unsaturated (85%) (Zhu et al.,
2018). In addition, they are also a good source of dietary fibers, min­
erals, and some vitamins (Sathe & Sze, 1997). The kernels from almonds
can be isolated and used as functional ingredients in various processed
foods (Sathe, 1993). Many desirable functional attributes of almonds are
linked to the proteins they contain (Young & Cunningham, 1991).
Almond protein isolate (API) has been reported to have good emulsi­
fying properties in previous studies (Sze-Tao et al., 2000). The produc­
tion of API from whole intact almonds is not economically viable
because they are too expensive. However, damaged almonds or
side-streams from almond production can be utilized for the production
of almond proteins. The API ingredients obtained can then be used as
emulsifiers to fabricate food emulsions, such as beverages, dressings,
creams, sauces, and desserts (Singh & Sarkar, 2011).
Owing to the perceived benefits of lipids enriched with poly­
unsaturated fatty acids (PUFAs) on human health, they are commonly
incorporated into emulsion-based foods and beverages. These lipids are,
however, prone to oxidation, thereby having deleterious effects on the
aroma, taste, texture, color, and nutrient value of emulsified foods (Ma
et al., 2012). Both animal and plant proteins have been reported to be

* Corresponding author.
** Corresponding author.
E-mail addresses: yijianhua1971121@126.com (J. Yi), mcclements@foodsci.umass.edu (D.J. McClements).

https://doi.org/10.1016/j.foodhyd.2020.106136
Received 13 May 2020; Received in revised form 23 June 2020; Accepted 26 June 2020
Available online 29 June 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
S. Zhang et al.
effective antioxidants in emulsions (Gumus, Decker, & McClements,
2017; Qiu, Zhao, Decker, & McClements, 2015; Yang & Xiong, 2015).
The antioxidative activity of these proteins has been ascribed to their
potential to sequester and inactivate pro-oxidant transition metals (e.g.
iron and copper), to scavenge free radicals, and to generate thick coat­
ings around the droplets that separate the unsaturated lipids from the
prooxidants in the aqueous phase (Elias, Kellerby, & Decker, 2008;
Villiere, Viau, Bronnec, Moreau, & Genot, 2005).
Antioxidant proteins slow down the rate of lipid oxidation in foods,
but they are often oxidized themselves during this process (Villiere et al.,
2005). Moreover, the products of lipid oxidation often have proox­
idative effects on proteins (Estevez, Kylli, Puolanne, Kivikari, & Hei­
nonen, 2008; Refsgaard, Tsai, & Stadtman, 2000). Oxidation causes
many physical and chemical alterations in proteins, including confor­
mational changes, formation of carbonyl derivatives, destruction of
amino acids (especially lysine, cysteine, methionine, histidine, and
tryptophan), crosslinking, polymerization, and β-scission (Mestdagh,
Kerkaert, Cucu, & Meulenaer, 2011; Schaich & Karel, 1976). These al­
terations typically result in changes in the functional attributes of pro­
teins, such as a reduction in solubility, a loss of essential amino acids,
and the generation of possibly toxic reaction products (Chen, Zhao, Sun,
& Zhao, 2013; Obando, Papastergiadis, Li, & De Meulenaer, 2015;
Schaich, 2014). Protein oxidation also influences the emulsifying and
interfacial properties of proteins. For instance, highly oxidized whey
protein has been reported to form less elastic interfacial layers compared
to the non-oxidized version (Berton-Carabin, Schroder,
Rovalino-Cordova, Schroen, & Sagis, 2016), which may account for the
reported decrease in the resistance of oxidized emulsions to droplet
coalescence (Muijlwijk et al., 2017). Consequently, it is desirable to
retard both protein and lipid oxidation in food emulsions to improve
their stability and extend their shelf lives.
In multiphase foods, like emulsions, the oil-water interface is a pri­
mary location of lipid and protein oxidation reactions (Berton-Carabin,
Ropers, & Genot, 2014). In this location, amphiphilic lipid hydroper­
oxides can interact with hydrophilic transition metals to form free rad­
icals, thereby propagating the lipid oxidation reaction (Berton-Carabin,
Genot, Gaillard, Guibert, & Ropers, 2013; Berton-Carabin et al., 2014;
McClements & Decker, 2000). Moreover, adsorbed proteins are more
readily oxidized than non-adsorbed ones (Yi et al., 2019). In the recent
decades, numerous scientists have focused on improving the stability of
emulsions by using emulsifier combinations rather than single emulsi­
fiers. For example, proteins and other oppositely charged biopolymers
have been successfully used to formulate multilayer emulsions by
layer-by-layer electrostatic deposition (Dickinson, 2011; Evans, Rat­
cliffe, & Williams, 2013; Guzey & McClements, 2006). Multilayer
coatings can be designed to prevent lipid oxidation In many respects: (i)
including antioxidant components within them; (ii) creating a steric
barrier; and (iii) making them positively charged so they repel cationic
transition metal ions (Chaprenet, Berton-Carabin, Elias, & Coupland,
2014; Lesmes, Sandra, Decker, & McClements, 2010).
The functional attributes of protein emulsifiers can often be extended
by using them in combination with synthetic or natural surfactants. The
nature of the mixed interfacial layers formed at the droplet surfaces
depends on the surface activities, interactions, and concentrations of the
proteins and surfactants used (Miller, Fainerman, Makievski, Krgel, &
Sinyachenko, 2000). For instance, it is possible for two emulsifiers to
form homogeneously mixed interfaces, phase-separated interfacial do­
mains, multilayer interfaces, or interfacial aggregates (Mackie & Wilde,
2005; Pugnaloni, Dickinson, Ettelaie, Mackie, & Wilde, 2004). The kind
of interfaces formed mainly depends on the interactions (attractive or
repulsive) between the two emulsifiers, their order of addition, and their
affinity for the oil-water interface. In some cases, one emulsifier may
displace another one from the interface due to competitive adsorption,
which depends on the relative concentrations and surface activities of
the two emulsifiers (Dickinson, 1999). Consequently, it is important to
optimize the proteins and surfactants used to form mixed interfaces with
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Food Hydrocolloids 109 (2020) 106136
the desired physicochemical and functional attributes (Dan, Gochev, &
Miller, 2015; Dan et al., 2012). In principle, the composition, thickness,
packing, and charge of the droplet coatings can be modulated by
selecting appropriate protein-surfactant combinations, which would be
postulated to influence the physicochemical stability of emulsions. For
instance, it may be possible to form thick, closely packed, charged in­
terfaces that contain antioxidant emulsifiers (e.g., with free radical
scavenging or chelating properties) that are effective at inhibiting
oxidation in emulsions. This could be achieved by judicious choice of
emulsifier types, concentrations, and orders-of-addition.
Here, we examined the possibility of using a natural plant-based
surfactant, camellia saponin (CS), to enhance the emulsification and
antioxidant properties of almond proteins. Camellia saponins are sec­
ondary metabolites of Camellia seeds (Cui et al., 2018; Yu & He, 2018).
Historically, Camellia has been cultivated in East Asia for the extraction
of commercial tea oil from its seeds. After oil extraction, the seed
pomace was typically discarded without further use. Recently, however,
it has been realized that these agricultural waste products contain sub­
stantial quantities of triterpenoids (such as saponins) that have potential
for application as functional ingredients within foods and other prod­
ucts. Camellia triterpenoids have already been marketed in some coun­
tries as therapeutic agents for the treatment of liver-related diseases,
diabetes, and as wound-healing agents (Cui et al., 2018). However, there
commercial success in food applications will depend on them being
approved for use in each target market. Camellia saponins contain both
hydrophilic groups (e.g., xylose, arabinose, galactose, and glucuronic
acid) and hydrophobic groups (e.g., triterpene), and therefore have
surface activity (Guo, Tong, Ren, Tu, & Li, 2018). Consequently, these
natural plant-based surfactants are receiving more attention as func­
tional ingredients in foods due to the desire to replace chemically syn­
thesized or animal-derived ingredients with more “label-friendly”
alternatives (McClements & Gumus, 2016; Ozturk & McClements,
2016).
In our previous study, we found that CS can form fine oil droplets at
low surfactant-to-oil ratios, which are highly resistant to alterations in
system environmental conditions, such as temperature, pH, or ionic
strength (Zhu et al., 2019). Moreover, we found that CS was negatively
charged across the pH ranges typically found in foods (pH 2–9). Other
researchers, have found the isoelectric point (pI) of almond protein to be
around pH 4.5 (Sathe, 1993). Consequently, API is positive at pH＜4.5,
whereas CS is negative. This means that it may be possible to form mixed
API-CS interfaces based on electrostatic attraction under sufficiently
acidic conditions. Conversely, API and CS both have a negative charge at
pH > 4.5. Under these conditions, it may still be possible to form mixed
interfaces, but the nature of the interactions involved should be
different, leading to the creation of interfacial layers with different
functional attributes. In particular, one would expect hydrophobic in­
teractions to be much more important under conditions where the two
types of emulsifier electrically repel each other.
The aim of the current work was to establish if a combination of
almond protein (API) and tea saponins (CS) could be used to create
plant-based emulsions that were resistant to lipid and protein oxidative
reactions. In addition, the influence of pH on the physicochemical sta­
bility of the emulsions was evaluated as this would be expected to alter
the nature of the interfacial complexes formed. The results of this study
may aid in the development of plant-based functional ingredients for
formulating vegan or vegetarian products enriched with omega-3 fatty
acids.
2. Materials & methods
2.1. Materials
Almond kernels were obtained from a local market. API was pre­
pared according to the method of Sze-Tao & Sathe (Sze-Tao et al., 2000).
The resulting API powder was characterized according to AOAC (1990)
S. Zhang et al.
methods (Zhu et al., 2018), and found to contain 89.51% protein, 0.88%
fat, and 0.66% ash. The walnut oil was isolated and purified according to
our previous method (Yi et al., 2019), where the fatty acid composition
was reported to be: C16:0, 7.1%, C16:1, 0.10%, C18:0, 3.5%, C18:1,17.8%,
C18:2, 61.9%, C18:3, 9.3%, C20:0, 0.12%, C20:1, 0.22%; tocopherols,
non-detectable; and hydroperoxides, 1.8 mmol/kg oil. Camellia saponin
was donated by Qing Han Bio-technology Co. Ltd. (Hunan, China). It
was reported to contain saponins (51.8%), total carbohydrates (36.1%),
crude proteins (5.6%), ash (2.7%), and water (3.7%). This product was
further dialyzed to removed proteins. Briefly, the Camellia saponin was
placed in a dialysis bag (molecular mass cutoff, 8 kDa, Shengya Co.,
Xi’an, China) and dialyzed for 24 h. The external solution was collected
and replaced with fresh solution, and then dialysis was repeated two
more times (3 in total). The dialysates were pooled together for each
sample and was then concentrated and lyophilized. The dried sample
contained saponins (68.4%), total carbohydrates (25.3%), crude pro­
teins (0.2%), ash (3.6%), water (2.4%), and total polyphenols 12 mg/g.
Isooctane, isopropanol, methanol, 2-propanol, ethyl acetate,
ammonium thiocyanate, 1-butanol, and guanidine hydrochloride 5,5-
dithio-bis (2-nitrobenzoic acid) were obtained from Aladdin Reagent
(Shanghai, China). Cumene hydroperoxide, Trichloroacetic acid (TCA),
thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane, and 2,4-dinitro­
phenylhydrazine (DNPH) were obtained from Adamas Reagent
(Shanghai, China). All other reagents were purchased from the Yuanye
Biotechnology Co. Ltd. (Shanghai, China). All chemicals were of
analytical grade unless specified otherwise. Solutions and emulsions
were prepared using double-distilled and deionized water.
2.2. Emulsion preparation
Preparation of stock emulsions: API and CS were dispersed separately
into phosphate buffer solutions (10 mM, pH 3 or 7) and stirred slowly
overnight at 4 � C to fully hydrate the ingredients. 20 wt % walnut oil was
dispersed into 80 wt% emulsifier solution (containing 2.0 wt% API
based on the final stock emulsion weight) with a hand-held homogenizer
(Model F6/10-10G, FuLuke Fluid Machinery Manufacturing Co. Ltd.
Shanghai, China) for 2 min and coarse emulsions were obtained. Stock
emulsions (pH 3 or 7) were produced by further homogenizing these
coarse emulsions by using a high-pressure homogenization as described
previously (Yi et al., 2019). The stock emulsions were stored at 4 � C
prior to use to inhibit lipid oxidation.
Two sets of primary emulsions (pH 3 and 7) were produced. These
two sets were produced by diluting the stock emulsions (pH 3 or 7) into
phosphate buffer solutions (10 mM, pH 3 or 7) at a mass ratio of 1:4 (w/
w), respectively. The final composition of both primary emulsions was 5
wt % walnut oil and 0.5 wt % API.
Two sets of secondary emulsions (pH 3 and 7) were also produced by
diluting the stock emulsions (pH 3 or 7) into CS solution (containing 1.0
or 2.0 wt % CS, based on the final secondary emulsion weight) at the
proportion of 1:4 (w/w) and subsequently adjusting pH to 3 or 7. The
final composition of these secondary emulsions was 5 wt % walnut oil,
0.5 wt % API, and 0, 1.0, or 2.0 wt % CS. These samples were fully mixed
by gently stirring for 4 h and then stored overnight at 4 � C. The oxidation
stability experiments were carried on by incubating the samples at 45 � C
in the dark to accelerate lipid and protein oxidation.
2.3. Emulsion characterization
Laser diffraction was used to measure the particle size distributions
of the emulsions (MasterSizer 2000; Malvern Instruments, Worcester­
shire, UK) at 25 � C after incubation for 0, 3and 6 days. Small volumes of
emulsions were added into the test chamber using a pipette. The test
chamber was filled with phosphate buffer solution at the appropriate
sample pH. Sufficient emulsion was added to obtain a good light scat­
tering signal without introducing multiple light scattering effects. The
d4,3 and d3,2 (the volume and surface weighted mean particle diameters,
3
Food Hydrocolloids 109 (2020) 106136
respectively) were calculated from the particle size distributions. The
droplet ζ-potential in the emulsions were measured by electrophoresis
(Zetasizer Nano ZS90, Malvern Instruments, UK). Emulsions were
dispersed into phosphate buffer at a ratio of 1: 200 before measurements
to obtain a good signal while avoiding multiple scattering effects.
Microstructural images of the emulsions was acquired by confocal
laser scanning microscopy (CLSM, TCS SP2, Leica, Germany) using a
65� immersion objective lens with a numerical aperture of 1.0. Emul­
sions were stained with a mixture of 0.02% (w/v) Nile Red (lipid stain)
and 0.1% (w/v) Nile Blue (protein stain). The CLSM device was run in
fluorescence mode with excitation being carried out at 633 nm for Nile
Blue and 488 for Nile Red.
The interfacial protein concentration (mg/m2) in freshly made
emulsions was measured using a published method (Yang et al., 2015).
Briefly, emulsions were centrifuged at 4 � C for 1 h at 35,000 g to separate
the serum phase from the cream phase. The non-adsorbed proteins were
then recovered from the cream, by washing the cream phase twice with
buffer solution, and then centrifuging using the same conditions stated
above. The serum phases were then combined and passed through a
0.45 μm Millipore filter to remove any residual oil droplets. The protein
content of the serum phase was detected by the previous method
(Bradford, 1976). The interfacial protein concentration was obtained by
deducting the non-adsorbed serum protein from the proteins initially
used to produce the emulsions.
2.4. Determination of lipid and protein oxidation
The methods used to monitor lipid and protein oxidative reactions in
emulsions during storage are the same as those described in detail in our
previous study (Zhu et al., 2018). Briefly, lipid oxidation was followed
by monitoring the generation of lipid hydroperoxides and 2-thiobarbitu­
ric acid reactive substances (TBARS) during storage. Protein oxidation
was followed by measuring carbonyl formation, free sulfhydryl groups,
intrinsic tryptophan fluorescence, and electrophoresis (SDS-PAGE)
during storage.
2.5. Statistical analysis
All experiments were carried out in triplicate. Statistical analyses
were performed utilizing a software package (SPSS 17.0, SPSS Inc.,
Chicago, IL, USA). Data are reported as means � standard deviations,
and mean values were compared using Duncan’s multiple-range tests to
identify significant differences (p < 0.05).
3. Results and discussion
3.1. Physical properties of emulsions
3.1.1. Electrical characteristics of emulsion droplets
Preliminary experiments were carried out by measuring droplet size
versus API concentration to determine the minimum amount that could
be used to form small droplets under the homogenization conditions
used. These experiments showed that 0.5% API could form stable
emulsions with little non-absorbed proteins in the aqueous phase. This
level of API was therefore used to formulate the emulsions in the
remainder of the study. Preliminary experiments were also carried out to
establish whether stable emulsions could be formed by adding CS to API-
coated lipid droplets. At pH 3, stable emulsions could only be formed
from 0 to 2 wt% CS, above which appreciable droplet aggregation was
observed, presumably attributed to electrostatic charge neutralization
and bridging flocculation effects, i.e., the sharing of anionic CS mole­
cules or micelles between the surfaces of two or more cationic droplets.
At pH 7, the emulsions were stable even at the highest CS concentration
added (5 wt %), which was probably ascribed to the strong electrostatic
repulsion between the droplets. For this reason, emulsions were pre­
pared containing 1.0 and 2.0 wt % CS so that the effects of pH could be
S. Zhang et al.
compared under similar surfactant conditions.
At pH 3, the ζ-potential of the emulsions stabilized by 1 and 2 wt %
CS alone were 11.1 and 13.2 mV, respectively (data not shown).
Meanwhile, the ζ-potential of the emulsion stabilized by API alone was
þ16.6 mV (Fig. 1a). The oil droplets were positively charged because the
pH was appreciably below the pI of the adsorbed almond proteins. The
ζ-potential became less and less positive as more and more CS was
added. This result is consistent with either the formation of interfacial
CS-API complexes through electrostatic attraction or the partial
displacement of cationic API by anionic CS due to competitive adsorp­
tion. For this reason, we measured the interfacial protein concentrations
to provide more insights into the origin of this effect. The addition of 1 or
2 wt % CS did not change the interfacial protein concentration (data not
shown), which suggests that the observed changes in ζ-potential were
due to the formation of interfacial CS-API complexes. Other researchers
have also reported that protein-surfactant complexes are formed when
ionic surfactants are added to oppositely-charged protein-coated drop­
lets (Dan et al., 2015; Dan et al., 2012).
At pH 7, the ζ-potential of the emulsions stabilized by 1 and 2 wt %
CS alone were 41.2 and 41.5 mV, respectively (data not shown).
Meanwhile, the ζ-potential of the emulsion coated by API alone was
24.3 mV (Fig. 1b). The ζ-potential became more negative as the CS
level was increased, suggesting that some of CS adsorbed to the almond
protein-stabilized oil droplets, despite the fact that both the surfactant
and protein were negatively charged. The interfacial protein
Fig. 1. Changes in the ξ-potential of emulsions during storage. (a) pH 3.0; (b)
pH 7.0. Key: API: emulsions stabilized by API alone; APIþ1%CS or APIþ2%CS:
emulsions stabilized by API and either 1% or 2% CS. Samples with different
lowercase superscripts (a–c) differ significantly (p < 0.05) when different
emulsions are compared at the same oxidation time; Samples with different
uppercase superscripts (A–C) differ significantly (p < 0.05) when the same
emulsion is compared at different oxidation times.
4
Food Hydrocolloids 109 (2020) 106136
concentration measurements indicated that the almond proteins were
not removed from the oil-water interface when CS was added (data not
shown), suggesting the formation of interfacial API-CS complexes or the
co-adsorption of API and CS. One would anticipate an electrostatic
repulsion between the anionic API and anionic CS molecules, indicating
that other types of attractive interactions are important. CS contains
both hydrophobic and hydrophilic regions on the molecule, and so it
could interact with API through hydrophobic and/or hydrophilic
attraction (McClements & Jafari, 2018). Other researchers have also
reported that anionic proteins can form complexes with anionic sur­
factants under neutral conditions, such as pea proteins and quillaja sa­
ponins (Reichert, Salminen, Leuenberger, Hinrichs, & Weiss, 2015) or
β-lactoglobulin and SDS (Magdassi, Vinetsky, & Relkin, 1996), Alter­
natively, the CS may adsorb directly onto the oil-water interface leading
to a mixed interfacial layer with surfactant-rich and protein-rich do­
mains (Pugnaloni et al., 2004).
There were some changes in the droplet potentials in the various
emulsions during storage (Fig. 1). In the absence of CS, the magnitude of
the ζ-potential increased during incubation at pH 3 (becoming more
positive), but decreased at pH 7 (becoming less negative). These changes
in surface potential are indicative of modifications in the interfacial
composition of the emulsions. Oxidation alters the molecular structure
of the adsorbed proteins (discussed later), which may change the elec­
trical properties of the amino acid residues at the droplet exteriors
(Gumus et al., 2017). Alternatively, changes in the droplet aggregation
state during storage (discussed later) might alter the magnitude of the
ζ-potential determined by the electrophoresis instrument (McClements,
2015). Other researchers have also reported that the ζ-potential of oil
droplets coated by plant proteins (lentil, pea, and faba bean) changed
throughout storage under neutral conditions, however, in that case the
ζ-potential became more negative (Gumus et al., 2017). The different
trends observed in various studies may be attributed to the different
protein types used, which may undergo different oxidation reactions. In
the presence of CS, the changes in surface potential during storage were
much smaller (Fig. 1a–b). This suggests that the presence of the saponins
may have inhibited any chemical changes at the droplet surfaces.
3.1.2. Aggregation state of emulsion droplets
The volume-weighted mean particle diameter (d4,3) is highly
dependent on the existence of any large particles in colloidal disper­
sions, and so it is useful for characterizing the aggregation stability of
emulsions (McClements, 2015). As shown in Fig. 2, all of the emulsions
had smaller d4,3 values at pH 7 than pH 3 (p＜0.05), suggesting that less
droplet aggregation occurred at the higher pH. This is probably because
the absolute value of the ζ-potential was higher at pH 7, resulting to a
stronger electrostatic repulsion between the droplets. Addition of CS
into the initial emulsions caused a significant reduction in d4,3 at both
pH 3 and 7 (p＜0.05), but little change in d3,2 (data not shown). This
suggests that the addition of the saponins promoted the dissociation of
some of the flocs in the emulsions. At pH 7.0, the incorporation of CS
would have increased the droplet electrostatic repulsion by increasing
their negative surface potentials. At pH 3.0, however, the added CS
actually decreased the magnitude of their surface potential, which
means this phenomenon cannot account for the observed results.
Instead, the presence of the saponins may have reduced any hydro­
phobic attraction between the protein-coated droplets by covering any
exposed non-polar regions, or they may have increased the steric
repulsion by forming thicker interfacial layers.
The mean particle size of all emulsions increased during storage,
indicating that some droplet aggregation occurred (Fig. 2a–b). However,
the increase was much smaller in the emulsions containing CS than in
the ones without CS, and was considerably less at pH 7 than at pH 3. Our
results demonstrate that the API-CS coatings enhanced the aggregation
stability of the emulsions, when compared to API coatings alone.
S. Zhang et al.
Fig. 2. Changes in mean particle diameter (d4,3) during storage: (a) pH 3.0; (b)
pH 7.0. See caption to Fig. 1 for description of sample codes and statisti­
cal analysis.
3.1.3. Microstructure of emulsions
Images of the microstructures of emulsions containing either 0 or 2%
CS were acquired by CLSM (Fig. 3). At pH 3, the microscopy images
show that the oil droplets were highly flocculated in the fresh emulsions
stabilized by API alone but less so in the ones stabilized by API-CS
(Fig. 3a). At longer storage times, however, extensive aggregation was
observed in all the emulsions under acidic conditions. The instability
observed in these emulsions may be a result of the relatively low surface
potential of the droplets or due to the impact of oxidation on their
physical stability (discussed later). At pH 7, all emulsions were consid­
erably more stable to droplet aggregation than at pH 3 (Fig. 3b), which
may have been due to the strong electrostatic repulsive forces acting
between them under neutral conditions. Interestingly, the microscopy
images indicated that there was a substantial increase in droplet floc­
culation in the emulsions containing CS during storage (Fig. 3), which
was not observed in the light scattering experiments (Fig. 2). The most
likely reason is the different principles of the light scattering and mi­
croscopy measurements. Light scattering measurements require dilution
and stirring of the samples that can disrupt weak flocs, whereas micro­
scopy simply involves placing an emulsion on a slide that might leave
these flocs intact.
3.2. Lipid oxidation
Lipid oxidation was investigated by monitoring the generation of
primary (lipid hydroperoxides) and secondary (TBARS) reaction prod­
ucts during storage (Fig. 4). Both the primary and secondary products
increased progressively throughout storage in all emulsions, suggesting
that lipid oxidation occurred. At both pH 3 and 7, the formation of lipid
5
Food Hydrocolloids 109 (2020) 106136
oxidation products was significantly lower in the emulsions containing
CS (p < 0.05). Our results indicate that the adsorbed API-CS complexes
were better interfacial antioxidants than the API alone, which may have
been due to various reasons. First, the adsorbed CS molecules have some
inherent antioxidant activity, including free radical scavenging and
ferrous ion chelating (Joshi, Sood, Dogra, & Mahendru …, 2013;
Xiao-Ling, Qiu, Sun, & Zhao-Jiang, 2005). Second, the API-CS may form
a physical barrier that sterically inhibits water-soluble pro-oxidants
reaching the emulsified lipids. Interfacial complexes have also been
reported to improve the oxidative stability of other types of emulsions
due to their ability to form thicker coatings around the oil droplets, such
as pectin/fibroin (Chen, Li, Ding, & Rao, 2011) and pec­
tin/β-lactoglobulin (Katsuda, McClements, Miglioranza, & Decker,
2008). An alternative explanation is that the non-adsorbed CS could
have some antioxidant activity. Previous studies with proteins have
shown that non-adsorbed proteins can bind pro-oxidants (such as tran­
sition metal ions) and therefore inhibit their ability to adsorb to the
droplet surfaces and promote oxidation (Gumus et al., 2017).
The droplets in the emulsions were positive at pH 3 but negative at
pH 7 (Fig. 1). Consequently, one might expect slower oxidation at pH 3
than pH 7 due to electrostatic repulsion of the cationic transition metal
ions from the cationic droplets under acidic conditions (McClements
et al., 2000). In practice, however, the emulsions were more resistant to
oxidation under neutral pH conditions. For example, the lipid hydro­
peroxide concentration of the API-emulsions increased around 81%
(6.05–10.95 mmol/L) after 6 days storage at pH 3 (Fig. 4a), but only
61% (5.56–8.93 mmol/L) at pH 7 (Fig. 4b). Similar findings have been
reported for lipid droplets coated by other kinds of proteins, such as
whey proteins and casein (Haahr & Jacobsen, 2008; Yesiltas,
Garcia-Moreno, Sorensen, & Jacobsen, 2011). These authors proposed
that the higher rate of oxidation observed under acidic conditions was
primarily a result of the higher water-solubility and chemical reactivity
of the iron at low pH, as well as pH-dependent changes in interfacial
composition. In our study, we also found that the interfacial protein
composition in the emulsions was markedly different at pH 3 and 7
(discussed below). This phenomenon may therefore have contributed to
the differences in oxidation stability observed at the different pH values.
In addition, non-adsorbed proteins can play a critical role in lipid
oxidation under neutral conditions. Anionic non-adsorbed proteins can
bind cationic transition metal ions, inhibiting their interactions with the
encapsulated lipids (Gumus et al., 2017).
3.3. Protein oxidation
Proteins adsorbed to oil droplet surfaces may be oxidized more
readily than the oil itself, thereby altering their susceptibility to oxida­
tion (Berton, Ropers, Guibert, Sol�e, & Genot, 2012; Yang et al., 2015).
For instance, reactive species generated during lipid oxidation, such as
free radicals, can interact with proteins, thereby promoting their
oxidation (Stadtman, 2006, pp. 22–38). For this reason, protein oxida­
tion within the emulsions was also monitored with and without CS.
3.3.1. Protein-bound carbonyls
The formation of carbonyl derivatives is one of the first stages of
protein oxidation (Berton et al., 2012). The carbonyl content of the
emulsions was therefore measured to evaluate protein oxidation (Fig. 5).
The protein-bound carbonyls increased progressively during 6 days of
storage in all the emulsions, which is indicative of the proteins being
progressively oxidized. No significant differences in carbonyl levels
were observed between the initial emulsions in the absence or presence
of CS at both pH 3 and 7 (p＞0.05). During storage, however, the rate of
carbonyl formation was appreciably less in the presence of CS at both pH
values, suggesting it inhibited protein oxidation. Carbonyls can arise
from the direct attack of arginine, lysine, proline, and threonine, which
is catalyzed by transition metal ions. Carbonyls can also be produced by
reactions between lipid oxidation products and functional groups on the
S. Zhang et al.
Fig. 3. Confocal micrographs of emulsions with 0 or 2% added CS at different oxidation times. (a) pH 3.0; (b) pH 7.0.
proteins, such as lysyl, cysteyl, and histidyl residues (Chen, Chen, Ren, &
Zhao, 2011). The presence of the saponins at the oil droplet surfaces may
have inhibited protein oxidation due to their antioxidant properties such
as their capacities for chelating transition metal ions and scavenging free
radicals or the ability of API-CS complexes to form a thick impermeable
coating that acted as a steric barrier against the contact between pro­
teins and the pro-oxidants in the aqueous phase. Moreover, the reduced
generation of lipid oxidation products in the emulsions containing CS
(Fig. 4) may have reduced the rate of protein oxidation. Same as lipid
6
Food Hydrocolloids 109 (2020) 106136
oxidation, the rate of protein oxidation was greater at pH 3 than pH 7
(Fig. 5a–b), which again may be due to the increased water-solubility
and chemical reactivity of the transition metals under acidic conditions.
3.3.2. Free sulfhydryls
Changes in the number of free sulfhydryl groups within proteins can
be used as a measure of their oxidation state: the lower the number, the
greater the extent of oxidation (Zhu et al., 2018). The free sulfhydryl
concentration decreased significantly (p < 0.05) in all the emulsions
S. Zhang et al. Food Hydrocolloids 109 (2020) 106136

Fig. 5. Formation of protein carbonyls in different emulsions during storage.

(a) pH 3.0; (b) pH 7.0. See caption to Fig. 1 for description of sample codes and
statistical analysis.

during storage (Fig. 6a–b), indicating that the proteins were undergoing
oxidation. Again protein oxidation was significantly faster at pH 3 than
pH 7, and was slower in the emulsions containing CS (p < 0.05).

3.3.3. Intrinsic tryptophan fluorescence

A decrease in the intrinsic tryptophan fluorescence of proteins is also
indicative of their oxidation (Berton et al., 2012), which is a result of a
modification of the tryptophan indole ring through free radical re­
actions, as well as changes in the environment of the tryptophan groups
caused by conformational changes (Rampon et al., 2003). The loss of
intrinsic tryptophan fluorescence of the emulsions was therefore moni­
tored during oxidation (Fig. 7a–b). The fluorescence intensity in the
primary emulsions decayed faster than in the secondary emulsions at
both pH 3 and 7. In contrast, the reduction of intrinsic tryptophan
fluorescence was less marked in the secondary emulsion stabilized by
the API-CS complexes. For instance, at pH 3, the intrinsic tryptophan
fluorescence intensity at the peak of emission decreased 18% after 6
days of incubation, while it only reduced 11% in the secondary emulsion
stabilized by the API-CS complexes (Fig. 7a). These observations
therefore further supported the above results that the proteins in the
secondary emulsions were more oxidatively stable than their counter­
parts in the primary emulsions. In addition, a greater decrease in tryp­
tophan fluorescence intensity was observed in the emulsions at pH 7
than at pH 3 (Fig. 7a and b), which is consistent with the results dis­
cussed earlier.

Fig. 4. Formation of lipid hydroperoxides (a, b) and TBARS (c, d) in different 3.3.4. SDS-PAGE analysis
emulsions during storage. (a, c) pH 3.0; (b, d) pH 7.0. See caption to Fig. 1 for Information about the impact of CS addition on the characteristics of
description of sample codes and statistical analysis. the almond proteins in the emulsions was obtained using gel

7
S. Zhang et al.
Fig. 6. Changes in free sulfhydryls in different emulsions during storage. (a) pH
3.0; (b) pH 7.0. See caption to Fig. 1 for description of sample codes and sta­
tistical analysis.
electrophoresis. Proteins isolated from emulsions stabilized by API or
API-CS were characterized by non-reducing SDS-PAGE after 0, 3 and 6
days storage in the dark at 37 � C (Fig. 8a and b). Interestingly, the nature
of the proteins in the emulsions was strongly pH-dependent. At pH 3, the
proteins formed a number of bands with molecular weights below 40
kDa. Conversely, at pH 7, the proteins formed five major bands with
molecular weights of 62, 40, 29 27, and 15 kDa. The reason for this big
difference in protein composition is unknown. It is possible that an
acidic environment promoted the disruption of some chemical bonds
between or within the protein molecules, thereby generating a series of
smaller polypeptides. This difference in protein composition may at least
partially account for the difference in physical and chemical stability of
the emulsions observed at pH 3 and 7.
The band intensities for all the emulsions decreased during incuba­
tion, suggesting that the proteins had become oxidized and degraded.
Before incubation, the emulsions had similar SDS-PAGE profiles under
the same pH conditions in the absence or presence of CS, indicating that
the surfactant itself did not directly alter protein composition. However,
the reduction in band intensities during storage was slower in the
presence of CS, again demonstrating its ability to inhibit protein
oxidation. For example, at pH 3, there were only two obscure protein
bands remaining in the absence of CS after 6 days storage, while there
were four relatively clear bands in the presence of CS (see the green
square in Fig. 8a). The reduction in band intensities was also slower at
pH 7 than at pH 3, again indicating that protein degradation was faster
under acidic conditions.
8
Food Hydrocolloids 109 (2020) 106136
Fig. 7. Changes in intrinsic tryptophan fluorescence in different emulsions
during storage. (a) pH 3.0; (b) pH 7.0.
4. Conclusion
To summarize, we have shown that adsorbing tea saponins onto
almond protein-coated lipid droplets improves their physical and
chemical stability. In particular, the mixed protein/saponin-coatings
inhibit lipid and protein oxidation more effectively than protein-
coatings alone. The driving force for saponin adsorption depends on
pH. Both electrostatic and hydrophobic attraction are likely to be
important when the proteins and saponins have opposite charges (pH <
pI), but hydrophobic attraction is likely to be the most important when
they have similar charges (pH > pI). There are at least two possible
mechanisms of action for the antioxidant properties of the saponins: (i)
the saponins have free radical scavenging and metal chelation proper­
ties; (ii) the protein-saponin coatings create a steric barrier that prevents
water-soluble pro-oxidants reaching the lipids. Oxidation was faster at
pH 3 than at pH 7, which was mainly attributed to the relatively high
water-solubility and chemical reactivity of the metal ions under acidic
conditions. In addition, there were differences in the protein composi­
tion at the different pH values, which may also have contributed to
differences in oxidative stability. Our results may lead to the formulation
S. Zhang et al.
Fig. 8. SDS-PAGE electrophoretic profiles of proteins from different emulsions
under nonreducing conditions. M: marker; 0%CS: emulsions stabilized by API
alone; 1%CS: emulsions stabilized by API and 1% CS; and, 2%CS: emulsions
stabilized by API and 2% CS.
of plant-based foods fortified with polyunsaturated lipids that are suit­
able for vegans and vegetarians.
Declaration of competing interest
The authors state that they have no conflicts of interest related to this
study.
CRediT authorship contribution statement
Shulin Zhang: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology. Li Tian: Conceptualization, Data curation,
Formal analysis, Investigation, Methodology. Jianhua Yi: Conceptual­
ization, Data curation, Formal analysis, Investigation, Methodology,
Writing - original draft. Zhenbao Zhu: Conceptualization, Funding
acquisition, Writing - original draft. Eric Andrew Decker: Conceptu­
alization, Writing - review & editing. David Julian McClements:
Conceptualization, Writing - review & editing.
Acknowledgements
Financial support was received from the National Natural Science
Foundation of China under Grants 31671888 and science and technol­
ogy plan project of Shaanxi under Grants 2019NY-122. This material
was also partly based upon research supported by the National Institute
of Food and Agriculture, USDA, Massachusetts Agricultural Experiment
Station (MAS00491).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodhyd.2020.106136.
9
Food Hydrocolloids 109 (2020) 106136
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