ability of whey protein isolate andorfish gelatin to inhibit physical separation

8/13/2019 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]


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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

A.R. Taherian et al. / Food Hydrocolloids 25 (2011) 868e878 869


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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

A.R. Taherian et al. / Food Hydrocolloids 25 (2011) 868e878870


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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.



6/11

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.



8/11

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.



9/11


10/11

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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ability of whey protein isolate andorfish gelatin to inhibit physical separation

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