Influence of whey protein concentrate on the functionality of egg white and bovine serum albumin

Influence of whey protein concentrate

on the functionality of egg white and bovine serum

albumin

K. J. Aryana,1 Z. Z. Haque1* & P. D. Gerard2

1 Department of Food Science and Technology, South-east Dairy Foods Research Center, MAFES, Mississippi State

University, Mississippi State, MS 39762, USA

2 Experimental Statistics Unit, MAFES, Mississippi State University, Mississippi State, MS 39762, USA

(Received 21 December 2000; Accepted in revised form 15 November 2001)

Summary The objective was to elucidate the functionality of various combinations of egg white

(EW), whey protein concentrate (WPC) and bovine serum albumin (BSA) and compare the

microstructures of their gels. Dispersions of individual proteins (1%, w/v, in imidazole-

HCl pH 7.0) were prepared. The BSA, EW and WPC and were combined in the ratios of

0 : 0 : 1, 1 : 0 : 0, 0 : 1 : 0, 0.5 : 0 : 0.5, 0 : 0.5 : 0.5, 0.5 : 0.5 : 0, 0.33 : 0.33 : 0.33,

0.42 : 0.16 : 0.42, 0.16 : 0.42 : 0.42 and 0.42 : 0.42 : 0.16. The functionalities studied were

oil holding capacity (OHC), water holding capacity (WHC), thermostability (TS), foam

activity (FA), foam stability (FS), foam density (FD), emulsion stability (ES) and

emulsifying activity index (EAI). Gels with a protein concentration of 10% (w/v) in 0.1 M

NaCl at pH 7 were used for determination of stress, strain values and microstructural

analyses. The R2 was in excess of 0.95 for all attributes studied except FD which had an R2

of 0.85. The results indicate that WPC apparently impacted water entrapment in an

additive manner proportional to its concentration. The combination of WPC with EW or

BSA resulted in a synergistic effect for TS and FS and an additive effect for OHC, WHC,

FD, EAI, gel stress and strain. On the contrary, an antagonistic effect was observed for

FA. If a multifunctional combination was to be picked, it would be a 1 : 1 ratio of WPC

and BSA as it had the highest number of attributes with synergistic effects. Data indicated

there were multifunctional advantage of using WPC.

Keywords Gelation, ingredient, microstructure.

Introduction

Food systems are usually complex compositions of

various ingredients. The manner in which these

ingredients interact affects the overall characteris-

tics of the food product. Arntfield & Bernatsky

(1993) reported that depleting egg albumin of

the lysozyme naturally found in it, adversely

affected the gel strength of egg albumin, reducing

its textural contribution to food products.

Matsudomi et al. (1994) observed gels formed

from mixtures of the major whey protein,

b-lactoglobulin, and bovine serum albumin (BSA)

to be stronger than b-lactoglobulin or BSA gels,

implying synergistic effects.

Bovine serum albumin is a well characterized

protein. It has a good ability to bind fatty acids to

hydrophobic sites (Boyer et al., 1947; Spector

et al., 1969). Although very well studied, BSA is

underutilized. Bovine serum albumin and egg

white (EW) are comparatively expensive. Whey

proteins are similar to EW with regard to their

heat coagulability, high sulphydryl and disulphide

contents (To et al., 1985). Substituting whey

*Correspondent: Fax: 662-325-8728/2474;

e-mail: [email protected]

Approved for publication as Journal Article no. J-9767 of

the Mississippi Agricultural and Forestry Experiment

Station, Mississippi State University.

International Journal of Food Science and Technology 2002, 37, 643–652 643

� 2002 Blackwell Science Ltd

proteinsforEWhasbeenreportedearlier (Wiechers,

1952; Kumetat & Beeley, 1954). Arunepanlop

et al. (1996) reported that whey protein isolates

could replace a maximum of 25% of the EW

proteins in angel food cake without adversely

affecting its physical and sensory characteristics.

Although effects of protein interactions have

been observed in food products, little has been

done to facilitate understanding of basic function-

al attributes and structure–function relationships.

Whey protein concentrate (WPC) is a comparat-

ively inexpensive source of functional proteins. It

would be interesting to determine the effect of the

addition of WPC on the functionalities of expen-

sive ingredients like BSA and EW. This study was

conducted to evaluate and compare the function-

alities of various combinations of BSA, EW and

WPC and to compare the microstructures of their

gels.

Materials and methods

Materials

Spray-dried EW protein was from Hygrade Egg

Products (Elizabeth, NJ, USA). Spray-dried WPC

was from New Zealand Milk Products (Santa

Rosa, CA, USA). Freeze-dried BSA (catalog no.

A-7906) and glutaraldehyde were from Sigma (St

Louis, MO, USA). Imidazole, petroleum ether,

potassium phosphate, osmium tetroxide, uranyl

acetate were from Fisher Scientific (Fair Lawn,

NJ, USA). Ethanol, propylene oxide, formvar,

double-sided carbon sticky tape, aluminium stubs,

vinylcyclohexene dioxide, D.E.R 736 epoxy resin,

noneyl succinic anhydride and dimethyl-amino-

ethanol were all from Electron Microscopy Sci-

ences (Fort Washington, PA, USA).

Experimental design

The design was that of a mixture experiment with

three replications of each combination of

BSA : EW : WPC, respectively, in the following

ratios 0 : 0 : 1, 1 : 0 : 0, 0 : 1 : 0, 0.5 : 0 : 0.5,

0 : 0.5 : 0.5, 0.5 : 0.5 : 0, 0.33 : 0.33 : 0.33,

0.42 : 0.16 : 0.42, 0.16 : 0.42 : 0.42 and 0.42:

0.42 : 0.16. A {3,2} lattice design (Myers &

Montgomery, 1995) augmented by four additional

combinations was used. The four combinations

were equal proportions of the three components

and the combinations with 42% of two compo-

nents and 16% to the remaining component.

Proximate analysis of sample components

Moisture content of the samples were determined

by the Association of Official Analytical Chemists

(AOAC) method no. 927.05 (Association of Offi-

cial Analytical Chemists, 1990) (Table 1).

Fat was determined by ether extraction using a

Goldfisch extraction apparatus (Labconco, Kan-

sas city, MO, USA). Samples used for moisture

determination were re-used for fat determination.

Samples in Alundum crucibles (Fisher scientific,

Pittsburgh, PA, USA) were placed in stainless steel

sample containers fitted to a condenser (Lab-

conco, Kansas city, MO, USA). Fat was extracted

in petroleum ether (Fisher scientific, Pittsburgh,

PA, USA). After 4 h of extraction samples were

allowed to cool at 21 �C for a 15-min period.

Alundum crucibles were allowed to air dry for

45 min. These crucibles were placed in an air oven

at 100 �C for 15 h, transferred to a desiccator to

cool and then weighed. This weight was the gross

dry fat-free weight which was subtracted from the

gross dry weight and divided by the original

weight of the sample. The resulting number was

multiplied by 100 to give weight of fat in per cent

(Table 1).

Protein content of the samples were determined

by AOAC method no. 930.29 (Association of

Official Analytical Chemists, 1990). Percent nitro-

gen obtained was multiplied by 6.25 for EW

samples and 6.38 for the remaining samples to

obtain per cent protein (Table 1).

Sample Protein Fat Moisture Ash

WPC 74.82 ± 0.100 0.53 ± 0.005 7.83 ± 0.020 3.23 ± 0.060

EW 78.03 ± 0.430 0.38 ± 0.190 7.77 ± 0.010 5.85 ± 0.020

BSA 91.90 ± 0.050 0.31 ± 0.018 7.19 ± 0.035 0.84 ± 0.030

Table 1 Composition of samples

(% w/w). Mean ± s.e.

Functionalities of protein combinations K. J. Aryana et al.644

International Journal of Food Science and Technology 2002, 37, 643–652 � 2002 Blackwell Science Ltd

Ash was determined by AOAC method no.

945.46 (Association of Official Analytical Chem-

ists, 1990).

Preparation of protein dispersions

Dispersions of 1% (w/v) BSA, WPC and EW were

prepared in 10 mM imidazole buffer pH 7. Var-

ious combinations of the BSA, EW and WPC

dispersions were prepared (v/v) according to the

following ratios BSA : EW : WPC, 0 : 0 : 1,

1 : 0 : 0, 0 : 1 : 0, 0.5 : 0 : 0.5, 0 : 0.5 : 0.5,

0.5 : 0.5 : 0, 0.33 : 0.33 : 0.33, 0.42 : 0.16 : 0.42,

0.16 : 0.42 : 0.42 and 0.42 : 0.42 : 0.16.

Functionalities

Emulsion stability (ES), emulsifying activity index

(EAI), thermostability (TS), and oil holding

capacity (OHC) were conducted according to

Haque & Mozaffar (1992). Water holding capacity

(WHC) was conducted according to Handa et al.

(1998).

Foams were generated using a swinging arm

agitator specially designed to study foaming by a

constant energy input by shaking. Angle of swing

was 140�, number of strokes were 102 strokes

min)1. Agitation time was 4 min. Height of the

swinging arm was 53 cm. Height within the

measuring cylinder over which the dispersion

shook back and forth was 22.5 cm and the internal

diameter of the measuring cylinder was 2.7 cm.

Foam activity (FA), foam stability (FS) and foam

density (FD) were calculated according to Haque

& Kito (1983).

Preparation of gels.

Proteins were hydrated (10% w/v protein) in 0.1 M

NaCl at pH 7. The pH was adjusted to 7.00 with

0.1 N NaOH. Gelation of proteins was as des-

cribed earlier (Lee et al., 1997). Dispersions were

stirred for 1 h with a magnetic stirrer at medium

speed. Air incorporation was kept to a minimum

and degassing was conducted in vaccuo. Two 15-

cm long glass tubes (22 mm OD · 1.5 mm thick)

were sprayed with cooking oil-based spray and the

inside of the tubes were brushed with a test-tube

brush for complete coverage. Degassed protein

dispersion was stirred for uniformity before pour-

ing in the tubes whose bottom ends had been

closed with a cap (heat resistant plastic of 2.22 cm

diameter). The top end was closed with a cap that

was punctured to make a (1.5 mm diameter) vent.

Tubes were placed vertically in holding racks in a

water-bath such that water level was slightly above

the sample line and heated at 90 �C for 15 min.

Tubes were then immediately cooled in cold water

(10 ± 1 �C) for 20 min and left overnight in a

refrigerator (4 ± 1 �C). Three replications were

conducted.

Gel stress and strain

Textural attributes, stress and strain, of the

protein gels were determined using an Instron

Universal Testing Machine (Series IX Automated

Materials Testing System V 401C; Instron Cor-

poration, Canton, MA, USA). From each end of

the tube 1 cm was discarded and samples were

cut into uniform size of 19 · 19 mm (diameter ·length). These samples were placed on a flat plate

and a yield compression test was carried out at

21 �C. The crosshead speed was 50 cm min)1

in both upward and downward directions and

the cell load was 50 kg. The Instron was inter-

faced with a computer which was instructed to

calculate stress and strain at the point of failure

manually indicated by a cursor at the peak of

the curve. This was done to be sure that the

stress and strain output being given out by the

computer was exactly in reference to the point of

failure.

Stress at failure ¼ F ½1� ðDÞh=h�=pr2ðkNm�2Þ;

where, F is the compressive force, h the sample

height, r the radius of sample crossection, and

Dh the displacement. Strain at failure (80%) was

given by the expression, Strain¼Dh/h.

Scanning electron microscopy (SEM)

Sample preparation was according to Aryana &

Haque (2001). Five representative blocks of each

of the three replications were mounted as one

replication per stub. From these five blocks per

stub three representative blocks were selected at

random. On these three blocks many fields

were quickly examined before a representative

field of 15 · 15 lm was randomly selected and

Functionalities of protein combinations K. J. Aryana et al. 645

� 2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 643–652

photographed. This was done for each of the three

replications. Samples were viewed using a Cam-

bridge S 360 SEM (Leo, Electron Microscopy Inc.

Thornwood, NY, USA) at 15 kV. Images were

recorded on Polaroid Type 55 P/N film (Polaroid

Corp. Cambridge, MA, USA) and/or stored in

electronic media.

Transmission electron microscopy (TEM)

Gels were cut in approximately 1 mm cubes and

fixed in 1% glutaraldehyde in 0.1 M potassium

phosphate buffer and placed at 4 �C overnight.

This was followed by three rinses of 0.1 M

potassium phosphate buffer (pH 7.2). Post-fixa-

tion was by 2% OsO4 in the same buffer. Samples

were dehydrated in a graded series of ethanol 35,

50, 70, 95% v/v for 15 min each and in three

changes of 100% ethanol over 1 h. Samples were

transferred to propylene oxide (PO) (two changes

over 1 h) Spurr’s resin (Spurr, 1969) was pre-

pared using vinylcyclohexene dioxide, D.E.R 736

epoxy resin, noneyl succinic anhydride and dim-

ethyl-aminoethanol. Infiltration using 50% PO:

50% resin (2 h), 25% PO: 75% resin (2 h), and

100% resin change was done overnight on a

rotator, and 100% resin change (8 h) on a

rotator. Samples were placed in 100% resin in

labelled BEEM (Better Equipped for Electron

Microscopy) capsules. These were then placed

overnight in an oven at 70 �C for 15 h. The

resulting resin blocks were trimmed with blades

and thin-sectioned using a Reichert-Jung ultra-

microtome (Cambridge Instruments Inc. Buffalo,

NY, USA). Thin sections were collected on

formvar coated copper grids and stained with

uranyl acetate and Reynold’s lead citrate

(Reynolds, 1963). Sections on the grid were

washed in distilled water, air-dried and examined

at 60 kV in a JEOL JSM 100 CX II transmission

electron microscope (JEOL, Peabody, MA,

USA). Five resin blocks were prepared per

combination for each of the three replications.

From these five resin blocks two were randomly

selected for thin sectioning. Seven ± one sections

were picked up per grid. Two grids were prepared

(one per resin block). Many fields were viewed on

both grids before a representative field of

10 · 10 lm was photographed. This was done

for each of the three replications.

Statistical analyses

Data of this mixture experiment was analysed

using a regression model without an intercept term

as described in Myers & Montgomery (1995).

Linear terms for each component of the mixture as

well as all cross product terms were included. Data

was analyzed using PROC REG of the SAS (SAS,

1999). Coefficients of linear terms for BSA and

EW were compared with WPC, which is essen-

tially a comparison of effects when pure blends

were employed. Significance of cross product

terms were examined to evaluate possible syner-

gistic or antagonistic effects. Significant positive

coefficients for the cross product terms imply the

components acted synergistically, significant neg-

ative coefficient imply antagonism, while non

significant coefficients imply additive effects. A

significance level of 0.05 was used for all hypo-

thesis tests.

Results and discussion

Functionalities

Bovine serum albumin had the highest oil holding

capacity (OHC) and WPC appeared to have the

least OHC (Table 2). No two-way interactions

(Table 3) were significant (at the 5% level) indi-

cating additive effects (Myers & Montgomery,

1995). Bovine serum albumin has numerous

hydrophobic sites that can bind hydrocarbon

chains (Boyer et al., 1947; Spector et al., 1969).

This may have caused the additive effect as seen in

the interactions.

Water holding capacity (WHC) of WPC was the

maximum, while BSA appeared to have least

WHC (Table 2). There were no significant two-

way interactions (Table 3) implying additive

effects.

Thermostability is an important attribute in

thermally processed foods. Bovine serum albumin

was the least thermostable (Table 2). Comparing

the effects of substitution of WPC with EW or

BSA, WPC had TS similar to EW (Table 3). The

WPC in combination with EW or BSA resulted in

synergistic effects (Table 3). Estimates of all two-

way interaction effects were positive and signifi-

cant (at 5% level) indicating synergistic effects

(Myers & Montgomery, 1995).



Foam activities of BSA and EW were compar-

able with each other but were about six times

higher than WPC (Table 2). Bovine serum albu-

min and EW in combination with each other

resulted in a positive estimate which was not

significant, implying an additive effect. The WPC

acted antagonistically with both BSA and EW

(Table 3).

Foam stability was the least for WPC (Table 2).

The WPC acted synergistically with both BSA and

EW (Table 3), However, BSA combined in an

additive fashion with EW.

A high FD would imply smaller bubble size,

larger number of bubbles and hence greater

interfacial area. Comparing the effects of substi-

tution of WPC with EW or BSA, WPC had dense

foams similar to both EW and BSA (Table 3). All

two-way interactions showed additive effects for

FD (Table 3).

The WPC formed stable emulsions comparable

with both EW and BSA (Table 3). The WPC,

when combined with BSA, resulted in a synergistic

effect for emulsion stability (Table 3). The EW

combined additively with both WPC and BSA.

The EAI of WPC was similar to BSA and EW

(Table 3). All two-way interactions revealed

additive effects for EAI (Table 3). The major

proteins in WPC are b-lactoglobulin and a-lactal-bumin. Klemaszewski et al. (1992) reported that at

2% w/w concentration at pH 7 b-lactoglobulinemulsified slightly more oil than BSA while the

least amount of oil was emulsified by a-lacta-lbumin.

The WPC gels had the lowest strain values

(Table 2). Additive effects were observed for all

two-way interactions (Table 3).

Maximum force per unit area (stress) was

required to fracture BSA gels (Table 2) while

WPC gels appeared to need the least stress, i.e.

they fractured easily with less force per unit area

of the gel. Additive effects were observed for all

two-way interactions. Tang et al. (1994) reported

EW gels have higher gel stiffness compared with

WPC gels. Gezimati et al. (1996) reported that the

Table 2 Functionalities of the combinations of the various proteins. Mean ± s.e.

Sample OHC WHC TS FA FS

WPC 6.04 ± 0.21 43.59 ± 1.72 60.50 ± 5.01 23.75 ± 11.25 11.43 ± 0.32

EW 7.96 ± 0.00 3.87 ± 1.48 75.83 ± 13.0 146.25 ± 3.75 94.21 ± 1.62

BSA 27.11 ± 0.12 1.35 ± 1.07 15.50 ± 5.40 153.75 ± 8.75 88.35 ± 3.07

B50W50 16.00 ± 0.42 5.96 ± 1.22 77.67 ± 14.08 45.25 ± 0.75 85.65 ± 6.09

W50E50 6.91 ± 0.14 13.13 ± 1.25 94.50 ± 5.50 58.75 ± 6.25 76.65 ± 3.35

B50E50 17.03 ± 0.10 2.87 ± 1.37 83.16 ± 10.13 202.5 ± 2.50 92.18 ± 0.43

B33W33E33 13.36 ± 0.16 4.23 ± 1.96 92.83 ± 3.59 62.5 ± 2.50 75.92 ± 10.29

B42W42E16 14.96 ± 0.02 5.23 ± 0.13 90.00 ± 5.92 52.25 ± 7.25 85.02 ± 5.88

B42W16E42 15.26 ± 0.04 3.04 ± 0.63 81.83 ± 14.81 67.50 ± 10.0 81.90 ± 0.18

B16W42E42 9.39 ± 0.21 5.42 ± 1.40 93.50 ± 3.27 60.63 ± 3.13 85.35 ± 2.59

FD ES EAI STRESS STRAIN

WPC 31.05 ± 13.39 70.0 ± 0.00 222.19 ± 13.28 0.20 ± 0.066 0.01 ± 0.008

EW 23.15 ± 6.48 72.0 ± 0.00 196.24 ± 17.38 0.28 ± 0.007 0.08 ± 0.007

BSA 20.21 ± 5.5 70.5 ± 0.29 190.82 ± 21.12 0.41 ± 0.007 0.11 ± 0.014

B50W50 17.36 ± 0.99 76.75 ± 0.14 194.41 ± 15.79 0.25 ± 0.003 0.28 ± 0.018

W50E50 14.65 ± 1.35 74.25 ± 0.75 216.48 ± 3.87 0.32 ± 0.052 0.12 ± 0.017

B50E50 12.27 ± 3.19 73.33 ± 0.67 195.23 ± 2.72 0.26 ± 0.050 0.23 ± 0.031

B33W33E33 18.00 ± 0.76 74.92 ± 0.42 212.16 ± 9.58 0.32 ± 0.019 0.13 ± 0.011

B42W42E16 16.29 ± 1.89 72.83 ± 0.33 217.54 ± 6.77 0.23 ± 0.027 0.23 ± 0.007

B42W16E42 32.21 ± 11.70 72.67 ± 0.58 207.08 ± 9.02 0.23 ± 0.014 0.26 ± 0.027

B16W42E42 16.38 ± 4.31 72.00 ± 0.25 210.44 ± 21.08 0.32 ± 0.052 0.12 ± 0.017

OHC¼oil holding capacity, WHC¼water holding capacity, TS¼ thermostability, FA¼ foam activity, FS¼ foam stability, FD¼ foam

density, ES¼ emulsion stability, EAI¼Emulsifying activity index, WPC¼whey protein concentrate, EW¼ egg white, BSA¼bovine

serum albumin, B50W50¼BSA 50% and WPC 50%, W50E50¼WPC 50% and EW 50%, B50E50¼BSA 50% and EW50%,

B33W33E33¼BSA 33.33% WPC 33.33% and EW 33.33%, B42W42E16¼BSA 42% WPC 42% EW 16%, B42W16E42¼BSA 42% WPC

16% EW 42%, B16W42E42¼BSA16% WPC 42% EW 42%.



BSA gels were stiffer than gel made with

b-lactoglobulin, the major functional protein in

WPC. Wang & Damodaran (1991) reported that

BSA gelation involved transconformation of

a-helix and aperiodic structures into b-sheetconformation. They observed gel strength to

decrease under conditions that decreased forma-

tion of a-sheet structures.The R2, which is an indication of goodness of fit

of the model, was in excess of 0.95 for all

attributes studied except FD which had an R2 of

0.85 (Table 3). The raw means (Table 2) of BSA,

EW and WPC were closely represented by

their estimates (Table 3) for the attributes viz.

OHC, TS, FA, FS, FD, ES, EAI indicating that

the model is a good fit. However, the raw means

for WHC, strain and stress were not closely

represented by their estimates. Moreover for

EW, negative values were estimated for WHC

and stress implying that the fit of this model is

questionable for these two attributes.

Microstructure of the gels

The BSA gels are represented in Fig. 1A. The

pores (arrow heads) represent the spaces in the

matrix within which the liquid phase/water was

held prior to dehydration during sample prepar-

ation for electron microscopy, while the phase

other than the pores/water entrapment spaces

represent the protein aggregate matrix. The BSA

gels had a compact microstructure. There was a

uniform distribution of the spaces and aggregates.

Comparatively, the EW matrix had larger water

Table 3 Quadratic mixture model coefficient estimates

Coefficient

Attribute Statistic BSA EW WPC BSA 3 EW BSA 3 WPC EW 3 WPC R2

OHC Estimate 27.1* 7.9* 6.0 )1.7 )1.7 )0.8 0.999

s.e. 0.2 0.2 0.2 0.9 0.9 0.9

P-value – – – 0.0682 0.0695 0.3538

WHC Estimate 58.2* )12.5 7.8 )80.0 )108.3 61.9 0.964

s.e. 25.7 25.7 25.7 54.3 54.3 54.3

P-value – – – 0. 1787 0.0812 0.2872

TS Estimate 15.9* 76.1 61.1 131.7 156.0 93.8 0.967

s.e. 9.5 9.5 9.5 39.6 39.6 39.6

P-value – – – 0.0028 0.0006 0.0262

FA Estimate 157.2* 149.8* 29.4 54.2 )248.1 )175.2 0.959

s.e. 17.7 17.7 17.7 74.0 74.0 74.0

P-value – – – 0.4759 0.0047 0.0329

FS Estimate 89.1* 95.1* 12.7 )27.5 125.1 82.0 0.994

s.e. 5.4 5.4 5.4 22.7 22.7 22.7

P-value – – – 0.2460 <0.0001 0.0026

FD Estimate 19.5 22.5 29.7 0.6 )20.6 )35.7 0.856

s.e. 7.1 7.1 7.1 29.5 29.5 29.5

P-value – – – 0.9837 0.4956 0.2460

ES Estimate 70.8 72.3 70.3 8.3 17.5 4.7 0.999

s.e. 1.0 1.0 1.0 4.2 4.2 4.2

P-value – – – 0.0589 0.0003 0.2697

EAI Estimate 189.9 194.7 221.0 32.5 )9.1 44.7 0.991

s.e. 12.8 12.8 12.8 53.6 53.6 53.6

P-value – – – 0.5502 0.8663 0.4131

STRAIN Estimate 0.2 0.7 0.3 0.3 1.1 )0.1 0.995

s.e. 0.5 0.5 0.5 1.1 1.1 1.1

P-value – – – 0.7536 0.3429 0.9441

STRESS Estimate 0.4* )0.1 )0.2 )0.2 0.03 0.6 0.983

s.e. 0.2 0.2 0.2 0.5 0.5 0.5

P-value – – – 0.7413 0.9459 0.1810

*Significantly different from WPC. Abbreviations as in Table 2.



entrapment spaces (Fig. 1B). Aggregates were in

small strands and groups. Similar observations of

EW gels at pH 7 were reported earlier (Handa

et al., 1998). The WPC gels had the largest sized

water entrapment spaces (Fig. 1C) in the matrix.

Aggregates were in large strands and groups

compared with the EW gels. Similar observations

of large ‘voids’ (spaces) were also seen in WPC gels

reported by Hung & Smith (1993). The gel

resulting from one third concentration of WPC,

EW and BSA (Fig. 1D), had reduced water

entrapment spaces compared with WPC gel

(Fig. 1C). As the concentration of WPC increased

to 50%, the sizes of the water entrapment spaces

also increased (Fig. 1E, F). Absence of WPC

resulted in complete disappearance of large water

entrapment areas (Fig. 1G). A reduction in WPC

from 50 to 42% resulted in further decline in size

of large water entrapment spaces (Fig. 1H, I).

Reduction in WPC concentration to 16% (Fig. 1J)

Figure 1 Scanning electron photomicrograph of 10% protein gel of (A) bovine serum albumin (BSA), p¼ protein aggregate

matrix, arrowhead¼water entrapment space/pore; (B) Egg white (EW); (C) Whey protein concentrate (WPC);

(D) BSA33.3% EW33.3% WPC33.3%; (E) BSA50% WPC50%; (F) WPC50% EW50%; (G) BSA50% EW50%;

(H) BSA42% WPC42% EW16%; (I) BSA16%WPC42%EW42%; (J) BSA42% WPC16% EW42%.



was comparable with the absence of WPC

(Fig. 1G) as regards the size of the water entrap-

ment areas. In the combination, as the concentra-

tion of WPC declined, and BSA increased from

one-third (Fig. 1D) to 42 (Fig. 1J) and then to

50% (Fig. 1G), the compactness or close packing

of the aggregates, i.e. tight packing of the gels,

appeared to increase.

The matrices of the BSA gels comprised of a

fine-stranded network of protein aggregates. The

BSA gel matrix was compact with small water

entrapment areas (Fig. 2A). Similar observations

related to BSA gels (8%) have been reported

earlier (Neiser et al., 2000). On the other hand EW

gel matrix had comparatively larger water entrap-

ment spaces (Fig. 2B) while the largest water

entrapment spaces were exhibited by the WPC

gels (Fig. 2C). This substantiated our SEM related

observations. The WPC gel matrix was comprised

of aggregates that were more densely clustered

(darker) and appeared more tightly fused com-

pared with EW or BSA aggregates. Equal combi-

nations of WPC, EW and BSA yielded gels

(Fig. 2D) with water entrapment spaces smaller

than the WPC gels (Fig. 2C) but larger than

the BSA gels (Fig. 2A). As observed by SEM,

Figure 2 Transmission electron photomicrograph of 10% protein gel of (A) bovine serum albumin (BSA), p ¼ protein

aggregate matrix, arrowhead¼water entrapment space/pore; (B) Egg white (EW) (C) Whey protein concentrate (WPC);

(D) BSA33.3% EW33.3%WPC33.3%, p¼protein aggregate matrix, arrowhead¼water entrapment space/pore, Bar¼ 2 lm;

(E) BSA50% WPC50%; (F) WPC50% EW50%; (G) BSA50% EW50%; (H) BSA42% WPC42% EW16%; (I) BSA16%

WPC42% EW42%; (J) BSA42% WPC16% EW42%. All bars¼ 2 lm.



as the concentration of WPC increased the sizes of

the water entrapment spaces also increased

(Fig. 2E, F). Conversely, reduction and absence

of WPC resulted in gels with decreased size of

water entrapment areas (Fig. 2G–J). On the other

hand, as observed by SEM, BSA gave compact

gels. Gels of a 1 : 1 combination of BSA and EW

(Fig. 2G) were more compact (aggregates in a

closer proximity to each other) compared with EW

gels (Fig. 2B). Transmission electron microscopy

corroborated the SEM observations that increase

in concentration of WPC increased sizes of water

entrapment spaces in gels.

The low water holding capacity of BSA

(Table 2) may be the reason why BSA formed

such compact gels (Fig. 1A) with high gel stress

(Table 2). Conversely, the high WHC of WPC

(Table 2) resulted in large water entrapment and

reduced gel stress values (Table 2). This low gel

stress reflected a weaker matrix that caused the

gels to cave into the large entrapped water

containing spaces on application of the slightest

force.

Conclusions

The results indicate that WPC affected the size of

water entrapment areas, apparently in an additive

manner, proportional to the concentration of

WPC. The WPC in combinations with EW or

BSA resulted in synergistic effect for TS and FS.

The WPC in combination with EW or BSA

resulted in additive effects for OHC, WHC, FD,

EAI, gel stress and strain. An antagonistic effect

was observed for WPC in combination with EW or

BSA for foam activity. If multifunctional combi-

nations were to be picked out, it would be a 1 : 1

ratio ofWPC and BSA as it had the highest number

of attributes with synergistic effects. Data indicated

a multifunctional advantage of using WPC.

Acknowledgements

Assistance for Instron provided by Paul Lee and

Chad Winter of the Agricultural and Biological

Engineering Department at Mississippi State Uni-

versity, Mississippi State, MS 39762 are greatly

appreciated. Funded by Mississippi Agricultural

and Forestry Experiment Station project no.

343010.

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Influence of whey protein concentrate on the functionality of egg white and bovine serum albumin

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