ability of whey protein isolate andorfish gelatin to inhibit physical separation
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Ability of whey protein isolate and/or sh gelatin to inhibit physical separationand lipid oxidation in sh oil-in-water beverage emulsion
Ali R. Taherian*, Michel Britten, Hassan Sabik, Patrick Fustier
Agriculture and Agri-Food Canada, Food Research and Development Center, 3600 Casavant West, St-Hyacinthe, Quebec J2S 8E3, Canada
a r t i c l e i n f o
Article history:
Received 31 May 2010
Accepted 11 August 2010
Keywords:
Whey protein isolate
Fish gelatin
Lipid oxidation
Physical separation
Rheology
Beverage emulsions
a b s t r a c t
The effect of pH on the capability of whey protein isolate (WPI) and sh gelatin (FG), alone and in
conjugation, to form and stabilize sh oil-in-water emulsions was examined. Using layer-by-layer
interfacial deposition technique for WPIeFG conjugate, a total of 1% protein was used to prepare 10% sh
oil emulsions. The droplets size distributions and electrical charge, surface protein concentration, ow
and dynamic rheological properties and physiochemical stability of emulsions were characterize at two
different pH of 3.4 and 6.8 which were selected based on the ranges of citrus and milk beverages pHs,
respectively. Emulsions prepared with WPIeFG conjugate had superior physiochemical stability compare
to the emulsions prepared with individual proteins. Higher rate of coalescence was associated with
reduction in net charge and consequent decrease of the repulsion between coated oil droplets due to the
proximity of pH to the isoelectric point of proteins. The noteworthy shear thinning viscosity, as an
indication ofocculation onset, was associated with whey protein stabilized sh oil emulsion prepared
at pH of 3.4 and gelatin stabilized sh oil emulsion made at pH of 6.8. At pH 3.4, it appeared that lower
surface charge and higher surface area of WPI stabilized emulsions promoted lipid oxidation and
production of hexanal.
Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Among the functional ingredientsu-3 andu-6 fatty acids in sh
oil which contain eicosapentaenoic acid (EPA), and docosahex-
aenoic acid (DHA) has been claimed for their health benets. These
benets include reduced susceptibility to mental illness, protection
against heart disease, and improved brain and eye function in
infants (Krutulyte et al., 2008; Ritter-Gooder, Lewis, Barber-Heidal,
& Waltz-Hill, 2008; Sir, Kpolna, Kpolna, & Lugasi, 2008). As
a result, food products containing these polyunsaturated fatty acids
which positively affecting human health can be classied as so-
called functional food (Kolanowski, Swiderski, & Berger, 1999).
However, theu-3 andu-6 fatty acids are subject to rapid and/orextensive oxidation and other chemical changes by exposure to air,
light or heat during processing (Jacobsen, Bruni Let, Nielsen, &
Meyer, 2008; Medina, Cascante, Torres, & Pazos, 2008). The
outcomes are production of aldehydes, ketones, alcohols and
hydrocarbons (Coupland & McClements, 1996) that render unac-
ceptable colours, odours and avours in polyunsaturated fatty acid
(PUFA) containing foods and nutraceutical products. In addition,
products of lipid oxidation, such as hexanal, propanal, acrolein and
malonaldehyde, among others, possess adverse health effects due
to their cytotoxic and genotoxic effects (Giroux, St-Amant, Fustier,
Chapuzet, & Britt, 2008; Huber, Vasantha Rupasinghe, & Shahidi,
2009).
Therefore, successful incorporation ofu-3 fatty acids into pro-
cessed foods would most likely be in the form of lipid dispersions
which are referred to as oil-in-water emulsions (Dalgleish, 2006).
Small spherical oil droplets, in an oil-in-water emulsion, could be
stabilized in the aqueous phase by surface-active hydrocolloids
such as proteins, arabic gum and modied starch (Sun &
Gunasekaran, 2009; Taherian, Fustier, Britten, & Ramaswamy,2008). The surface-active hydrocolloid is adsorbed at the inter-
face between oil and the aqueous phase to lower surface tension,
increase force of repulsion and prevent oil droplets from aggrega-
tion. Proteins extracted from a variety of natural sources can be
used as emulsiers in foods because of their ability to facilitate the
formation, improve the stability, and produce desirable physico-
chemical properties in oil-in-water emulsions (Surh, Decker, &
McClements, 2006; Surh, Ward, & McClements, 2006).
Owing to its hydrophobic and hydrophilic regions, whey protein
isolate has been widely used as an emulsier for its ability toadsorb
rapidly at the oilewater interface and provide protection for oil* Corresponding author. Tel.: 1 450 768 3329; fax:1 450 773 8461.
E-mail address:[email protected](A.R. Taherian).
Contents lists available at ScienceDirect
Food Hydrocolloids
j o u r n a l h o m e p a g e : w w w . e l s e v i e r .c o m / l o c a t e / f o o d h yd
0268-005X/$e see front matter Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2010.08.007
Food Hydrocolloids 25 (2011) 868e878
mailto:[email protected]://www.sciencedirect.com/science/journal/0268005Xhttp://www.elsevier.com/locate/foodhydhttp://dx.doi.org/10.1016/j.foodhyd.2010.08.007http://dx.doi.org/10.1016/j.foodhyd.2010.08.007http://dx.doi.org/10.1016/j.foodhyd.2010.08.007http://dx.doi.org/10.1016/j.foodhyd.2010.08.007http://dx.doi.org/10.1016/j.foodhyd.2010.08.007http://dx.doi.org/10.1016/j.foodhyd.2010.08.007http://www.elsevier.com/locate/foodhydhttp://www.sciencedirect.com/science/journal/0268005Xmailto:[email protected] -
8/13/2019 Ability of Whey Protein Isolate Andorfish Gelatin to Inhibit Physical Separation
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droplets through a combination of electrostatic and steric interac-
tions (Matsumiya, Takahashi, Inoue, & Matsumura, 2010; Sun &
Gunasekaran, 2009). Such adsorbed layers around the surface of
oil droplets are responsible for stabilizing the vast majority of food
emulsions against occulation and coalescence. The unfolding of
protein molecules at the oilewater interface leads to changes in
secondary and tertiary structure, and to the exposure of residues
which would normally be buried within the native globular
structure (Dickinson & Matsumura, 1991).
Gelatin, a derivative of animal collagen, is a relatively high
molecular weight protein which is prepared by sweltering animal
tissues in the presence of either acid (Type A, pIw7e9) or
alkaline (Type B pI w5). The relatively high isoelectric point
(pI 7.0) of Type A gelatin allows the creation of oil-in-water
emulsions with positively charged droplets. As a result, Type A
gelatin may be suitable for preparing oil-in-water food emulsions
with high oxidative stability since it could repel iron ions from oil
droplet surfaces over most of the pH range typically found in foods
(Surh, Decker, et al., 2006; Surh, Ward, et al., 2006). Gelatin as an
emulsier has been subject of several studies (Cheng, Lim, Chow,
Chong, & Chang, 2008; Lobo, 2002; Ries, Ye, Haisman, & Singh,
2010; Surh, Gu, Decker, & McClements, 2005).
The aims of this work were rst to nd the evidence for pref-erential adsorbtion of the WPI over FG using deposition technique
and characterize the physiochemical properties of the omega-3 sh
oil emulsions as an inuence of pH and understand the factor that
determine the efciencies of WPI and FG, alone and conjugated, for
providing the steric and electrostatic stabilization against coales-
cence and occulation.
2. Material and methods
2.1. Materials
Fish oil (OmegaPure, Houston, TX) containing 32e37% omega-3
fatty acid was kindly donated by NEX-XUS (Montreal, PQ). Based onthe claim by OmegaPure the sh oil contains 35.2% omega-3 fatty
acids and fatty acid prole was as follow:
Right after receiving the sh oil, 36 30 g sh oil was weighed
in 36 screw cap bottles and store at 18 C. Fish gelatin (275 FG 30)
and whey protein isolate (Hilmar 9400) were respectively
provided by Rousselot Inc (Wisconsin, WI) and Hilmar Ingredients
(Hilmar, CA). Food grade citric acid and disodium phosphate
dehydrate (donated by Canada Colors and Chemicals Limited,
Brampton, ON) were used to adjust the acidity and 0.02% sodium
azide to reduce the risk of contamination in prepared emulsions.
2.2. Preparation of stock solutions
Buffer solutions were prepared based on the method by
Colowich and Kaplan (1995). The pHs of buffer solutions were
adjusted at 3.4 (juice beverage) and 6.8 (milk beverage) using citric
acid (0.1 M) and dibasic sodium phosphate (0.2 M) solutions mixed
in appropriate ratios.
2.3. Preparation of emulsions
Prior to preparation of emulsions, sh oil was thawed in
a refrigerator at 4 C for 12 h. Pure protein emulsions were then
prepared by slow addition of 3 g WPI or FG to 267 g buffer solution
in a pre-homogenize vessel and successive blending at high speed
for 2 min using a commercial blender (Waring, ON, Canada).
Protein solutions were then placed in a screw cap bottle and kept
overnight at 4 C (WPI) or room temperature (sh FG) for complete
hydration. Fish gelatin was stored at room temperature to prevent
low temperature gelation. Following day, pre-weighed 30 g sh oil
was slowly added into a 500 ml beaker containing hydrated WPI or
FG solution while blending at low speed. A coarse emulsion
(300 mL total volume) was then made by blending sh oil and
protein solution for 3 min at high speed. Oil droplets size wasfurther reduced with the aid of high pressure homogenizer (Emu-
lisiex-C5, Avestin, ON, Canada) at 4000 psi for 3 passes. Final
protein and fat content in the emulsions were respectively 1 and
3%. The prepared emulsions were transferred into screw capglasses
bottle and tested right after preparation. The rest of emulsion was
loaded with 0.02% sodium azide and stored at 4 C before con-
ducting the second series of test.
For the preparation of WPIeFG conjugate emulsions, the
method ofAoki et al. (2005)was adopted with slight modications.
WPI (1.5 g) and FG (1.5 g) were separately hydrated in 133.5 g buffer
solutions. Fish oil (30 g) was rst added into the hydrated whey
protein, while agitating, and blended for 3 min at high speed. The
coarse emulsion was homogenized at 4000 psi for 3 passes to
prepare primary emulsion. The primary emulsion was then dilutedin hydrated FG following by blending for 3 min at high speed and
high pressure homogenization at 4000 psi for 3 passes. Prepared
emulsions were tested right after preparation and within 3 weeks
for assessment of size growth kinetics.
2.4. Electrical charge and oil droplet size
The electrical charge (z-potential) and mean diameter of
emulsion droplets were determined using a commercial instru-
ment capable of electrophoresis and dynamic light scattering
measurements (Zetasizer Nano-ZS, Malvern Instruments, Worcs.,
UK). Prior to conducting the measurements, emulsions were
diluted 1: 250 (using double distilled water) in order to prevent
multiple scattering effects in size measurement. Viscosity of dilutedemulsion was measured at constant shear rate of 0.1 s1 and 25C
for 1 min to consider viscosity effect in z-potential assessment.
Each individual z-potential data point was calculated from the
average of at least 6 readings made on the duplicate samples.
2.5. Assessment of emulsion protein load: effect of protein
concentration
Emulsions wererst prepared at 4 level of protein concentrations
(0.2, 0.6, 1, and 1.5 wt%) using the identical preparation methods
provided earlier. The concentration of adsorbed and free protein at
the interface in the emulsionswas thendetermined by centrifugation
of emulsions at 25,200 g during 60 min at 5 C using Beckman
model J2.21 and rotor model JA-20.1 (Beckman Centrifuge, USA) to
Fatty acid Area% of total fatty acid
Myristic, C14:0 8.2
Palmitic, C16:0 19.1
Palmitoleic, C16:1 11.7
Stearic, C18:0 3.0
Oleic, C18:1 13.2
Linoleic, C18:2 (n-6) 2.2
Alpha Linoleic, C18:3 (n3) 1.6
Stearidonic, C18:4 (n-3) 3.5
Arachidonic, C20:4 (n-3) 1.7
Eicosapentaenoic, C20:5 (n-3) 13.8Docosapentaenoic, C22:5 (n-3) 2.2
Docosahexaenoic, C22:6 (n-3) 11.8
Other 7.0
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separate the oil droplets from the serum phase. With the aid of
a syringe, serum phase was collected and thequantity of protein was
assessed using kjeldahlmethod(Kjeltec auto 1030 analyser, Kjeldahl,
Tecator). Using a Zetasizer (Nano-ZS, Malvern Instruments, Worcs.,
UK) the mean particle size was determined as the surface-weighted
mean diameter, d32 P
nid3i=P
nid2i , where ni is the number of
particles with diameter di. The surface protein concentration(mg/m2)
wasthen calculatedfrom the meandiameter (d 32) of theoil droplets
and the difference in the amountof protein used to prepare emulsion
and those measured in the subnatants and sediment after
centrifugation.
2.6. Measurement of surface protein composition
The sodium dodecyl sulphate polyacrylamide gel electropho-
resis (SDS-PAGE) technique was used to identify the proteins
subunits present in dried cream, based on their molecular weights,
under denaturation condition.
Oil droplets in emulsions were rst isolated to measure the
adsorbed or incorporated proteins. Cream layers of emulsions were
separated by centrifugation at 25200 gfor 40 min at 5 C using
a Beckman centrifuge (Beckman model J2.21, and rotor model JA-20.1, Beckman Instruments, Fullerton, CA). The separated cream
was transferred into another sample tube, dispersed well in the DI
water and centrifuged at 87,000 gfor 30 min at 4 C. The washing
procedure was repeated again and the resultant cream layer was
freeze-dried prior to quantication.
Approximately 1 mg of each protein was accurately weighed,
and an exact volume of sample buffer (Bio-Rad No. 161-0791) was
added to obtain a concentration of exactly 1 mg protein per milli-
liter. For the washed cream samples the mixture was stored over-
night at room temperature to allow the separation into a cream
layer at the top and an aqueous serum at the bottom. The serum
was then recovered and loaded with 50ml of 2-mercaptoethanol
(Bio-Rad No. 161-0792). The solution was mixed well and
immersed in boiling water for 5 min. The resulting samples weremicrocentrifuged to remove any insoluble matter.
2.6.1. Assessment of protein
A Bio-Rad Criterion Cell was used with Bio-Rad 4e12% BiseTris
Gel as the medium, and dilution was performed using XT MOPS
buffer (Bio-Rad No. 161-0788) at a constant voltage of 200 V in
accordance with the manufacturers recommendations. A volume
of 20ml of the sample was used, and the molecular weights were
estimated with a low-range standard. The proteins were visualized
by staining with Coomassie Blue R-250 (Bio-Rad No. 161-0436).
2.6.2. Optical characterization of emulsion stability
Emulsion stability was quantied based on the method
provided byTaherian, Fustier, Ramaswamy (2007a, 2007b). Emul-sions were subjected to stability test by pouring 6 ml of each
emulsion into a at-bottom cylindrical glass tube (100 mm height,
16 mm internal diameter) and subjecting to an optical scanning
instrument (Quick Scan, Coulter Crop., Miami, FL). The transmission
of monochromatic light (l 850 nm) from the emulsions was
measured as a function of their height. Separation rate was quan-
tied by conducting a total of 10 scans (each scan was repeated 5
times throughout 10 min) within 15 days for each tube. This
quantication was based on the migration rate of the oil droplets
from the bottom to the top of the sample which induces
a progressive fall in concentration at the sample bottom (clari-
cation) and therefore increases the transmission. The resulting
positive peaks were then transferred to Microsoft Excel and sepa-
ration rate was calculated as slope of transmission mean values
over 15 day storage (complete information is available inMengual,
Meunier, Cayr, Puech, & Snabre, 1999).
In addition, 60 ml of each emulsion sample was placed in
a 100 ml Wainthropp tube to observe the separation in parallel
with instrumental assessment.
2.7. Flow and dynamic rheological measurements
Measurement of rheological parameters was based on the
methods by Taherian et al. (2007a, 2007b) using an AR1000
Rheometer (TA Instrument, New Castle, DE, USA) equipped with a 2
degree cone of 60 mm diameter. Emulsions were subjected to ow
test, measuring shear viscosity (hg) as a function of shear rate
ranging from 0.1/s to 100/s at 22 C. Flow behaviour index (n) and
consistency coefcient (m) were calculated using the power law
model. Prior to dynamic rheology assessment a stress ramp at
0.01e100 Pa and 1 Hz frequency was conducted to nd out linear
region. Dynamic rheological properties tests were then conducted
at constant temperature of 22C, 0.5 Pa stress and a range of
frequency from 1 to 25 rad/s to assess storage modulus (G0), loss
modulus (G00) and delta degree (G00/G0). The duplicate samples along
with 3 measurements for each emulsion were considered.
2.8. Optical microscopy
Using a glass test tube, emulsions were gently agitated to ensure
homogeneity prior to analysis. A drop of emulsion was then placed
on a microscope slide and covered with a cover slip. Images of
freshly prepared emulsions were taken using a Nikon Eclipse E600
microscope coupled with a Nikon digital DXM 1200 camera (Nikon
Corporation, Japan). The Nikon ACT-1 version 2.12 software was
used to process the images. Pictures were taken from three
differentelds on each slide and representative micrographs were
presented.
2.9. Assessment of oxidative stability
A volatile secondary oxidation compounds (hexanal, purchased
from SigmaeAldrich, Oakville, ON, Canada) was selected as an
indicator for sh oiloxidation and was extracted from emulsions by
solid-phase microextraction (SPME). Emulsions were stored in
screw cap bottles at 25 C and exposed to 150 W UV light using
a 40 cm 50 cm 60 cm open box (Macbeth, Judge II, New
Windsor, NY).
At the day rst and after 3 and 6 months, 1 g of sample was
transferred into a 10-mL screw-top headspace clear vial; the vial
was sealed with a magnetic screw cap containing a polytetra-
uoroethylene (PTFE)/silicone septum (Varian, Mississauga, ON,
Canada). The SPME ber (85mm Carboxen/PDMS, Supelco, Oakville,
ON, Canada) was inserted into the headspace of the vial for 44 min
at 40
C. The SPME operations were automated using an MPS2multipurpose sampler (Gerstel Inc., Baltimore, MD). Volatile
compounds were desorbed by inserting the ber into the injection
port 1078/1079 of a Varian CP-3800 gas chromatograph (Palo Alto,
CA) in splitless mode (Glass insert SPME, 0.8 ID; Varian, Mis-
sissauga, ON, Canada) for 3 min at 300 C. The GCeMS system used
in this study consisted of an ion trap mass spectrometer equipped
with an electronic impact (EI) ionization source controlled with
Saturn 2000 mass spectrometry detector (Varian Inc., Palo Alto,
CA). A Varian CP-Sil 8CB-MS capillary column (5% phenyl/95%
dimethylpolysiloxane; 30 0.25 mm; 25mm lm thickness) was
used with Helium as carrier gas at a ow rate of 1.0 mL/min. The
column oven was set initially at 35 C for 3 min, heated to 80 C at
a rate of 6C/min, increased to 280 C at a rate of 20 C/min and
then held at 280
C for 2 min. The total time of analysis was
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22.5 min. The mass spectrometer was operated in the mass range
from 30 to 200 at a scan rate of 1.00 s/scan. Calibration curves were
done using standards of hexanal and propanal in a media of whey
and gelatin at pHs 3.4 and 6.8. Hexanal retention time was around
6.4 min. The quantication was realized by total ion current (TIC)
mode.
2.10. Statistical analysis
All experiments were repeated at least 6 times and the data
were analyzed usingT-test and differences were considered to be
signicant at p 0.0005. Statistical analysis was done using
Microsoft Excel and experiments were performed in duplicate.
3. Results and discussion
3.1. Effect ofz-potential on emulsion stability
The droplet charge quantication is normally done by applying
an electrical voltage to the particle and measuring the speed of
induced movement (Malhotra & Coupland, 2004).
Fig.1indicates that the intensity of zeta potential of oil droplets,
for all studied emulsions, was notably inuenced by pH. As the pH
increased from 3.4 to 6.8 the net electrical charge of adsorbed
protein on the surface ofsh oil droplets changed from positive to
negative. The electrical charge at low pH is below the isoelectric
points of both WPI (pIz4.6e5.6)and FG(pIz7e9) indicating that
H and OH ions are potential determining ions for z-potential
dependency on pH (McClements, 2005). At pH of 6.8 the net
negative charge intensity of FG-coated droplets is 3.5 0.1 mV
which is greatly lower than that of whey protein isolate
(56 2.1 mV). The low negative charge intensity of FG-coated
droplets is related to the balancing of positive charges by the
negative charges due to proximity of its isoelectric point to the pH
of the system as indicated by Kittiphattanabawon, Benjakul,
Visessanguan, Kishimura, and Shahidi (2010). Conversely, the net
positive charge intensity of FG stabilized droplets at pH 3.4 is42.71.1 mV and that of WPI is 18.0 0.4 mV.
The surface charge of the droplets in both WPI and FG-coated
droplets is governed by the ionization degree of amino groups
(eNH2) and carboxyl groups (eCOOH) of protein molecules
depending on the pH of the surrounding aqueous phase ( Surh,
Decker, et al., 2006; Surh, Ward, et al., 2006). At the pH closed to
the isoelectric point, z-potential became zero, indicating that the
number of cationic charged groups was equal to the number of
anionic charged groups and therefore the net surface charge of the
droplets is neutral. A further increase in pH causes the droplets to
gain a net anionic charge, which increases as the number of anionic
charged group increases and cationic charged group decreases
(Kulmyrzaev & Schubert, 2004). The negative charge of the gelatin
covered emulsion droplets, therefore, might be due to the nega-
tively charged amino acid surrounding the oil droplet (Aewsiri
et al., 2009). The relatively higher negative z-potential of whey
proteinisolate coated droplets at pH 6.8 as well as higher positive z-
potential ofsh gelatin coated droplets at pH 3.4 may account for
greater intensity of the electrostatic repulsion force and hence
superior stability of emulsion.
Study by Gu, Decker, and McClements (2007) indicated that
the z-potential of the droplets covered with b-lactoglobu-
linecarrageenanegelatin as tertiary emulsions at pH 6 was
38 1 mV. The negative zeta potential was related to the
adsorbtion of cationic gelatin molecules to the surfaces of the
anionic b-lactoglobulinecarrageenan coated droplets. The z-
potential of the droplets coated with WPIeFG conjugates were
44 1.0 mV and 64.41.7 mV for pH 3.4 and pH 6.8 respectively,
which may account for adsorbtion of the cationic gelatin molecules
to the surfaces of anionic whey protein coated droplets.
3.2. Protein concentration at the interface
The purpose of conducting this test was to determine directly
the amount of protein adsorbed at the oilewater interface in
emulsions by analyzing the cream phase. Fig. 2 illustrates the
surface concentrations of protein in emulsions as a function of WPI,
FG, and WPIeFG concentrations in deionized (DI) water deter-
mined by Kjeldahl method. Increasing the concentrations of WPI
and FG in emulsions made with either individual or conjugated
proteins augmented the interfacial protein load. The results are in
agreement with Ye (2008) and Raikos (2010) which indicated
that proteins will adsorb to the oil interfaces in proportion to their
concentrations in the aqueous phase. At all concentrations,
however, FG appeared to be, somewhat, less readily adsorbed and
the total surface protein concentrations of emulsions made with
WPI were slightly higher, compare to those made with FG. This may
also be related to higher proportion of hydrophobic residues, short
peptides, and the open molecular structure in whey protein isolate
compared to sh gelatin.
The surface sh gelatin and whey protein isolate concentrations
increased from 4.16 mg/m2 up to 7.23 mg/m2 and from 4.30 mg/m2
to 7.75 mg/m2, respectively, as the total protein concentration
increased from 0.2% to 1.5%. Beyond 1%, the surface whey protein
isolate and sh gelatin slightly increased. Conjugating WPI and FG
increased interfacial protein concentration from 4.92 mg/m2 up to
8.84 mg/m2 and, similarly, slightly increased as concentration
Fig. 1.Zeta potential of whey protein isolate (WPI), sh gelatin (FG) and conjugates as
an in
uence of pH.
Fig. 2. Interfacial protein load of emulsions made with whey protein isolate (WPI),sh
gelatin (FG) and whey protein isolate
sh gelatine (WPI
FG) in DI water.
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augmented from 1% to 1.5%. The outcomes suggested that whey
proteins adsorbed in preference to sh gelatin, especially when
protein concentrations were below 1% for preparing the emulsions.
This was also in agreement with Koupantsis and Kiosseoglou
(2009) and Ye (2008) as the adsorption behaviours of whey
protein in the emulsions formed with whey protein and poly-
saccharide or whey protein and sodium caseinate were similar.
Hunt and Dalgleish (1994)also suggested that a protein concen-
tration of 1% is adequate to provide monolayer coverage of the
interfacial area for 20 wt% soy oil during homogenization.
Increasing the protein concentration, however, increases the
unadsorbed protein concentration leading the enhancement of
depletionocculation and physical separation.
The different amounts of individual proteins adsorbed at the
surface may also be attributed mainly to different states of protein
molecular structure at the surface. Greater adsorbtion of whey
proteins at low concentration may be due to less spreading of the
globular whey protein molecules on the surface; in particular,
b-1actoglobulin may have been adsorbed at the surface as a dimer
structure as stated byYe (2008).
Fig. 3, presenting SDS-PAGE patterns of adsorbed proteins in
emulsions prepared with WPI or/and FG in deionized (DI) water
(pHw6) and buffers at pH of 3.4 and 6.8 with a total proteinconcentration of 1 wt%. The low molecular weight bands related to
whey protein (b-1actoglobulinw18 kDa anda-lactalbuminw14.4
kDa) appeared as 4 bands in the base of the gels whereas high
molecular weights proteins, which occupy the large fractions ofsh
gelatin, are observed on the top of the gels. Thepatterns of adsorbed
proteins for emulsions prepared with conjugate of WPIeFG in DI
water and at pHs of 3.4 and 6.8 look more clear and similar. Similar
gel quality was observed byTaylor et al. (2006) after addition of
whey protein isolate into the gelatin sample. The patterns for
emulsion prepared with either protein and in DI water and pH of 6.8
are also resemblance.
The concentrations ofb-1actoglobulin were 62.88%, 73.92% and
86.62% for WPI added emulsions at pHs of 3.4, 6.8 and DI water
respectively. Conjugating WPI and FG slightly increased the surfaceconcentration ofb-1actoglobulin up to 68.58% and 76.07% at pH 3.4
and 6.8, correspondingly, even though the concentration of WPI
was half of that used for emulsions made with WPI alone (0.5% vs
1%). The concentration of a-lactalbumin remains unchanged
(w11%) for emulsions containing WPI and prepared in buffer
solutions. The whey protein subunits concentrations for the same
emulsion prepared with DI water were slightly lower.
The 3 intense bands appeared on the top of the gels for emulsion
prepared with either FG or WPIeFG are associated with high
molecular weight proteins of 166 kD, 125 kD and 115 kD present in
FG. The concentrations of proteins at 166 D and 125 kD were found
to be considerably greater for emulsions made with FG at pH 3.4
compare to those prepared at pH 6.8 (20.67 vs 14.48 and 41.63% vs
22.85% respectively). Accordingly, concentrations of high molecular
weight proteins (166 kD and 125 kD) were greater in WPI-FG added
emulsions at pH 3.4 (7.58% and 3.77% vs 5.54% and 1.81% corre-
spondingly), whereas 115 kD protein concentrations were compa-
rable for emulsions made with WPIeFG at either tested pH (w14%).
Overall concentrations of these proteins were comparable for
emulsions prepared with DI water or buffer at pH of 6.8. This
suggest that pH and its intimacy to the isoelectric point of protein
play a major role in the amountof adsorbed protein to thesurface of
oil droplets. In addition, more exibility of amphiphilic whey
protein isolate compare to sh gelatin (Jiang, Li, Chai, & Leng, 2010)could cause faster adsorbtion during homogenization and makes
this protein the dominant species among the stabilizing layers.
These above results show evidence for preferential adsorption
of the b-1actoglobulin and a-lactalbumin where it appear in the
base of SDS-gel for emulsion prepared with either whey protein
isolate or conjugate of whey protein isolate and sh gelatin.
3.3. Effect of particle size distribution on emulsion stability
Britten and Giroux (1991)stated that the coalescence becomes
a slow destabilizing mechanismwhen preparing the o/wemulsions
with low concentrations of proteins as the only emulsifying agent
and under quiescent conditions. On this basis, emulsions were aged
under quiescent conditions at room temperature. The mean
average droplet size (Z-average size) was considered to compare
emulsions stability and compute the rate of coalescence (Dc).
Changes in Z-average were monitored during 3 weeks consid-
ering duplicate measurements and a total of 3 tests for each
measurement (Table 1). Studies bySherman (1983),Ye, Hemar, and
Singh (2004), Paraskevopoulou, Boskou, and Kiosseoglou (2005)
and Taherian et al. (2008) pointed out that the rate of coales-
cence of emulsion droplets (Dc) mainly follows the rst-order
kinetics (Eq.(2))
Nt N0expDct (1)
whereN0 and Ntare the numbers of droplets per unit volume of
emulsion initially and time t, respectively, and Dc is the rate ofdroplets coalescence. Using mean average droplet size measured
after preparation and 3 consecutive weeks the rate of coalescence
(Dc) can be determined by plotting 3(ln (Dt/D0)) vs. time (t) using
Eq.(2):
lnDt lnD0 Dct
3 (2)
whereD0andDtare the average droplet sizes initially and at timet,
respectively.
As are shown inTable 1, emulsion stabilized by whey protein
isolate at pH 3.4 indicated the highest rate of particle growth and
coalescence. Deposition of gelatin on whey protein coated droplets
appreciably (r 0.0005) reduced the rate of coalescence by 6.4
times at the identical pH. The reduction in the rate of coalescence
Fig. 3. SDS-PAGE patterns of the aqueous serum of whey protein isolate (WPI), sh
geletin (FG) and mixture of why protein isolate sh gelatine (WPI FG) after sepa-
ration and centrifugation, in DI water and pHs of 3.4 and 6.8, ranging in size from
175.66 kDa at the top to 14.40 kDa at the bottom.
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for whey protein coated droplets at pH 6.8 and after deposing
gelatin was also predominant (r 0.0005). The fact that the
emulsion droplets were coated by whey protein with appreciable
electrical charge suggests that electrostatic repulsion may play an
important role in stabilizing them against droplet aggregation
(McClements, 2005). Nevertheless, statistical analysis indicated
that viscosity ofsh gelatin is signicantly higher (r 0.0005) than
that of whey protein isolate covering oil droplets. This suggests that
steric interactions have also played an essential role in retarding the
droplets coalescence. Study byHernndez-Balada, Taylor, Phillips,
Marmer, and Brown (2009)also indicated that addition of a smallamount of gelatin to whey protein isolate resulted in a dramatic rise
of viscosity, higher gel strengths, and the appearance of high
molecular weight bands due to inter-protein crosslinking in SDS-
PAGE gelpatterns compare to eithergelatin or whey protein treated
separately. They suggested that the reducing environment partially
unfolds the whey proteins, increases access to glutamine and lysine
side chains, and the gelatin chains crosslink the whey proteins to
form a network.
Comparing the rate of coalescencesfor emulsions prepared with
FG or WPI and sh oil at identical pH of 3.4 and volume ratio
(4z0.1) shows thatsh gelatin coated droplets are less susceptible
to coalesce, while at elevated pH of 6.8 the rate of coalescence is
inferior for whey protein coated droplets. This phenomenon is
directly related to the both changes in the conformation of the
protein molecule and the net charge of the adsorbed protein layers
at the interface as inuence of the pH. As Pearce and Kinsella (1987)
stated, the ability of the protein to be adsorbed at the surface of oil
droplets depends upon its capacity to unfold and spread over the
interface to stabilize the new area created. As a result, the confor-
mational changes of the protein molecule are extremely important,
because they are related to properties such as surface hydropho-
bicity, protein exibility, solubility, degree of disulphide bonds,
degree of hydrogen interactions and other stabilizing forces.
Furthermore, Fachin and Viotto(2005) studied the effects of pH and
heat treatment on emulsifying properties of whey protein
concentrate and concluded that emulsifying properties were
considerably improved when the heat treatment was done after
ultraltration at pH 7 compared to pH 6. They related this
improvement to the greater denaturation of whey proteins at thispH. Likewise, study byYamauchi, Shimizu, & Kamiya (1980)spec-
ied that the stability of whey protein emulsions enhanced when
pH is increased from 5 to 7 and noted that this is, most likely, due to
an increase in electrostatic repulsion of the charge protein. There-
fore at pH 3.4, closedto the isoelectric point of whey protein and pH
6.8 near to the isoelectric point of sh gelatin, the net charge is
reduced and consequently the repulsion between the oil droplets
coated by these proteins is decreased resulting in higher rate of
coalescence.
3.4. Effect of rheological properties on stability of emulsion
3.4.1. Flow rheology
Flow behaviours of emulsions were characterized by measuringthe shear-rate dependent viscosity over 21 days time period. The
ow curves were tted to the power law and the determination
coefcients (R2) were more than 0.98 (not shown), indicating a high
level of linear relation between measuring points.
Table 2 illustrates representative data for the dependency ofow properties on applied shear rate for the studied emulsions as
inuences by pH and aging time. In all cases, the emulsions showed
shear thinning behaviour and the viscosity decreased along with
increasing the shear rate. WPI stabilized emulsions at pH 3.4,
among the other emulsions, demonstrated the highest apparent
viscosity and lowest ow behaviour index after 21 days aging at
room temperature. For this emulsion the apparent viscosity
decreased from 9828.04 mPa s at 0.1 s1 to 22.42 mPa s at 100 s1
at day-21 with the ow behaviour index of 0.12.
WPI stabilized emulsion under acidic condition also showed the
highest rate of coalescence suggesting time dependent induction of
weak cold-set gelation and reduction of electrostatic repulsion due
to intimacy of the solutions pH to the isoelectric point (pI) of
protein. Cavallieri and Lopes da Cunha (2008)reported that gela-
tion process of whey protein involves two distinct stages; the rst
stage is associated with an initial setting upof the gel network upto
the gel point, and the second stage is linked to a structural devel-
opment through bond strengthening and/or local rearrangements,
which is time and pH dependant. Our results also indicated that
loss in viscosity for protein solutions with pH close to isoelectric
point is time dependent but structural development is non-
homogenous causing creation of two dissimilar phases, one rich in
occulated and coalesced droplets trapped in a weak gel network
and the other rich in soluble solid with water like viscosity. Thehigher amount of gel, which is directly related to the higher degree
of coalescence, caused more increase in viscosity at shear rate of
0.1 s1 following by sharp decrease as shear rate develop to 100 s1.
Table 2
Changes in consistency coefcient (m) and ow behaviour index (n) of emulsions as function of pH and storage time.
Emulsion pH m(mPa) day-1 m(mPa) day-7 m(mPa) day-14 m(mPa) day-21 nday-1 nday-7 nday-14 nday-21
WPI 3.4 13.0 0.3 62.0 1.1 634.0 10.0 1290.0 11.5 0.72 0.01 0.49 0.01 0.24 0.01 0.12 0.00
WPI 6.8 3.6 0.2 4.50 0.2 5.1 0.5 5.4 0.2 0.97 0.02 0.95 0.01 0.93 0.01 0.90 0.01
FG 3.4 38.0 1.2 48.0 1.5 62.0 1.9 72.0 2.3 0.75 0.01 0.70 0.01 0.66 0.01 0.60 0.01
FG 6.8 28.0 0.8 65.0 1.3 102.0 2.3 128.0 4.2 0.81 0.01 0.70 0.01 0.56 0.00 0.46 0.00
WPI FG 3.4 20.8 0.1 23.5 0.2 27.8 0.4 31.3 0.2 0.99 0.02 0.97 0.01 0.96 0.01 0.92 0.02
WPI FG 6.8 24.1 0.6 26.9 0.2 29.3 0.4 33.9 0.1 0.97 0.00 0.95 0.01 0.94 0.01 0.93 0.01
Table 1
Comparison of droplet size growth for sh oil emulsions.
Emulsions pH Average particle size (nm) Dc
Day-1 Day-7 Day-14 Day-21
WPI 3.4 408 5.5 1138 11.5 3299 24.7 4999 35.4 0.064
WPI 6.8 338.9 5.3 440.87 14.3 597.6 9.4 698.6 11.1 0.021
FG 3.4 612.5 13.5 808.4 13.5 1224 24.5 1364 18.8 0.022
FG 6.8 1110.3 15.8 2462.2 19.7 3098.6 25.4 4098.6 22.7 0.031
WPI FG 3.4 593.7 10.6 677.2 17.6 822.3 22.1 856.1 23.2 0.01WPI FG 6.8 474.3 9.2 518 12.6 568.5 8.5 655.7 13.1 0.008
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Moreover, previously reported data have shown that both increase
in viscosity and decrease in ow behaviour index of emulsion as an
inuence of aging is directly related to the onset of occulation,
close proximity of the droplets and enhancement of the coales-
cence (Dickinson & Stainsby, 1988; Taherian et al., 2007a, 2007b;
Vingerhoeds et al., 2009). When the ocs are small, increase in
viscosity is minimal and occulation is reversible upon application
of slight shear. In the case of strong occulation and coalescence of
droplets the aggregation is irreversible and seems to be driven
upon electrostatics interactions as was pointed out by Sillettia,
Vingerhoedsa, Nordeb, and van Aken (2007).
Contrary to WPI stabilized emulsions, rheological parameters
for FG stabilized emulsion were more affected at pH 6.8 and
increase in consistency coefcient of FG, as an inuence of aging,
was higher. The value of n decreased from 0.81 at day-1 to 0.46
at day-21 indicating unset of occulation and coalescence at
this pH. According to Bae et al. (2009), solubility of proteins is
the lowest near their isoelectric point since intramolecular elec-
trostatic interactions would be maximal at this point, causing the
dipolar molecule to adopt the most compact conformation. The
proximity of FG isoelectric point to the pH 6.8 and subsequent
increase in electrostatic interactions could, therefore, explain such
occurrence.
Diluting WPI-coated droplets into hydrated FG, at both pHs of
3.4 and 6.8, minimize the differences between apparent viscosities
at day-1 and day-21 and showed, practically, Newtonian ow
behaviour for all tested emulsions. This indicates that occulation
and coalescence, despite pH, were minimal for emulsions made
with conjugated WPI and FG.
3.4.2. Dynamic rheology
Dynamic rheological properties were assessed through frequency
development of delta degree (G00/G0) over 21 days time period.
Emulsions were rst pre-sheared at 100 s1 during 1 min to ensure
homogeneity of the sample before subjection to frequency sweep
(Fig. 4). The accuracy of storage modulus (G0) and loss modulus (G")
was assured by investigating the linear region, where the dynamic
parameters (G0,G" and phase shift angle d) are independent of the
magnitude of applied stress, and selection of proper measuring
parameters via conduction of a stress sweep test. As illustrated in
Fig. 4, theeffect of aging was prominent foremulsion made with WPI
in acidic pH. The delta degree, at the highest level of applied
frequency, increased from35.79 degree up to 54.27 degree indicating
loss of elasticity. Comparing all gures, it can be noted that the
emulsions stabilized with conjugated proteins show the lowest
diversity between delta degrees at all levels of applied frequency.
WPI pH 6.8
0
20
40
60
80
100
0 5 10 15 20 25 30
Frequency (rad/s)
)'G/"G(eergedatleD
Day 1
Day 7
Day 14
Day 21
WPI pH 3.4
0
20
40
60
80
100
0 5 10 15 20 25 30
Frequency (rad/s)
)'G/"G(eergedatleD
Day 1
Day 7
Day 14
Day 21
FG pH 6.8
0
20
40
60
80
100
0 5 10 15 20 25 30
Frequency (rad/s)
)'G/"G(eergedatleD
Day1
Day 7
Day 14
Day 21
FG pH 3.4
0
20
40
60
80
100
0 5 10 15 20 25 30
Frequency (rad/s)
)'G/"G(eergedatleD
Day1
Day 7
Day 14
Day 21
WPI-FG pH 6.8
0
20
40
60
80
100
0 5 10 15 20 25 30
Frequency (rad/s)
'G/"G(eergedatleD
)
Day1
Day 7
Day 14
Day 21
WPI-FG pH 3.4
0
20
40
60
80
100
0 5 10 15 20 25 30
Frequency (rad/s)
)'G/"G(eergedatleD
Day1
Day 7
Day 14
Day 21
Fig. 4. Frequency development of delta degree (G00/G0) for emulsions prepared with whey protein isolate (WPI), sh gelatin (FG) and conjugates of FG WPI as inuences of pH and
storage time.
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Furthermore, the corresponding frequency developments of delta
degree for emulsions stabilized with FG at pH 3.4 and WPI at pH 6.8
diverge in lower extend compare to FG stabilized emulsion at pH 6.8
and WPI stabilized emulsion at pH 3.4. The results are in agreement
with the outcomes of rheological ow tests indicating gain of
viscosityand loss of elasticcomponents foremulsions made at the pH
close to the isoelectric points of either protein.
In general, emulsions prepared with conjugated WPI-FG desig-
nated higher viscosity, elasticity, and physical stability compare to
those prepared with WPI or FG alone. This suggests diluting WPI-
coated droplets in hydrated FG enhance the interfacial lm
viscosity and elasticity due to segregative interactions between
WPI and FG(Fitzsimons, Mulvihill, & Morris, 2008) on the surface of
double coated emulsions droplets resulting in superior stability.
3.4.3. Effect of pH and aging on physiochemical stability of
emulsions
Emulsions droplets are in continual motion and frequently
collide with one another leading to occulation or coalescence.
Proteins lowersurface tension at the interface that is formed during
the emulsication process and form a macromolecular layer
surrounding the dispersed droplets which structurally stabilizes
the emulsions and reduce the rate of coalescence (Juliane Floury,
Desrumaux, & Lardires, 2000).
Fig. 5illustrates the rate of aggregations combined with visual
observation in 100 ml Wainthropp tubes for studied emulsions
during 18 days with 2 days time interval. Each point in the graph
represents the average of 3 measurements of transmittedlight from6 cm emulsion height which was placed in a at-bottom cylindrical
glass tube. The slopes indicate the rate of droplets migration rising
to the upper part of the tube and collide between each other.
Palazoloa, Sorgentinib, and Wagner (2005)and McClements (2005)
quoted that the total collision frequency between emulsion drop-
lets is the contribution of the Brownian movement and the shifting
of the droplets under gravitational force. Once cream phase is
formed, the coalescence process is mainly governed by uctuations
prole and interfacial lm movement due to a long contact
between oil droplets. Coalescence likelihood increases when uc-
tuations become large enough to form holes that can pass from one
droplet to another. At this situation the magnitude of the shapeuctuations is governed by the interfacial tension, lm rheology
and mechanical applied forces.
The steeper slope, therefore, signies lower resistance of drop-
lets interfacial lm againstocculation and coalescence during the
migration process. The shallower slopes, on the other hand,
represent more stable emulsions suggesting that the thickness of
the continuous phase around the droplets (interstitial continuous
phase) was enough to avoid contact between droplet lms, and
interfacialuctuations did not lead to the exclusion of interstitial
water. As the highest rate of aggregation is associated to whey
protein stabilized emulsion at pH 3.4, the results are in excellent
agreement with previously mentioned coalescence rate presented
inTable 1. The results obtained during stability studies conrmed
rheological observations, and once again the emulsion stabilized by
whey protein and sh gelatin conjugate at elevated pH demon-
strated the lowest aggregation rate and, hence, the greater stability.The microstructures of studied emulsions obtained after prep-
aration are compared inFig. 6. As can be observed, the micrographs
display evidence of aggregated droplets of WPI-coated droplets at
pH 3.4 and FG-coated droplets at pH 6.8 compare to whey protein
andsh gelatin stabilized emulsions at pH 6.8 and 3.4 respectively.
This conrms the effect of closeness of pH to the isoelectric point of
each protein. The micrographs for emulsion prepared with conju-
gation of both proteins show similarity between particle size
distribution and conrm the segregation effect of whey protein-sh
gelatin interfacial lm.
Fig. 7 shows the hexanal concentration for tested emulsions
after 3 and 6 months along with pictures of corresponding emul-
sions taken after 6 month. Hexanal is a characteristic secondary
oxidation product of linoleic acid (Ries et al., 2010) and wasmeasured to nd out extend ofsh oil oxidation in proteins coated
droplets. For emulsion made with whey protein at pH 3.4 the
hexanal production was above all the other emulsions with the
concentration of 2.54 ppm after 3 month which almost doubled up
to 4.21 after 6 month aging under ultraviolet light. Deposition of
sh gelatin on whey protein coated droplets at identical pH
reduced the hexanal production down to 0.19 and 0.70 after 3 and 6
months respectively. Conversely, formation of hexanal for sh
gelatin coated droplets was higher at pH 6.8 with concentrations of
1.74 ppm and 2.25 ppm compare to the one at pH 3.4 at 0.52 ppm
and 1.41 ppm after 3 and 6 months correspondingly. Whey protein-
sh gelatin coated droplets at pH 6.8 was the lowest at 0.06 ppm
after 3 month and 0.18 ppm after 6 month aging at ultraviolet and
room temperature conditions.
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14 16 18 20
Time (day)
)%(n
oissimsnarT
WPI+FG pH=3.4 (a)
WPI+FG pH=6.8 (b)
WPI pH=3.4 (c)
WPI pH=6.8 (d)
FG pH=3.4 (e)
FG pH=6.8 (f)
a b fc edWPI+FG pH=3.4 (a)
WPI+FG pH=6.8 (b)
WPI pH=3.4 (c)
WPI pH=6.8 (d)
FG pH=3.4 (e)
FG pH=6.8 (f)
Fig. 5. Creaming velocity proles for emulsions prepared with whey protein isolate (WPI), sh gelatin (FG) and conjugates of FG WPI as inuences of pH and aging.
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u-3sh oil, thereby enhancing lipid oxidation. Conversely, droplet
carries positive charge may repel co-ions and retard lipid oxidation.
Nevertheless, the solubility of mineral ions generally increases at
decreasing pH which could potentially promote lipid oxidation
(Jacobsen et al., 2008; Jongjareonraka, Benjakula, Visessanguanb, &
Tanak, 2008).
Study by Hu, McClements, and Decker (2003) indicated that
particle size, inuencing surface area, droplet charge, causing either
attraction or repulsion of transition metals, thickness of emulsier
layer at the interfacial region of emulsion droplet, impacting inter-
actions between lipids and aqueous phase prooxidant, and chemical
components of proteins that enabling to scavenge free radicals or
chelate prooxidant metals are the major factors affecting lipid
oxidation rate in oil-in-water emulsions.Jacobsen et al. (2008)sug-
gested that the antioxidative mechanism of protein in the interfacial
region, such as bindingtrace metal ions from the lipid phase andfree-
radical scavenging activity, may involve a dynamic exchange process
with protein molecules from the continuous phase.
Our study showed that, the hexanal production for emulsion
stabilized by whey protein was signicantly higher (r< 0.0005)
than that stabilized by sh gelatin. The higher oxidative stability of
sh gelatin stabilized emulsions at acidic pH could, therefore, be
due to the higher surface charge (42.7 1.1 mv vs 18.05 0.42 mv)and lower surface area (612.513.5 nm vs 408 5.5 nm) of
emulsions. Overall, the greatest stability was observed for WPI FG
at pH 6.8, which corresponds to the highest negative charge of this
conjugate (zeta potential w60 mV). Also there was a good
correlation between the hexanal concentration and emulsion
creaming velocity. In addition, physiochemical stability of whey
protein-sh gelatin stabilized emulsions were signicantly greater
(r< 0.0005) than either whey protein or sh gelatin stabilized
emulsions and, hence, the stabilization effects could be consider as
both steric and electrostatic.
4. Conclusions
This study showed that deposition of sh gelatin over whey
protein coated sh oil droplets has a major impact on the physi-
ochemical stability of emulsion. This was attributed to both steric
effect of whey protein-sh gelatin conjugate and electrostatic
repulsion between the sh oil droplets which prevents them from
coming into close proximity. Addition of whey protein could
improve the stability of emulsions by increasing the electrostatic
repulsion at pH 6.8 and is useful for delivering omega-3 sh oil into
the milk beverages. On the other hand, adding sh gelatin to cover
oil droplets perk up the stability of emulsions through steric
stabilization at pH 3.4 and could be valuable for delivering omega-3sh oil into the fruitsbeverages. The conjugate WPIeFGcanbe used
as an effective emulsier for formulating food emulsions under
acidic conditions and the results from this study may have practical
applications for the design of industrial dispersions to deliverfunctional ingredients into the beverages.
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