[advances in food and nutrition research] volume 33 || proteins in whey: chemical, physical, and...

ADVANCES IN FOOD AND NUTRITION RESEARCH, VOL. 33

PROTEINS IN WHEY: CHEMICAL, PHYSICAL, AND FUNCTIONAL PROPERTIES

J. E. KINSELLA AND D. M. WHITEHEAD

Institute of Food Science Cornell University

Ithacu, New York 14853

I. Introduction 11. Whey Products

Ill. Structure of Whey Proteins A. p-Lactoglobulin B. a-Lactalbumin C. Bovine Serum Albumin D. Immunoglobulins E. Proteose-Peptones F. Lactofenin G. Lysozyme Whey Protein Preparation and Isolation Functional Properties of Whey Proteins A. B. Extrinsic Factors C.

Thermal Properties of Whey Proteins A. Effects of Heat on Conformation B. Factors Affecting Heat Denaturation

VIII. Gelation A. Gel Structure B. Gelation of Whey Proteins C. Composition and Gelling Properties D. Whey Proteins as Gelling Ingredients Surface Activity of Whey Proteins A. Protein Adsorption B. Protein Structure and Film-Forming Properties C. Whey Protein Films D. Film-Forming Properties of Glycosylated p-Lactoglobulin

IV. V.

Intrinsic Factors and Functional Properties

Variability in Whey Protein Preparations VI. Hydration and Solubility

VII.

IX.

343 Copyright 8 1989 by Academic Press, Inc.

AII rights of reproduction in any form reserved.

344 J. E. KINSELLA AND D. M. WHITEHEAD

X. Foams A. Protein Structure and Foam Stability B. Whey Protein Foams C. Factors Affecting the Foaming Properties of Whey Proteins

XI. Emulsions A. Emulsifying Properties of Whey Proteins B. Molecular Flexibility, Surface Hydrophobicity, and Emulsifying

Properties C. Factors Affecting Emulsifying Properties Ligand Binding by Whey Proteins A. Lipid Binding B. Flavor Binding C. Ligand Binding by P-Lactoglobulin D. Interactions of Flavors with Whey Proteins

A. Enzymatic Modification B. Chemical Modification Nutritional Aspects of Whey Proteins

XV. Summary and Conclusions Research Needs References

X11.

XIII. Modification of Whey Proteins

XIV.

1. INTRODUCTION

The annual production of cheese whey is expected to exceed 23 x lo’ kg in the near future while current utilization of whey approximates 60% of this total. Whey represents a potentially significant source of functional protein ingredients for many traditional and novel food products. The unique structure and physicochemical properties of whey proteins govern their functional behavior in food systems and structure-function relationships must be elucidated and controlled in order to utilize this food re- source fully. This review focuses on the structural properties of whey proteins and how these properties are influenced by environmental factors (e.g., pH, temperature, salts) in manifesting characteristic functional properties, such as gelation, foaming, and emulsifying activity, which are important in food applications (Fig. 1).

Proteins are important structural components in many foods and they are also used as ingredients because of their nutritional value and physicochemical properties (Kinsella, 1976, 1982; Morr, 1984). The market for functional protein-rich ingredients is expanding and is currently supplied by various proteins (Table I). Proteins obtained from dairy products have traditionally been one of the main protein sources for humans. Currently, a selection of high-protein dairy powders, e.g., caseinates, coprecipitates, and whey powders are used, yet these display a highly variable range of

PROTEINS IN WHEY 345

PROTEIN STRUCTURE and

PRESENT CONFORMATION

OTHER MOLECULES

PR-INS tEMrEnlltunE

PR E DOM I N A N T

INTRA- AND INTERMOLECULAR FORCES HYDROPHOBIC IONIC (HYDROGEN BONDINO AND ELECTROSTATIC) VAN DER WML'S INTERCTIONS DlSULFlDE BONDINO

FIG. I. teins.

Factors and forces involved in determining the functional properties of food pro-

TABLE I APPROXIMATE AMOUNTS OF PROTEINS USED AS FUNCTIONAL INGREDIENTS IN FOOD

MANUFACTURING IN THE UNITED STATES

Estimated Commodity amounts used

source ( x lo6 kg)

Milk Whey EfB Meat Fish Cereals Oilseeds Yeast

I36 168

6.8

14.0 145.3

I .4

Functional component protein

Caseins, whey proteins Whey proteins Egg white proteins, lipoproteins Collagen, myosin Collagen, myosin Wheat and corn gluten Soybean concentrates, isolates, and flours Single-cell protein (SCP)


functional properties because of differences in composition and processing treatments (Kinsella, 1984a).

The anticipated market demand for functional ingredient proteins is expected to increase in the United States and worldwide as the food industry increases the formulation and fabrication of new foods from basic ingredients (Kinsella, 1985). To satisfy this demand, food processors require ingredient proteins with consistent functional properties that perform in food systems in a reliable manner and are compatible with auto- mation.

II. WHEY PRODUCTS

Production of fluid whey in the United States increased to 20 x lo’ kg (18 x lo9 kg sweet and 2 x lo9 kg acid whey) in 1985 and the volume is increasing annually (Clark, 1987). Approximately 60% of this total is processed principally for use in foods and animal feed, with dried whey powder being the predominant product (Table 11). Liquid whey, a by- product of cheese manufacture, contains approximately 20% of the origi-

TABLE I1

( x lo6 kg) DIRECT AND INDIRECT USES OF DRY WHEY POWDERS IN FOODS IN 1982 AND 1984

Dry sweet Whey with Whey protein whey lactose/salt concentrate

1982 1984 1982 1984 1982 I984

Bakery Dairy Blends Dry mixes Confectionery Margarine Processed meats

Chemicals Soft drinks Institutions Infant foods Other

soups

50.8 48.6 26.8 13.6 6.0 0.2 1.4 0.8 5.2 0.3 - - 6.0

46.3 72.6 35.4 18.6 7.7 2.4 2.6 2.8 0.4 2.8 0.2

19.0 -

- 4.9 I .5 0.8 0.3 -

- - 10.6 1.4

0.6 6.2 1.3 0.2 0.3 -

0.3 4.4 3.0

0.4 -

- 0.4 0.6 1.1

10.0

0.5 8.1 3.2 0. I 0.3

0.6 -

- 0.5

1 . 1 2.3

-


nal protein of milk ranging from 4 to 7 g/liter of which 3.7,0.6,0.3, and 1.4 g represent the P-lactoglobulin (P-Lg), a-lactalbumin (a-La), serum albumin (BSA), and “proteose-peptone” fractions, respectively (Marshall, 1982). In addition, it contains other proteins such as lactoferrin, immunoglobulins, ceruloplasmin, and such milk enzymes as lysozyme, lipase, and xanthine oxidase, which are present in low concentrations.

The major whey products, dry sweet whey (DSW), demineralized delactosed whey (DLMW), and whey protein concentrate (WPC), are used in several general food categories with dried whey predominantly being used in bakery, dairy, and dry mixedblends (Table 11). The recent data indicate increases in the use of dry whey and whey protein concentrate in certain food categories. Approximately 75% of total processed whey is used in the form of dry whole whey, i.e, whey condensed by evaporation and spray dried to a powder. About 2% of whole whey is demineralized by ion exchange or electrodialysis prior to spray drying and is suitable for use in infant formula and special nutritional products. About 14% of whey solids is used in the preparation of refined lactose and about 10% of total whey solids is subjected to ultrafiltration (UF) to produce WPC (Kosaric and Asher, 1985). A range of UF-derived, spray-dried WPC powders containing 35, 50, 60, and 80% protein; 46, 31, 21, and 3% lactose; and 8,6,4, and 3% ash have been produced (Kinsella, 1984a). Whey protein isolates (WPI), containing little else but protein, can be prepared by ion exchange and diafltration processes (Skudder, 1983; Zall, 1984). In addition, approximately 0.7 x lo6 kg of lactalbumin (heat-precipitated whey protein) is imported and used in the United States, mostly in pasta, bakery, soups, and cereal-based products.

The use of whey in certain products is restricted by some intrinsic components, for example, in breadmaking, the maximum amount of whey powder that can be incorporated is limited by the ash and lactose content, which can impair hydration, retard yeast growth, cause excessive browning, and impart an undesirable “salty” taste. In addition, the presence of free thiol groups and proteose-peptone or other components may reduce loaf volume. This effect may be minimized by appropriate preheat treatments of whey (75”C/30 min) prior to drying (Webb, 1970).

Whey proteins are very susceptible to denaturation. For example, during preparation of WPC, maximum care must be taken to minimize denaturation during preheating, evaporation, drying, and subsequent processing. The extent of denaturation is normally assessed by solubility loss at pH 4.5 (Morr, 1979a). Commercial whey protein concentrates may range in solubility from 24 to 93% and 65 to 100% at pH 6 and 7, respectively (Morr, 1979b). Maillard browning reactions can also result in gradual deterioration and loss of solubility.


A wide array of whey proteins, whey powders, and whey powder blends are increasingly utilized in a broad range of products (Table 111). In most of these applications, whey powders have been sold as replacements for nonfat dry milk (NDM). Vendors tend to make many claims concerning the value of using whey powders in products; however, in many applications whey powder is being used simply as a minor least- expensive “filler” ingredient, improving nutritional value by supplying essential amino acids and perhaps providing some quality attributes, e.g., enhanced crust browning of bread. The price advantage of whey powder over milk powder has contributed to its increasing use over the past de- cade, particularly in bakery applications. In most conventional uses, however, the high content of lactose and ash in whey powders limits their use at high application levels.

Upgrading and further refining of whey powder to reduce lactose and ash will become more important in order to expand markets. For the long- run utilization and marketing, whey proteins must be concentrated and processed to retain essential functional properties. For whey proteins to

TABLE 111 EXAMPLES OF TYPES OF WHEY POWDERS AND BLENDS USED IN A VARIETY OF FOODS

IN THE UNITED STATES

Type of ingredient” Typical applications

Dairy blend of WP + NDM + BM (16%

Fortifiedlmodified whey Whey + BM for flavor Modified whey Liquified whey Modified whey (20% protein, 15% ash)

protein)

Whey + NDM Fractionated cheese whey

Partially demineralized whey proteins Lactose hydrolyzed whey

Baked goods

Yeast-raised bakery products Breads; pancakes; biscuit mixes Potato-based products Beverages; processed foods Fortified snacks; flavored beverages;

puddings; frostings; bakery applications

Baked goods; beverages Whipped toppings; coffee whiteners;

soft-serve ice creams; confectioneries; cereals; baked goods, margarines

Baby foods; meat and sausage products Desserts; confectioneries; yogurts;

beverages, baked goods; baby foods; low-sodium foods; whipped toppings; ice cream mixes

“WP, Whey protein; NDM, nonfat dry milk; BM, buttermilk powder.


be fully utilized, it is necessary to understand the properties of the individual whey proteins in order to understand the structural features which contribute to their characteristic functional properties.

111. STRUCTURE OF WHEY PROTEINS

The amino acid composition, sequences, and structural characteristics of the major whey proteins, p-Lg, a-La, and BSA, have been described previously (Whitney et al., 1976; Swaisgood, 1982; Eigel et al., 1984), and some notable characteristics are listed in Tables IV and V. Whey proteins are globular proteins with a limited number of disulfide bonds (Fox and Mulvihill, 1982; Swaisgood, 1982) which confer a certain degree of structural constraint and impart stability. Compared with the caseins, whey proteins are more heat-sensitive, less calcium-sensitive, and can engage in thiol-disulfide interchanges to form oligomeric structures (McKenzie, 1971; Fox and Mulvihill, 1982; Swaisgood, 1982).

TABLE IV PHYSICOCHEMICAL CHARACTERISTICS OF WHEY PROTEINS OF BOVINE MILKo

Approximate percentage in

Protein skim milk protein Isoelectric Cystine component Molecular mass (dliter) PH groups

@-Lactoglobulin Q -Lactalbumin Serum albumin Immunoglobulin Proteose-peptones:

Component 5

Component 5

Component 8-fast

p -CN-SP (fl-105)

p -CN-SP (fl-107)

@ -CN-4P (fl-28) residual fragment

@ -CN-IP (09-107) Lysozyme Lactofemn

18,600 14,200 66,000

15.0-96.0 X lo4

1 I .500

13,000

4,100

- 18,000 76,500

7-12 2-5

0.7- 1.3 1.9-3.3

2-6

0.13-0.32 0.02-0.35

5.3 4.8 5.1 5.5-6.8

3.7 -

4.5

3.0

5.2 9.5

NIA

2 (1 -SH) 4

32 17 ( 1 -SH)

- - 0 -

0 -

0 -

0 - 3 -

19 -

“Data from Whitney ef a/. (1976); Eigel ef al. (1984); Reiter (1985); Kitchen (1985); Wang ef a/. (1984).


TABLE V AMINO ACID COMPOSITIONS OF THE MAJOR PROTEINS OF WHEY FROM BOVINE MILK'

Bovine serum

Acid p -Lactoglobulin a -Lactalbumin albumin Lysozyme Lactofenin

ASP Asn Thr Ser Glu Gln Pro GlY Ala 112 cys Val Met Ile Leu TYr Phe Trp LYS His Arg

I 1 5 8 7

16 9 8 3

14 5

10 4

10 22 4 4 2

15 2 3

9 12 7 7 8 5 2 6 3 8 6 1 8

13 4 4 4

12 3 I

41 13 34 28 59 20 28 I5 46 35 36 4

14 61 19 27 2

59 17 23

13 10 8 8

10 4 9

11 5 6 6 2

10 5 7 7 I

10 7

15

53 44 52 50 30 68 44 74 98 38 66 4

26 106 34 43 17 78 14 58

Total I62 123 58 1 I54 987

"Data from Swaisgood (1982); Brown (1977); Brew et al. (1968); Eitenmilter et nl. (1976); Wang et a/. (1984).

A. p-LACTOGLOBULIN

p-Lactoglobulin (p-Lg), the major protein of whey, is the most extensively characterized and best described of all food proteins (McKenzie, 1971; Swaisgood, 1982; Morr, 1982; Eigel et al., 1984). The amino acid sequence of p-Lg is depicted in Fig. 2 (Creamer ef al., 1983). The protein exists as a dimer in solution because of electrostatic interactions between

and Glu'" of one monomer with corresponding lysyl residues of another monomer (Creamer et al., 1983). The native conformation is sensitive to heat and pH; at temperatures below 25°C and pH values above 7.0, the protein forms octamers (Fig. 3) (Pessen et al., 1985). Native p- Lg possesses two disulfide bonds ( C y ~ ~ - C y s ' ~ and Cy~'"-Cys"~), and a free thiol group (Cys'*') which is inaccessible to solvent at or below neu-

FIG. 2. Primary sequences of bovine p-lactoglobulin B. The residues marked with an as- terisk vary from species to species with the genetic variant. The free sulfhydryl is shown as Cysl*'. [From Creamer el a/ . (1983).]

I I

I drnol. I rlor. wry

occrlorotrd rapld In cold

I I

82 - fi--@Irr?

I I

. . . 3.58nm I

I I

rrvrrtlblr 1 Ironsformallon

\ I

I I I I I I I I I I

I ' --- --- I 3.7 I 4.65 15.1 I I 7.5 1 I 9.7

2.0 3.0 4D 5.0 6.0 7.0 8.0 9.0 PH

FIG. 3. Schematic representation of changes in the structure of p-lactoglobulins as a function of pH. p. p-Lg. [Inset (molecular models)] (A) Dimer (B) Octamer, with square decahe- dral faces on top and bottom; 0, octamer bonds; x --- x , tetrad axis; circular lines indicate monomer equators and parallels perpendicular to the dimer axis; (C) Octamer, with square faces in front and back, tetrad axis perpendicular to plane of paper. [From Pessen el a / . (19851.1


tral pH (Papiz et al., 1986). At pH 7.5, a reactive carboxyl group which displays many unique chemical properties becomes exposed and signals a conformational change in the protein molecule, as observed by changes in the optical rotatory dispersion (ORD) spectrum (Tanford et al., 1959). The free thiol group normally occluded in the protein dimer also displays increased reactivity at pH values above 6.5 and appears to facilitate thiol-disulfide interchange reactions which allow the formation of new structures upon heating. Above 65”C, p-Lg undergoes a timehempera- ture-dependent denaturation which is accompanied by extensive conformational transitions (molecular expansions) that expose highly reactive nucleophilic groups in hydrophobic regions (McKenzie, 1971 ; Kella and Kinsella, 1988).

The secondary structure of p-Lg has been calculated from circular dichroism and infrared spectroscopy data and it contains approximately 15% a-helix, 51% p-sheet, 17% reverse turn, and 17% aperiodic structure (Table VI) (Creamer et al., 1983). The e-amino groups of Lys”’, Lys”*, and LysI4’ form a diagonal band which interacts ionically with the side- chain carboxyl groups of and G1ulM in the adjacent monomer to impart additional stability to an a-helical segment (Creamer et al., 1983). Recently, the three-dimensional structure of p-Lg was determined by high-resolution X-ray crystallography (2.8 A) (Papiz et al., 1986). The p- Lg molecule has an unusual protein fold composed of two slabs of anti- parallel p-sheet (Sawyer et al., 1985; Papiz et al., 1986). The core of the protein fold is an eight-stranded, cross-hatched p-barrel composed of apolar amino acid side chains lining the internal cavity. This hydrophobic core binds apolar ligands, e.g., p-Lg binds retinol (Fugate and Song, 1980) and there is a tryptophan residue associated with the binding (Papiz et al., 1986). This tryptophan at position 19 is highly conserved among different species of p-Lg (Pervaiz and Brew, 1985). The reported crystal structure of human plasma retinol-binding protein (RBP) (Newcomer et al., 1984) demonstrates a remarkable conformational homology to p-Lg, particularly with regard to the cross-hatched p-barrel region and location of an a-helical rod (Sawyer et al., 1985; Papiz et al., 1986; Monaco et al., 1987). Although p-Lg shows limited sequence homology to RBP, there are two regions which have four consecutive identical residues and both of these contain residues involved in disulfide bridging and retinol binding (Papiz et al., 1986). The conformational and chemical similarities between RBP and p-Lg have prompted speculation that the biological function of p-Lg is in vitamin A transfer from maternal milk to the neonate via specific receptors in the intestine (Papiz et al., 1986) and possibly facilitates vitamin A esterification (Ong, 1985).


TABLE VI SECONDARY STRUCTURE ASSIGNMENTS FOR SEGMENTS OF BOVINE p-

LACTOGLOBULIN B"

Gamier Most likely Possible analysis structural elements structural elements

Residues Structure Residues Structure Residues Structure

1-7

11-16

20-33

34-37 38-45 46-5 1

52-59

60-64 65-75 78-84 88-91 91-97 98-100

I0 I- 107 108-1 13 114-1 18 119-125 126- I28 129-1 43 144-149 150-1 55

p-Sheet

$-Sheet

$-Sheet - -

Random coil @-Sheet Random coil

a-Helix -

Reverse turn a-Helix p-Sheet Reverse turn $-Sheet Reverse turn p-Sheet Random coil Reverse turn p-Sheet Random coil a-Helix p-Sheet Random coil

1-6 7-10

11-16 - - -

39-45 - - -

61-64 65-76 80-85

- -

101-107 -

112-1 15 117-123

129-143 145-151

$-Sheet Reverse turn p-Sheet

17-20 -

25-28 29-34

p-S hee t -

49-52 -

54-58 Reverse turn a-Helix p-Sheet

- p-Sheet

Reverse turn P-Sheet

-

a-Helix p-Sheet

- - -

Reverse turn

Reverse turn p-Sheet

21-24

35-38

46-48 Reverse turn

53-60 p-Sheet

-

92-96 97-100

108-1 I 1 -

-

- 152-155 156-160 152-162

Reverse turn

Random coil

a-Helix or

p-Sheet Reverse turn

Random coil -

- - - - -

Reverse turn and a-Helix or random coil

"After Creamer el af. (1983).

(3-Lactoglobulin exists in a t least five different genetic variants (Eigel et al., 1984). The two most common genetic variants, known as A and B, differ at positions 63 and 118, where an Asp and a Val in the A variant are substituted by a Gly and an Ala in the B variant (Braunitzer et al., 1973). Both the A and B forms undergo a variety of conformational


changes and association states that have been diagrammatically presented in Fig. 3.

Pessen and co-workers, (1985) investigated the slow, irreversible denaturation and rapid dimer e octamer equilibrium of p-Lg, both occurring when the protein is subjected to temperatures below 2°C. The 36,700-Da dimer is the kinetic unit persisting over the pH range 3 to 7; between pH 3.7 and 5.1, self-association of the p-Lg dimer to octamer occurs as the temperature is decreased to 2°C (Timasheff and Townend, 1964; Kumosi- nski and Timasheff, 1966). It is postulated that changes in molecular water surrounding the protein enhance hydrophobic interactions, thus promoting protein association. Above pH 6.5 the dimer dissociates because of excessive electrostatic repulsion (Brown and Farrell, 1978). The carboxyl group of AspM is believed to be involved in the dimer e octamer transition in p-Lg A since the transition does not proceed as readily in the B variant, where a glycine residue is substituted for the aspartyl residue (Kumosinski and Timasheff, 1966; Townend et al., 1969; Swaisgood, 1982).

B. a-LACTALBUMIN

a-Lactalbumin (a-La) accounts for 25% of whey protein. The ratio of a-La to p-Lg in bovine milk is approximately 1 : 3. a-Lactalbumin is a compact globular protein (14,Ooo Da) and possesses an excellent essential amino acid profile, being rich in lysine, leucine, threonine, tryptophan, and cystine (Table VI). It has four disulfide bonds linking residues 6 to 220, 28 to 111, 61 to 77, and 73 to 91 (Fig. 4). The biological function of a-La is to modulate the substrate specificity of galactosyltransferase in the lactose synthetase complex, which is responsible for the synthesis of lactose in lactating mammary tissue (Hill and Brew, 1975). The association of a-La to the lactose synthetase complex catalyzes the addition of glucose to galactose in the Golgi apparatus (Jones, 1978).

a-Lactalbumin avidly binds calcium, which may stabilize the molecule against irreversible thermal denaturation (Hiraoka and Sugai, 1984). The protein also contains a distinct binding site for a zinc ion (Murakami and Berliner, 1983). Studies of the mode of binding of these metal ions to a- La suggest that a balance between calcium and zinc concentrations in the Golgi lumen controls the protein conformation and the pattern of ion binding affects the catalytic properties ( Vmx) of the lactose synthetase complex (Musci and Berliner, 1985). Removal of bound Ca2+ by acid treatment reduces the transition temperature and facilitates the irreversible thermal denaturation and aggregation of the protein (Bernal and Jelen, 1984). Calcium binding may also be important in controlling the


FIG. 4. Primary sequence of bovine a-lactalbumin, including the four disulfide bridges (labeled with Roman numerals). [From Vanaman et al. (1970).]

release of a-La from the Golgi membrane, an essential step in inducing lactation (Musci and Berliner, 1985).

The high resolution (1.7 A) X-ray structure of a-La reveals a Ca2+- binding fold which superficially resembles the EF-hand of other calcium- modulated proteins (Stuart et al., 1986). a-La displays a clear homology


in sequence and backbone conformation with hen egg-white lysozyme (L). The “parent” protein, c-type lysozyme, has a corresponding loop which binds three water molecules in place of the Ca“ ligand (Stuart et al., 1986). The significant structural homology shared between a-La and lysozyme is particularly interesting since both proteins apparently have a common ancestral origin but have evolved quite different functions (Shewale et al., 1984). Not only are the four disulfide bonds of a-La and lysozyme in similar locations but the discovery of a hydrophobic “box” region in a-La (Richardson and Brew, 1980; Sinha and Brew, 1981) that is homologous (both in types of amino acid residues involved and spatial orientation) to a similar hydrophobic region associated with the monosaccharide binding subsites D and E in lysozyme has prompted speculation on the biochemical significance of this region (Brew et al., 1968; Richard- son and Brew, 1980). It has been proposed that a-La interacts with galactosyltransferase at the hydrophobic box region, essentially forming a stronger, hybrid monosaccharide binding site and creating the lactose synthetase complex (Powell and Brew, 1976; Shewale er al., 1984).

In many previous studies on Ca” binding of a-La, it has been empha- sized that removal of the bound Ca” results in a pronounced conformational rearrangement (Kronman et al., 1981; Murakami et al., 1982). However, a recent study of Ca2’ binding by a-La using proton nuclear magnetic resonance spectroscopy suggests that the global structures of the folded apo-form and holo-form of a-La are essentially the same (Ku- wajima et al., 1986).

Thermal unfolding of bovine a-La at pH 8.0 has been monitored by circular dichroism (CD) and proton nuclear magnetic resonance (‘H NMR) spectroscopy (Hiraoka and Sugai, 1984, 1985; Kuwajima et al., 1986). The apo-form of a-La follows a three-state mechanism of unfolding, involving a stable intermediate which retains native secondary structure but no characteristic tertiary structure, while the native form of the protein follows the two-state unfolding mechanism similar to native lysozyme (Tanford, 1970; Ikeguchi et al., 1986a,b). Calcium binding stabilizes apo-a-La since the equilibrium intermediate state is not observed by CD, ‘H NMR, or calorimetric techniques during thermal unfolding of the protein molecule (Hiraoka and Sugai, 1985; Ikeguchi et d., 1986a). Thermo- dynamic characteristics of the thermal unfolding between bovine apo- and holo-a-La have been compared to evaluate the role of bound Caz+ in stabilizing the native protein. The heat capacity of the apoprotein is significantly lower than the Ca2+-bound protein and the enthalpy of unfolding is greater for the apoprotein than for the holoprotein (Hiraoka and Sugai, 1984). Apo-a-La assumes a native-like structure at low temperatures


(10°C) as suggested by the similarity in the CD spectrum to that of the native holoprotein, which has one Ca2+ bound per protein molecule (Hir- aoka and Sugai, 1985). These results would be expected if the intrinsic stability of the native form of the protein is attributable to calcium binding and the fact that lysozyme does not bind any calcium (Imoto et al., 1981).

C. BOVINE SERUM ALBUMIN

The bovine serum albumin (BSA) component of whey is a large globular protein (66,OOO Da) with a good essential amino acid profile (Table V). BSA has been extensively characterized and its chemical and physical properties are well known (Peters, 1975). The protein consists of a single polypeptide chain containing about 580 amino acid residues with 17 intra- chain disulfide bonds and one free thiol group at residue 34. Although the precise three-dimensional crystal structure of BSA is not known, the distribution of disulfide bonds and the location of specific residues throughout the polypeptide chain suggest that the albumin molecule folds to form three structural domains and nine subdomains (Brown, 1977). The multidomain structure of BSA is responsible for the anomalous behavior of the protein under denaturing conditions (Pace, 1975). The domains of BSA are dissimilar in hydrophobicity, net charge, and ligand binding sites and each has a distinct function apart from the others (Peters and Reed, 1977).

BSA avidly binds free fatty acids, other lipids, and flavors which can stabilize the molecule somewhat against thermal denaturation (Damo- daran and Kinsella, 1980, 1981a). Isolated BSA is a versatile, highly functional protein (Waniska et al., 1981). There is some evidence that between 40" and 50"C, BSA partially unfolds, exposing apolar residues to the molecular surface, facilitating reversible protein-protein interactions.

D. IMMUNOGLOBULINS

Four of the five classes of immunoglobulins in serum, i.e., IgG, IgA, IgM, and IgE occur in bovine milk in small amounts (gramdliter), although the concentration in colostrum is high (Whitney et al., 1976; Eigel et al., 1984). IgG is the principal type in bovine milk and comprises about 80% of the total content of immunoglobulins. All classes of immunoglobulins exist as either polymers or monomers of a basic unit composed of four polypeptide chains linked covalently by disulfide bridges. The monomeric form consists of two identical light (approximately 20,000 Da) and two identical heavy (50,000 to 70,000 Da, depending on class) polypeptide

358 J . E. KINSELLA AND D. M. WHITEHEAD

chains. Each of the light and heavy chains contains constant (C-terminal half) and variable (N-terminal half) regions of amino acid residues responsible for various functions, e.g., membrane transport, and antigen binding (Gally, 1973). These proteins are very thermolabile and their functional properties in the whey protein fraction have not been determined.

E. PROTEOSE-PEPTONES

The proteose-peptone (PP) fraction of bovine milk has been characterized as a heterogeneous mixture of heat-stable, acid-soluble (pH 4.6) polypeptides precipitated by 12% trichloroacetic acid (TCA) (Whitney et al., 1976). The PP fraction is actually a heterogeneous group of phospho- glycoproteins formed following proteolysis of the N-terminal region in the sequence of p-casein by plasmin (Eigel, 1981). Andrews (1978) demonstrated that proteins previously called proteose-peptone components 5 and 8-fast are identical with p-casein residues 1-105, 1-107, and 1-28, respectively. The remaining fragments of p-casein (residues 2-105 and 29- 107) released during plasmin degradation correspond to PP component 8 (Eigel and Keenan, 1979; Eigel, 1981). It has been recommended that fragments resulting from proteolytic cleavage be named as derivatives of the parent polypeptide from which they originated (Eigel et al., 1984).

Whey preparations contain highly variable amounts (from 2-20 giliter) of PP. These molecules are amphiphilic because of their charged phosphate groups and sequences of hydrophobic amino acid residues. The amount present may have an affect on the functional behavior of whey proteins, and more research is required to monitor the amounts and types present and their effects on specific functions, e.g., whipping and baking. There is some question as to whether this fraction is responsible for depression of egg white foams (Volpe and Zabik, 1975; Phillips et al., 1987). It is possible that the PP fraction may also contain other milk peptides or proteins, lipoproteins, and proteolipids that impart foam depressant activity.

F. LACTOFERRIN

Lactoferrin is present in bovine milk at 0.02 to 0.35 g/liter, while there are considerably larger quantities (approximately 2-5 giliter) in human milk (Table V). Lactoferrin is a single glycoprotein with an approximate molecular mass of 80,000 possessing two attached carbohydrate groups (Reiter, 1985). Lactoferrin is an iron-binding protein, containing two ferric ion (Fe3') binding sites per protein (Aisen, 1980; MetzBoutique et al., 1984). Limited proteolysis, amino acid sequence, and metal-binding


analyses have demonstrated that the polypeptide chain may be subdi- vided into two major domains, each of which has a nonidentical iron binding site, one glycan moiety, and a high degree of sequence homology to each other (Mazurier and Spik, 1980; MetzBoutique et af., 1984). Amino acid analysis reveals an unusually high half-cystine content (Table VI) (Wang et af., 1984).

Lactoferrin is structurally and functionally homologous with two other nonheme, iron-binding proteins, serum transfertin and ovotransferrin (Aisen, 1980). These proteins reversibly bind two iron atoms per molecule with the synergistic binding of two bicarbonate or carbonate anions (Mas- son and Heremans, 1966). Because of their ability to complex free iron, all three apotransfemns exert a bacteriostatic effect in v i m (Oram and Reiter, 1968; Parry and Brown, 1974; Reiter, 1985). They may function as iron transport proteins, and lactoferrins may also possess a mitogenic function and stimulate the development of intestinal mucosa in the neonate.

G. LYSOZYME

Bovine milk contains between 13 and 32 pg lysozyme/100 ml, depending on the stage of lactation, while human milk contains 10 mg/100 ml (Kitchen, 1985; Reiter, 1985). The enzyme is generally considered to be synthesized in the mammary gland (Reiter, 1985). Lysozyme isolated from bovine milk is apparently different both in molecular mass and amino acid composition than lysozymes from human milk or egg white sources (Tables IV and V ) (Eitenmiller et af., 1976; Kitchen, 1985). It has an isoelectric point of pH 9.5 and the pH of optimum enzyme activity is at 7.9, which is higher than that found for lysozymes from other sources. The enzyme is stable at low pH values (Eitenmiller et af., 1976). Reduc- tion of the enzyme from bovine milk with mercaptoethanol followed by reoxidation resulted in a 3-fold increase in the specific activity. Reforma- tion of disulfide bonds different from the original native structure, or an altered tertiary structure arising from new amino acid interactions are likely explanations for this effect (Friend et al., 1972). Lysozyme is considered to be an important component of the antibacterial system of milk, possibly affecting the general immune system as well (Reiter and Oram, 1982; Reiter, 1985). Its bactericidal and lytic action depends not only on the concentration of enzyme and the prevailing ionic strength but also on the nature of the anions; SCN- and HCO; promote bacterial cell wall lysis more effectively than C1- or F- (Goodman et af., 1981; Tortosa et al., 1981). There is a renewed interest in the bactericidal effect of lysozyme for applications in cheese-making, infant formula, and pharmaceuti- cal products (Reiter, 1985).


IV. WHEY PROTEIN PREPARATION AND ISOLATION

Knowledge of protein structure and the relationship to functional properties (combined with an awareness of the various factors affecting functional properties) and recognition of the critical functional requirements in different products are important in developing practical methods for preparing functional ingredients from whey. The methods employed in the isolation and fractionation of whey protein preparations should minimize denaturation and avoid some of the undesirable consequences discussed above. As a functional ingredient per se, whey powder contains too little protein for most uses requiring a protein-rich ingredient; hence a number of approaches have been used to recover or enrich the protein content of whey. The principle methods currently used for fractionation of whey are summarized in Table VII. The details of these methods have been reviewed (Marshall, 1982; Matthews, 1984; Pepper and Pain, 1987).

In the conventional process, whey is centrifuged to recover fat, condensed by evaporation to increase solids content, then spray dried or used for lactose crystallization and, after desludging, the residual liquor is dried. This yields whey powder containing about 11-14% protein which may be variably denatured, particularly during the evaporatiodcondensa- tion step.

Several methods have been used for concentration of the proteins from whey. Lactalbumin, a denatured, protein-rich product, is made by heating whey liquor at pH 5-6, 85" to 100°C (steam injection) for 15-20 min, acidification to pH 4.5, recovering the precipitated protein by centrifuga-

TABLE VII SUMMARY OF CONVENTIONAL METHODS USED FOR PREPARATION OF

VARIOUS WHEY PRODUCTS FROM RAW WHEY

Method

Centrifugation Evaporation and crystallization Steam injection (90°C, 20 min) Electrodial ysis Reverse osmosis Ultrafiltration Gel filtration Diafltration Ion-exchange chromatography

~~

Whey product"

Fat Lactose Lactalbumin Whey powder (low ash) Whey powder (up to 24% protein) WPC (30-50% protein) WPC WPI (5690% protein) WPI

"WPC, Whey protein concentrate; WPI, whey protein isolate.

PROTEINS IN WHEY 36 1

tion, and either spray- or drum-drying to give a product with 85,4,1.5, and 3% protein, lactose, ash, and lipid, respectively (Marshall, 1982; Mat- thews, 1984). This product is low in lactose and ash and the proteins are a rich source of essential amino acids, e.g., lysine, methionine, cystine. Lactalbumin is relatively insoluble but has useful water sorption characteristics (Kinsella and Fox, 1986). Because of its nutritive value, it is frequently added to cereals, baked goods, confectionery foods, and snack foods. There may be some merit in examining the possibility of improving the solubility and functional attributes of lactalbumin by enzymatic and/ or chemical modification.

A number of chromatographic methods have been used to prepare concentrated whey proteins (Morr, 1979b; Marshall, 1982; Matthews, 1984). Gel filtration on Sephadex proved to be effective but impractical because of column plugging, effluent problems, etc. (Melachouris, 1984). Sphero- sil ion-exchange chromatography (microporous silica beads with high exchange capacity, good flow rates, and low compressibility) has been used (Skudder, 1983; dewit, 1984) to prepare a protein isolate (90% protein) and to prepare whey protein concentrate (66% protein) (Nichols and Morr, 1985). Both protein preparations have good solubility. However, the problem of concentrating the protein eluate by UF, the cost of materials, and volume of solvents required render this method impractical. Ion- exchange chromatography is being used to prepare whey protein isolates (>90% protein) with excellent functional properties (Houldsworth, 1979; Marshall, 1982; Matthews, 1984).

With the advances in membrane and separation technologies, ultrafiltration has emerged as the principle method for increasing the protein concentration of whey (Matthews, 1984; Modler, 1985). The whey is clari- fied at 50°C, subjected to UF (and perhaps diafiltration, depending on the final protein concentration desired) and then spray dried ( 175"-2OO0C in- let, 8O"-9O0C outlet). This method can provide preparations with protein concentrations of 30 to 90% and denaturation is minimal when heat treatments are carefully controlled. The use of reverse osmosis (RO) rather than evaporation to concentrate protein solutions reduces protein denaturation and combined RO-UF is a feasible method for preparing undenatured proteins with good functional properties (Pepper and Pain, 1987). This technology, with appropriate control, may be successfully used by whey processors in generating products with consistent functional properties that will compete more effectively with other functional protein ingredients.

The differences in heat sensitivities of whey proteins can be exploited, e.g., the thermal separation of the major whey proteins a-La and p-Lg has been accomplished by choosing conditions for maximum precipitation of


a-La. Thus heat treatment of whey at 65"C, in the pH range 4.1 to 4.3 for 10 to 20 min should selectively precipitate a-La while the supernatant is enriched in p-Lg and the aggregated a-La may then be resolubilized above pH 5.0 (Table VIII) (Pearce, 1983). This method may provide a practical approach for preparing concentrated a-La or p-Lg with low levels of contamination from lactose or ash.

The addition of ferric chloride (4 mM) to whey at pH 3.0 precipitated all the proteins except p-Lg (Kuwata et al., 1985). The ferric chloride was removed from the resolubilized proteins by ion-exchange chromatography. The fraction containing p-Lg was recovered by heat denaturation (92*C, 15 min, pH 3.0) and isoelectric precipitation and was soluble at pH 6.8. When acid whey was treated with ferric chloride (7.5 mM, pH 4.3, 4"C), 90% of the p-Lg was precipitated with BSA while 70% of the immunoglobulins and 95% of the a-La remained in solution and could be recovered following precipitation of iron at pH 8-9 at 4°C (Kuwata et al., 1985).

Following neutralization of cottage cheese whey, the addition of Mg2+ or Zn2+ salts (at 4 and 22 g/dl, respectively) resulted in the precipitation of 20 to 35% whey protein nitrogen at pH 6.7 and 10.5, respectively (Cer- bulis and Farrell, 1986). Precipitation of whey protein nitrogen by zinc acetate is nearly complete and is highly dependent on pH, i.e., maximal precipitation occurred near neutral pH. However, by adding magnesium acetate and calcium hydroxide (4 g/dl) at pH 10.5, over 90% of the total whey nitrogen could be precipitated (Cerbulis and Farrell, 1986).

A large-scale process for the separation of whey protein concentrates has recently been described in which the whey is cooled to 2°C at pH

TABLE VIII SELECTIVE AGGREGATION OF a-LACTALBUMIN FOLLOWING HEATING AT DIFFERENT

PHS AT 55°C FOR 10 mill"

Turbidity

a-Lactalbumin P-Lactoglobulin pH of heating

at 55OC I mg/d 2 mdml 2 mg/ml 4 mg/ml

3.0 3.5 4.0 4.5 5.0

0.01 0.02 0.01 0.01 0.01 0.09 0.01 0.04 0.02 - 1.10 0.05 0.08 0.06 0.80 0.05 0.09 0.03 0.04 0.00 0.06

"Data after Pearce (1983).


7.3 and the calcium content adjusted to 1.2 &kg (Maubois et a / . , 1987). Subsequently, the whey is rapidly heated to 50°C for 8 min to precipitate the lipid fraction, which is then removed by microfiltration. In order to separate individual proteins, the pH of the whey is adjusted to 3.8 and heated to 55°C for 30 min, which causes aggregation of a-La, while p-Lg remains in the supernatant (Maubois et al., 1987).

Rapid, reliable methods are needed to quantify the various protein components of whey and to differentiate between protein and nonprotein fractions. Proteins can be fractionated by gel chromatography, electro- phoresis, ion-exchange chromatography, and, more recently, it has been shown that HPLC may provide a rapid method for separating and quanti- fying components (dewit, 1984; Brooks and Morr, 1984; Morr, 1984; Nichols and Morr, 1985). Methods for quantitative analysis of the extent of protein denaturation and its correlation to functional properties are required. Methods to assess denaturation may include physical measurements of aggregation, electrophoretic analysis, gel permea- tion chromatography, calorimetric analysis, and immunological assays (Aschaffenburg and Drewry, 1957; Wyeth, 1972; Ruegg ef al., 1977; Harper, 1984; deWit, 1984; Harper and Zadow, 1984). Because these methods are based on different physical or chemical properties of the protein, difficulties are encountered in ascertaining whether measurements of the extent of denaturation are comparable under differing experimental conditions.

TABLE IX COMPOSITION OF SOME WHEY PRODUCTS PREPARED BY DIFFERENT METHODS‘

Percentage of solids Extent of denaturation

Methodh Protein Lactose Ash Fat (%)

WPC (35%) 35.0 WPC (70%) 76.0 Lactalbumin 86.0

Ultrafiltered 37.0 UF + diafiltered 83.0

Spherosil 66.0

Ion exchange 90.0 Whey powder 12.0 Demineralized powder 13.0

40-60 2-20 2-4 8.0 3.0 8.0 3.5 1.5 3.6 2.0 20.0 8.0

51.0 7.0 3.0 5.0 2.8 7.0 3.3 2.0 1 .o

74.0 9.0 1 .o 81.0 0.8 1 .o

? ?

95 10 16 22 21 ? ?

“Data after Marshall (1982); deWit (1984); Morr (1985); Nichols and Mom (1985). bWPC Whey protein concentrate; UF, ultrafiltrate.


Methods which involve the determination of protein by nitrogen content or the Lowry method may give falsely high values because of the presence of other polypeptides and PP components. Wide discrepancies in values determined by the traditional methods were observed by Harper, (1984) and the data in Table IX illustrate the variability in the composition of whey products prepared by different methods (Marshall, 1982; dewit, 1984; Morr, 1985; Nichols and Morr, 1985).

V. FUNCTIONAL PROPERTIES OF WHEY PROTEINS

The possession of a range of functional properties is as important as cost in selecting proteins for use in specific products. Functional properties of proteins are those physicochemical properties which govern the performance and behavior of proteins in food systems during their preparation, processing, storage, and consumption, i.e., properties affecting the final quality attributes of foods. Examples of the structural and functional roles of proteins include the caseins in cheese curd; the myofibrillar proteins which impart structure, water holding, juiciness, and texture to meat; gluten proteins in leavened bread; egg white proteins in whipped toppings, etc. (Kinsella, 1976, 1982, 1984b).

The functional properties of food protein preparations reflect the manner in which the proteins interact with each other and other components in the system as determined by conditions of processing, storage, and perhaps by food preparation methods. Different proteins vary in their composition and properties and demonstrate different functional behavior, for example, caseins have good emulsifying properties but do not form gels whereas whey proteins have limited emulsifying properties but possess good gelling properties.

Traditionally, the food industry has relied almost exclusively on abundant commodity proteins, e.g., milk proteins, gluten, gelatin. However, food processors are increasingly selecting protein ingredients for their specific functional properties and performance in particular food products (Kinsella, 1984b, 1985). Thus, as the functional requirements of protein ingredients are defined and the functional behavior of various proteins in food systems is described, other protein preparations may be developed to compete with the traditional proteins. Food manufacturers are increasingly designing new products and then seeking functional ingredients to meet the specifcations for that particular product rather than allowing the type of product to be limited by the properties of traditional protein preparations. In view of these developments, the producers of whey proteins now recognize that the requisite functional properties in the final preparation must meet a set of decisive criteria. It is no longer sufficient


to locate a market after a by-product becomes available. Rather, the ingredient manufacturer must anticipate and recognize the specific needs of the food manufacturer and adopt and optimize manufacturing procedures for producing an array of ingredients with the requisite functional properties.

Different food applications each require a different set of functional properties. For example, in a beverage, solubility, storage stability, flavor compatibility, controlled viscosity, and turbidity may be required in a range of different pH values. In reformed meat systems, a range of functional properties that change in a desirable manner with processing and cooking, e.g., in sausage-type products, emulsion stabilization, subsequent gelation, good adhesive properties, and water holding are important. In a dough system, the protein should not absorb excessive water, but should align with gluten fibrils while not disrupting the gluten network, nor weakening the viscoelastic properties, nor reducing loaf volume. Thus, each application requires specific functional attributes to obtain the desired performance in each system (Table X).

In bakery applications, whey powders should readily hydrate and contain a minimum of PP and free thiol groups (which may exert a loaf depressant effect), not impair fermentation (low ash level), facilitate aeratiodleavening, and contribute flavor and color. In cake products, particularly in batter-based products, emulsifying activity and aeration capacity are necessary, and reasonable heat stability of the aerated matrix is also required. In regard to bakery uses, there is a tendency to promote whey powders as replacements for NDM; however, this may be incorrect because, in certain high-fat batters the presence of caseins, which impart emulsifying properties, is required for optimum performance (dewit, 1984). Interfacial film formation and foam stabilization during baking/ expansion are also criteria for functional ingredients in many air-leavened high-ratio cake products and meringues. Egg white proteins perform these functions optimally, whereas whey proteins lack suitable water- holding capacity and heat stability (devilbiss er al., 1974), despite claims that they are a suitable substitute for egg white.

Much has been reported about the excellent functional properties of whey proteins based on laboratory research. However, in some cases, their functional properties have been overrated and overpromoted for applications in which these whey preparations are unsuitable.

A. INTRINSIC FACTORS AND FUNCTIONAL PROPERTIES

Functional properties reflect the intrinsic physicochemical properties of the proteins, i.e., amino acid composition and disposition of amino acid residues, conformation, molecular size, shape, “flexibility,” net charge,

TABLE X FUNCTIONAL PROPERTIES REQUIRED OF PROTEIN INGREDIENTS IN DIFFERENT FOOD PRODUCTS

~ ~~

Functional Frozen Whipped Processed Coffee properties dessert Confectionery Bakery toppings meats whiteners

Solubility + Gelation - Emulsifying + Foaming + Adhesion - Flavor binding -

+ + + +

+ (+) - + +

+ -

+ ?

-

?


molecular hydrophobicity, substituent chemical groups (esterified phosphate, carbohydrate) and sulfhydryl groups (Table XII). Thus, knowledge of the relationship between intrinsic and extrinsic (temperature, pH, ion concentration, etc.) factors is critically important in elucidating and controlling functional behavior in different applications and in modifying proteins and/or processing conditions to optimize desirable functions (Table XI) (Kinsella, 1982, 1984a).

The amino acid composition determines the folding behavior and reactivity of proteins. Proteins with a high content of polar and/or charged amino acids, which tend to be exposed to the aqueous phase, bind more water and are useful as emulsion stabilizers (Table XIII). Proteins with a high content of apolar amino acid residues (greater than 30% of total amino acids) display good surface activity (Kato and Nakai, 1980), but generally do not possess good gelling properties because of their predis- position toward extensive self-association and coagulation (McKenzie, 1971; Fox and Mulvihill, 1982; Swaisgood, 1982; Morr, 1985). The presence of cysteine and cystine residues greatly affects heat-induced polypeptide association via thiol-disulfide interchange reactions and subsequent precipitation, e.g., p-Lg (Sawyer, 1968). However, it is the disposition of certain amino acids along the polypeptide chain, rather than their total content, which is more critical in governing protein conformation and hence functional properties.

The native structure of the globular proteins in whey represents a thermodynamic equilibrium, and protein conformation may fluctuate depend-

TABLE XI FACTORS INFLUENCING THE FUNCTIONAL BEHAVIOR OF PROTEINS IN FOOD

~ ~~

Process; treatments; Intrinsic features Extrinsic factors conditions

Composition of protein Composition of protein Monomeric or oligomeric Protein blends Rigidityhlexibilit y Hydrophobicit ylhydrohilicity Surface charge Bound flavor ligands

Temperature PH O/R status Salts, ions Water Carbohydrates Lipids Gums Surfactants Tannins

Heating Acidification Counterions Ionic strength Reducing conditions Drying Storage conditions Modification

Physical Chemical Enzymatic Genetic


TABLE XI1 INTRINSIC FACTORS THAT AFFECT PROTEIN STRUCTURE-

FUNCTION RELATIONSHIPS IN FOOD SYSTEMS

Amino acid composition Amino acid sequence (disposition of amino acid side groups) Secondary and tertiary structure (conformational energy) Size, shape (topography) Net surface charge, effective hydrophobicity Intramolecular stabilizing forces (ionic and hydrophobic interactions) Quaternary structures Secondary interactions (intra- and interpeptide) Substituent groups (phosphoryl and carbohydrate groups) Bound and/or prosthetic groups (iron, calcium, lipids) Disulfidelsulfhydryl content

ing on environmental conditions (Creighton, 1985; Karplus and McCam- mon, 1986). The noncovalent forces involved in stabilizing native protein structure include hydrogen bonding, hydrophobic, van der Waals’, and electrostatic interactions, while covalent disulfide bonding is important in maintaining the structural integrity of extracellular proteins (Schulz and

TABLE XI11 EXTRINSIC FACTORS WHICH CAN AFFECT COMPOSITION AND FUNCTIONAL BEHAVIOR

OF WHEY PROTEIN PREPARATION

Preparation Variables”

Milk composition

Cheese manufacturing

Whey handling

Whey concentration Dried whey Heat treatments Fractionation methods Distribution and content

of components Physical state of

components

Stage of lactation, sanitation, storage time and conditions, somatic cell count, proteolysis by milk enzymes

Sweet/acid whey; rennet type; calcium addition, fat separatiordwhey recovery

Storage time, temperature, sanitation, clarification efficiency, residual enzymes

Evaporation, ultrafiltration, dehydration conditions Storage, moisture content, chemicaVphysica1 changes Time, temperature, pH, calcium content Heat precipitation, UF/RO, chromatographic method Lipid, ash, lactose, proteins

Native/denatured, solubility, bound lipids

“UF, Ultrafiltration; RO, reverse osmosis.


Schirmer, 1979). Alterations in environmental factors which impact on these forces can alter the conformation of proteins: salt may weaken ionic interactions (Schulz and Schirmer, 1979); cleavage of intramolecular S-S bonds by thiol groups can facilitate protein unfolding and protein-protein interactions, resulting in coagulation or gelation (Schmidt, 198 1); acidic conditions can affect calcium binding (Bernal and Jelen, 1984); calcium binding may stabilize a particular conformation (Hiraoka and Sugai, 1984; Stuart et al., 1986); temperature treatments greatly affect protein conformation, as in the case of dissociation of p-Lg and exposure of the reactive thiol group of p-Lg upon heating (Watanabe and Kloster- meyer, 1976).

Whey proteins become denatured at temperatures above 65" to 70°C and may coagulate after heat treatment (Patocka et al., 1987). The nature, extent, and rate of denaturation can be influenced by a number of factors such as pH, ionic strength, protein concentration, time, and temperature of heating (Kilara and Sharkasi, 1986). The thermal denaturation of whey proteins is pH sensitive and the isoelectric pH of approximately 4.6 is used to recover heat-denatured whey proteins. The pH affects the rate of denaturation and coagulation by affecting the net charge of the proteins (Harwalkar, 1986). Thus, minimal coagulation occurs when whey proteins are heat-denatured above pH 6.5 (dewit, 1981) or below the critical pH range of 3.7 to 3.9 (Bernal and Jelen, 1985). The presence of calcium enhances protein aggregation following heating at particular pHs and this is attributed to neutralization of electrostatic repulsions (dewit, 1981 ; de- Wit and Klarenbeek, 1984; Bernal and Jelen, 1984; Patocka et al., 1987).

Thus, the net effects of noncovalent forces and interactions play major roles in the functional behavior of whey proteins. For example, in gelation, a balance of attractive (hydrophobic and electrostatic interactions and hydrogen bonding) and repulsive (electrostatic) forces is necessary in discrete regions of interacting molecules for adequate network formation (Schmidt, 1981 ; Mulvihill and Kinsella, 1987, 1988). Surface hydrophobicity is important in flavor binding and effective hydrophobicity greatly affects film formation, which is required for foaming and emulsifying properties (Kinsella, 1981; Kato and Nakai, 1980; Nakai, 1983).

B. EXTRINSIC FACTORS

The functional properties manifested collectively by whey proteins are largely determined by a number of extrinsic factors: methods of preparation, isolation, drying, storage; extent of refining and purification, content and concentration of proteins, and environmental conditions; tempera-


ture, pH, and solute concentration (Table XIII). For example, the method and conditions of drying can affect the extent of denaturation, particle size, rehydration, and dispersibility (Kinsella, 1984b). The efficiency of lipid removal may affect many properties related to surface phenomena (foaming, binding of flavor compounds, etc.) and sensory qualities of whey proteins.

C. VARIABILITY IN WHEY PROTEIN PREPARATIONS

Variability in whey composition reflects the different sources, e.g., total solids may range from 5.8 to 7.0% in sweet and acid whey, and minerals like calcium and phosphate may range from 0.5 to 1.4% and 0.6 to 0.8% in different sweet and acid whey samples, respectively (Marshall, 1982). Generally, minerals are much higher in acid whey and differences in mineral content can markedly affect the functional performance of whey proteins; calcium can render proteins more susceptible to thermal precipitation, thereby affecting gelation, water sorption, viscosity, etc. (Melachouris, 1984). The composition of WPC can range from 30 to 95% protein, 1 to 80% lactose, 1 to 18% ash, and 1 to 9% fat (Marshall, 1982). Typical ranges of protein concentrations in commercial WPCs are 29 to 60% (Morr, 1979a).

The protein content of cheese whey can vary in amounts of total solids in sweet (cheddar) and acid (cottage) whey by 11-15% and 10-15%, respectively. This can have a marked influence on functional behavior, especially of whey protein concentrates and isolates. The variability in composition is a challenge in producing whey with consistent functional properties. The protein distribution markedly affects the functional properties of a particular preparation; thus, greater amounts of p-Lg may increase sensitivity to heat precipitation and relative amounts of PP may adversely affect whipping and bakery uses (Jelen, 1973; Kinsella, 1984b; Phillips et al., 1987). In addition, whey fractions may contain sizable amounts of p-casein which dissociates from the micelle, particularly during cold storage of milk prior to cheese making, affecting the foaming and emulsifying properties of whey proteins.

The concentration of whey proteins in milk and hence in whey varies with season and stage of lactation (Morr, 1982). Heat treatment of milk, e.g., pasteurization, ultrafiltration, and thermalization, may result in the enhanced association of p-Lg and K-casein, thus reducing the protein concentration in whey (Kilara and Sharkasi, 1986).

The efficiency of cheese-making and whey separation influences the amount of protein in whey, which can range from 8 to 15% of total solids


(Melachouris, 1984). Since proteins are the major functional components, this variability must be reduced in order to produce the consistent functional ingredients required by the food industry. Ultrafltration (UF) and diafiltration (DF) are techniques presently used to routinely manipulate the protein content of whey in a consistent manner (Matthews, 1984).

High microbial counts (especially of psychotropic bacteria), somatic cells, and plasmin activity can all cause significant proteolysis in stored milk and thus affect whey composition. The content of PP is much higher in milk containing high numbers of somatic cells or plasmin activity (Schmidt et al., 1984).

The presence of lipid materials, which can range from 0.4 to 1.0% of total solids (Melachouris, 1984), exerts a deleterious effect on most of the desirable functional properties, e.g., solubility and whipping of whey protein prepartions. Lipids also undergo extensive oxidation during storage, causing off-flavor development in most whey preparations. In addition, lipids cause problems in the fractionation and refining of whey proteins, especially by chromatographic and U F methods (Melachouris, 1984). Thus, removal of lipid by efficient centrifugation, clarification, or complexation techniques is important to ensure elimination of unpredict- able and deleterious components. Polyphosphate treatment at pH 5.1 prior to clarification at pH 7.0 improves lipid removal (Grindstaff, 1977).

The state of the component proteins in the preparation, i.e., native/ denatured, soluble/insoluble, is perhaps the major factor determining the functional behavior of whey proteins. Denaturation, mostly from thermal processes, is the principal factor limiting the application of whey proteins and represents the major challenge for the preparation of whey concentrates and isolates with consistent and reliable functional properties.

The extent of protein denaturation significantly affects the utilization of whey proteins as functional ingredients (Melachouris, 1984). Denatured proteins have limited solubility, a primary prerequisite for most other functional uses. Thus, denaturing heat treatments have the greatest deleterious effect on functional properties of whey protein preparations. Even moderate heat treatments (45" to 60°C) may cause unfolding and partial denaturation of p-Lg, resulting in protein aggregation and loss of solubility. There is a close relationship between heat-induced alterations and denaturation temperatures of p-Lg and whey proteins. Generally, the rate of heat denaturation decreases with protein concentration, particularly for p-Lg (deWit and Klarenbeek, 1984). The extent of protein denaturation in whey samples ranges from 12 to >90% and depends on the heating procedures. Melachouris reported that 40,25,20, and 10% of various whey samples from the same plant contained 20-30, 10-20, 30-40, and


40-50% denatured protein, respectively (Melachouris, 1984). Denatur- ation must be minimized for whey proteins to compete successfully in the functional ingredients market. Advances in UF combined with rigorous control of spray-drying temperatures can overcome this problem in manufacturing functional proteins. Lactose tends to reduce whey protein aggregation during heat treatment, particularly in the isoelectric pH range (Hillier and Lyster, 1979). Sucrose inhibits whey protein thermal coagulation, although the disaccharide apparently promotes a conformational change in proteins (Garrett et al., 1988). Whey proteins are more stable in the neutral pH range (pH 6-7) and calcium facilitates heat-induced aggregation (dewit, 1981).

Commercial whey preparations should consistently meet minimum standards in terms of protein content, extent of denaturation, and requisite functional properties. Manji and Kakuda (1987) recently reported the effective use of fast protein liquid chromatography (FPLC) in determining the extent of whey protein denaturation in variously heat-treated milk samples. Results from this method were compared with literature values obtained from differential scanning calorimetry (DSC), whey protein nitrogen index (WPNI), and Kjeldahl nitrogen (KN) analyses and generally showed that the WPNI method consistently yields lower percent denaturation values as compared to those obtained with either FPLC or KN. These investigators conclude that FPLC is an equivalent method to KN, and that the FPLC method may yield data showing the extent of denaturation of individual whey proteins (Manji and Kakuda, 1987).

deWit and Klarenbeek (1984) demonstrated the effect of subtle differences in composition and degrees of denaturation of the functional performance of various whey protein preparations in different food systems. For example, in a meringue system, the presence of fat in the whey preparation was detrimental to foaming; in a madeira cake, the presence of a small amount of fat seemed to improve the performance of whey protein, indicating that perhaps emulsifying activity, in addition to aeration, is important in the madeira cake. This research indicated the sensitivity of different functional applications to variations in composition, and also re- vealed the potential for making whey preparations with different functional attributes which might be ideal for specific applications. Based on knowledge of functional criteria required in an ingredient, the conditions of preparation can be manipulated to optimize those functional properties.

Data are needed to assess systematically the cumulative effects of heating during processing operations (e.g., pasteurization, evaporation, concentration, spray drying) on the extent of denaturation. Generally, conventional heat treatments are not severe enough to cause extensive


denaturation, but modest heating around 55"-60"C can cause conformational changes in p-Lg which may subsequently slowly aggregate and lose solubility during storage.

Whey proteins can undergo deterioration in functional performance during storage. The Maillard reaction results in discoloration, some polymerization, and loss of solubility. Lipid oxidation can result in cross-linking of the proteins and loss of functionality. The moisture content (water activity) must be controlled to minimize browning and appropriate pack- aging should be employed in storing whey proteins.

For consistent quality control of the various factors affecting functional properties, WPC should be routinely analyzed for protein, nonprotein nitrogen (NPN), calcium, phosphorus, lipid content, and extent of protein denaturation. The processor can then be assured of consistent composition and quality. In addition to quality control, it is desirable to develop rapid tests for assessment of functional properties of each batch of WPC. This is critical for reliable functional performance of the product in food applications. In this regard, solubility, water adsorption, heat-induced gelation, and surface active properties such as foaming and emulsifying characteristics should become standard tests.

In food applications, a range of functional properties may be required in ingredients during processing and preparation. These vary with the food in question and usually a mixture of proteins is required to provide the desired range of functional properties. Thus, water sorption, rapid solubilization, viscosity, emulsifying properties, gelation, and all functional properties involving protein-water interactions are frequently required in food products. Several aspects of the functional properties of whey proteins have been discussed and reviewed (Morr, 1979b, 1984; Harper ef al., 1980; deWit, 1984; Kinsella, 1984a; Melachouris, 1984; Mangino, 1984; Kilara, 1984; Modler, 1985). In general, whey proteins show good solubility and gelling properties plus adequate whipping properties in certain applications, but these are critically dependent on the content of undenatured protein, salts, and lactose.

VI. HYDRATION AND SOLUBILITY

Hydration, wetting, dispersibility, and dissolution are terms describing the interactions of proteins with water in different systems and are important criteria for assessing the suitability of whey protein preparations. Some of the physical characteristics (particle size, shape, state of agglomeration, porosity, etc.) and chemical properties (surface hydrophilicity, net charge, molecular hydrophobicity, adsorbed wetting agents, etc.)


which affect wettability and dispersibility have been discussed (Kinsella, 1984a). Rapid dispersibility and dissolution are required for most applications. Control of spray drying to yield particle sizes of 150-200 pm diame- ter, agglomeration, and/or use of food-approved wetting agents can facilitate wetting and dispersibility (Neff and Morris, 1968). The water sorption behavior of whey powders and proteins is important in optimizing storage conditions (Kinsella and Fox, 1986).

The water-binding capacity of pure proteins can be estimated from their amino acid composition. However, protein conformation, surface polar- ity, ionic strength, ion species, pH, and temperature all affect the water- binding capacity of proteins (Kinsella and Fox, 1986). In addition, the particle size of the protein in a food system, its porosity, molecular surface features, and interactions with other food components can affect the extent and rate of water binding and/or hydration. Such effects may su- persede the basic compositional factors in determining water binding. Whey proteins show great variability in water binding, but generally have low water-holding capacity (Melachouris, 1984).

Solubility is a prime requisite for a functional ingredient protein and is critically necessary for products such as beverages (Kinsella, 1976; Da- modaran and Kinsella, 1980). Generally, whey proteins are the nitrogen- ous fraction remaining soluble in the supernatant at pH 4.6 after precipitation of casein. Thus, the loss of solubility at this pH is commonly used to assess the extent of protein denaturation (Guy et al., 1967). Operation- ally, solubility connotes the amount of protein that goes into solution at pH 6.5-7.0 at 25°C and is not sedimented by relatively low centrifugal forces. Usually, low concentrations (<lo mg/ml) of protein are tested (Morr et al., 1985). Whey protein concentrations for commercial users show a wide range of solubilities, ranging from 5 to 100% (Melachouris, 1984). This reflects the method of manufacture and, in many cases, the lack of precise control during the drying process. Thus, methods and procedures for the preparation of functional whey ingredients must employ conditions to minimize heat denaturation and insolubility of the component proteins. Most of the current methods used in the preparation of whey proteins entail a heating process during drying. While spray-drying conditions can be controlled to minimize denaturation, many whey preparations become partially denatured at or before this stage of processing. This is the principal cause of variability in the functional properties of many whey protein preparations.

Several qnvironmental conditions affect the solubility of whey proteins: pH, solute concentration, ionic strength, ion valency, surface/shear effects and, most acutely, heat treatment. Knowledge of these and their interacting effects is critical in the isolation and processing of whey pro-


teins while avoiding denaturation. For example, in a beverage, solubility under a variety of pH, temperature, and ionic strength conditions may be required.

The pH of the dispersing medium affects the net charge of proteins. Proteins with a net negative or positive charge tend to bind more water and are more soluble than if the net charge is minimal, i.e., at the isoelectric point, protein solubility is generally low, though whey proteins may remain hydrated and soluble at their isoelectric pH under certain conditions. Ionic strength affects protein solubility according to ion species and valency; salt concentrations up to 0.1 M enhance solubility but above 0.15 M can reduce it (Damodaran and Kinsella, 1981a). Ions in solution exert their influence by affecting the net charge of proteins, hydration, and electrostatic interactions. p-Lg displays the least solubility between pH 4 and 5 but in the presence of salts, the protein is soluble in this pH range. A high content of salts in whey and whey protein preparations limits their use in many foods, and electrodialysis and reverse osmosis have been employed to demineralize condensed whey preparations. Ion exchange is used less frequently (Houldsworth, 1979; Marshall, 1982). In acid whey, removal of salts by UF/DF may result in subsequent precipitation (acid coagulation) of p-Lg and some immunoglobulins in the pH range 4-5. Thus, precipitation of p-Lg in the isoelectric range at low ionic strength may be mistaken for denatured protein in determining the soluble protein content of whey samples.

Heat treatments alter the conformation of whey proteins and often result in denaturation, loss in solubility, and aggregation. The solubility of p-Lg following heating for 15 min at 80°C in the presence of 0.01 M sodium chloride showed a typical bell-shaped solubility curved with a minimum close to the pZ of 5.28 (Townend and Gyuricsek, 1974). Increasing the sodium chloride concentration to 0.1 M caused a decrease in the amount of protein precipitated (increased solubility) by 50% and shifted the apparent isoelectric pH to 6.0. At 0.5 M sodium chloride, precipitation of the protein was inhibited completely. In contrast, calcium chloride at 0.01 M caused complete precipitation of 0-Lg at pH 5.5 and 0.7 M calcium chloride caused precipitation above pH 6. Heating in the presence of milk salts yielded a typical bell-shaped curve with a minimum between pH 5 and 7 (Townend and Gyuricsek, 1974).

In contrast, Harwalkar and Kalab (1985b) reported that p-Lg unfolded but remained in solution upon heating at around 65°C and at low ionic strength (0.2 M). At higher ionic strengths and higher protein concentrations, denaturation upon heating was accompanied by aggregation in the form of either nonsyneresing gels or syneresing precipitates.


VII. THERMAL PROPERTIES OF WHEY PROTEINS

The effects of heat are greatly influenced by pH, ionic strength, the rate of heating, and, to a lesser extent, by protein and lactose concentrations. Upon mild heating (40"C), p-Lg undergoes a slight conformational change; further heating (50" to 60°C) causes unfolding and exposure of the thiol group (McKenzie, 1971; Kella and Kinsella, 1988). If p-Lg is allowed to cool, protein association and aggregation occur via disulfide bonding and entropic forces which are enhanced in the presence of calcium (Haque et al., 1987). This phenomenon may be encountered during lactose crystallization in whey liquors.

Heat denaturation of whey proteins at very low pH (pH C3.0) imparts some unique phy sicochemical and functional properties (Modler and Em- mons, 1977; Modler and Harwalkar, 1981). Modler and Emmons (1975, 1977) reported that heating at 90°C for 15 min at pH 2.5-3.0 yielded an isoelectric precipitate (Protolac, 60% protein) that was reasonably soluble, highly viscous, and capable of setting to form a soft coagulum. p-Lg has a stable conformation at pH 2 (Reddy et al., 1988), and Modler and Harwalker (1981) showed that concentration of whey by U F at pH 2.5-3.0 prior to or after heating altered some properties, i.e., viscosity and gelling of the isolated protein.

A. EFFECTS OF HEAT ON CONFORMATION

Several investigators have studied the heat-induced unfolding of whey proteins (Larson and Rolleri, 1955; Ruegg et al., 1977; Elfagm and Whee- lock, 1978; deWit, 1981). In this respect, differential scanning calorimetry (DSC) has provided much of the data on the thermodynamics of whey protein unfolding. Ruegg and co-workers (1977) showed that a-La and p- Lg (concentration range 3-9%, pH 7.0) had transition temperatures (T,) (maximum on DSC endotherm peak) at 65" and 73"C, respectively, during heating (from 20" to 110°C at 10°C per minute). The T, increased slightly with heating rate and the enthalpies of denaturation (AH) for a-La and p- Lg were 318 and 227 kJ/mol, respectively. Increasing the pH from 6.4 to 7.3 had negligible effects on AH, while the T, decreased from 79" to 74°C with the rate of denaturation of p-Lg increasing slightly. Harwalkar (1979) reported that increasing pH from 4.5 to 6.5 at zero ionic strength decreased AH for p-Lg from 0.85 to 0.60 J/g protein and the denaturation temperature from 83" to 77°C. The transition temperature also decreased with increasing ionic strength. Hegg (1980) demonstrated that the denaturation temperature of p-Lg increased from 78" to 83°C between pH 2 and 4 and then progressively decreased with increasing pH, being approx-


imately 78°C at pH 6, 67°C at pH 7, and 60°C at pH 8. The enthalpy of denaturation (AH,) decreased with pH from 0.83 to 0.23 J/g at pH 6 and 8, respectively. The discrepancies in the published data may reflect differences in protein preparations, ionic strength, bound lipids, etc. (Hegg, 1980; deWit, 1984; Bernal and Jelen, 1985). Some thermal characteristics of the major whey protein components are summarized in Table XIV (de- Wit, 1984).

Conflicting reports exist in the published literature concerning the kinetics of heat denaturation of p-Lg. While Gough and Jenness (1970) reported a mostly first-order process, Hillier and Lyster (1979) and Park and Lund (1984) described the thermal unfolding of p-Lg as following a second-order reaction rate; the denaturation temperature (83"-84"C) changed little with pH between pH 4.0 and 7.0, while AH increased from 0.95 to I . 11 J/g and the activation energy (E,) increased from 28.3 to 33.0 kJ/rnol. Harwalkar (1980) concluded that the heat denaturation of p-Lg occurs in two stages, both being first-order; the first proceeds relatively slowly and is reversible, while the second stage is rapid and irreversible. Recently, Harwalkar (1986) reported that p-Lg follows a pseudo first-order process in aqueous solution but displays a second-order process in whey as observed by O'Neill and Kinsella (1988).

Protein unfolding can be caused by increasing the pH and this augments electrostatic repulsive forces between component polypeptides. At alkaline pH, the thiol group of p-Lg becomes exposed (Kella and Kinsella, 1988) and the thiol-disulfide interchange reaction is accelerated (dewit, 1984). Increasing ionic strength masks exposed charged groups, thus en-

TABLE XIV

AND ENTHALPIES OF WHEY PROTEINS~'~

THERMAL DENATURATION TEMPERATURES ("C)

~

AH Td T,, (kJ/mol)

a-Lactalbumin 62 68 253 @-Lactoglobulin 78 83 31 I Bovine serum albumin 64 70 803 Immunoglobulins 72 89 500 Whey proteins 62-78 68-83 -

"Data after deWit (1984). bTd, Initial denaturation temperature; T,,, tempera-

ture at maximum peak on thermogram peak temperature in DSC; AH is the enthalpy of denaturation.


hancing hydrophobic interactions. Calcium is very effective in this respect (Thompson et al., 1969; Bernal and Jelen, 1984).

The temperature of denaturation of a-La is 65°C and the enthalpy of denaturation is 318 kJ/mol at pH 6.7 (deWit and Swinkels, 1980). a-La shows rapid and extensive renaturation, i.e., 80 to 90% reversibility at concentrations in the range of 3 to 9% (Ruegg et al., 1977). The a-La molecule is a relatively small protein (123 amino acid residues) with four disulfide bonds, which limits the number of conformations the protein can assume and may account for the resistance to denaturation and rate of renaturation. The enthalpies of denaturation of a-La in milk ultrafiltrate and phosphate buffer (pH 7) were 318 and 272 kJ/mol, respectively, indicating that lactose, calcium, and citrate may stabilize a-La. When a-La is heated for 10-30 min at lOO"C, only 40% of the protein molecules revert back to the native state; the remainder are irreversibly denatured (Schnack and Klostermeyer, 1980). On prolonged heating, various-sized oligomers of a-La are formed via rupture of disulfide bonds, which apparently permits thiol-disulfide exchanges between these molecules and new reactive groups that are simultaneously formed (Chaplin and Lyster, 1986).

In contrast to p-Lg, a-La is unstable at pH values below 4.0 (deWit and Klarenbeek, 1984). This was supported by Kronman and co-workers (1981) when it was observed that, below pH 3.75, a-La is subject to heat- induced aggregation but this can be reversed by adjusting the pH above 5.2. Acid pH causes unfolding of a-La and facile protein-protein interactions. This may involve exposure of apolar residues and enhanced intermolecular aggregation. The effect of acid pH on heat stability may be related to its effect on calcium binding to the protein ( K , = 3 x 10-6 M- 1) which stabilizes the molecule. Removal of calcium by acidification or chelation destabilizes a-La. Bernal and Jelen (1984) demonstrated that, following chelation of calcium, the T, and AH of a-La (holo-form) decreased from 61" to 41°C and 22 to 6 J/g, respectively, and eliminated renaturation of a-La (Table XV). It is conceivable that, on exposure of a-La to low pH (3.0), some bound calcium is removed from the molecule, rendering a-La more susceptible to irreversible heat denaturation. Thus, extensive dialysis of acidified whey (UF/DF) may destabilize a-La by re- moving calcium. This may contribute to the anomalous behavior of whey protein preparations, particularly under low-ionic-strength conditions.

B. FACTORS AFFECTING HEAT DENATURATION

During whey processing, proteins are subjected to many heat treatments which may be cumulatively deleterious. Calorimetry can be effectively used to monitor the extent of heat denaturation of whey proteins


(dewit, 1981, 1984). The method is sensitive to the progressive denaturation of whey proteins as evidenced by the progressive decrease in the principal endothermic peak with cumulative heating during whey processing (dewit, 1984).

Harper and Zadow (1984) reported that a measurable portion of BSA and immunoglobulin molecules were denatured, as determined by rocket immunoelectrophoresis, following heating of the proteins a t 72°C for 15 sec. Denaturation progressively increased for all whey proteins following heating at 72°C for 5 min. Extensive denaturation of whey proteins occurred after 5 min of heating, particularly as the temperature was increased from 70" to 85°C. UF concentration of whey enhanced the rate of thermal denaturation and the extent of denaturation increased with time of heating. Based on immunological and solubility (denaturation) data at pH 4.6, BSA and immunoglobulins (IgG fraction) were the most heat-sensitive of the whey proteins, followed by (3-Lg and a-La (Harper and Zadow, 1984).

Although the thermal denaturation behavior of whey proteins reflects the collective responses of the component proteins, particularly those of p-Lg (deWit and Swinkels, 1980; deWit and Klarenbeek, 1984), the behavior is complicated by the effects of pH, ash (especially calcium), and the presence of chelating agents such as citrate and phosphate (de Rham and Chanton, 1985). Hermansson (1979) observed that, at pH 2.0 to 5.0, the thermal transition of whey proteins in water displayed a sharp peak with maximum transition temperatures increasing from 80" to 90°C up to pH 4.5 and then decreasing back toward 80°C as the pH was increased to 9.0. Significantly, above pH 6.0, the endotherms showed a gradual slope

TABLE XV

PARAMETERS OF a-LACTALBUMIN"" THE EFFECTS OF CALCIUM REMOVAL ON SOME THERMODYNAMIC

Extent of renaturation

Td rc) AH (JM (%) pH of

medium A B A B A B

4.5-6.5 61 41 22 6 85 0 3.5 58 40 10.6 4 <so 0

"Data after Bernal and Jelen (1984). "a-Lactalbumin skim milk ultrafiltrate without (A) and with 0.1 M EDTA (B). Td,

Denaturation temperature.


rather than a peak. Using turbidometry, it was reported that, above pH 3.5, the apparent transition temperature of very dilute solutions (0.09% in 0.2 M sodium chloride) of whey proteins increased from 58" to 75°C at pH 4.0 and 6.0, respectively. However, below pH 3.5 these whey proteins were stable at 90°C (Hermansson, 1979). At pH 3.0, 6.0, and 7.5, p-Lg had peak denaturation temperatures of 82", 78", and 71°C and AH of 16.6, 15.9, and 11.2 J/g, respectively, indicating that increasing pH rendered p- Lg susceptible to thermal denaturation (dewit, 1981 ; deWit and Klaren- beek, 1984). This behavior is consistent with previously inaccessible hydrophobic patches becoming exposed to the aqueous exterior and resulting in a decrease in AH,,, i.e., as the molecule unfolds, increasing surface hydrophobicity causes a disruption in the hydrogen bonding network between polar groups on the protein and water molecules which surround the protein (Pace, 1983).

The addition of calcium after heating results in greater flocculation than if present during heating (de Wit, 1981). However, very high heat treatments (160°C) reduce the sensitivity to calcium, reflecting greater polymerization of the proteins and perhaps occlusion of the charged anionic groups (deWit and Klarenbeek, 1981). Normal whey contains 2-3 mM calcium, yet above pH 6.5, the proteins are not heat precipitated because the calcium is apparently chelated. However, UF/DF of whey may increase calcium sensitivity and decrease solubility.

De Rham and Chanton (1985) carried out a detailed study on the effects of ions in the heat-induced insolubilization of whey proteins of normal and demineralized whey (DW). The proteins in DW remained soluble following heating (90°C, 15 min) at pH 6.5 and above. Heating at pH 4.5 and 5.5 decreased solubility by 33 and 50%, respectively. Removal of lactose and nonprotein materials slightly increased thermal sensitivity below pH 6.0. When DW was heated at pH 7.2, a marked increase (2-fold) in solubility at pH 4.5 to 6.5 was observed, with 100% solubility at pH values greater than 5.5. The addition of calcium to DW before testing reduced solubility, and less calcium (2 mM) was needed at pH 6.0 than at pH 7.0. Chelation of calcium with citrate eliminated this effect. Sweet cheese whey contains approximately 8 mM Ca2+, 3 mM MgZ+, 15 mM Na+, 36 mM K', 8 mM citrate, 12 mM phosphate, and 32 mM C1- (de Rham and Chanton, 1985). The pH of whey during UF significantly affects the residual ash content; at lower pH values a reduction in the residual calcium content is obtained. Citrate is rapidly reduced by UF, irrespective of the pH. Generally, the heat stability of whey proteins above pH 6.0 is improved after U F at acidic pH (4.5-53, reflecting lower calcium levels in those products. The addition of citrate to chelate calcium improves solubility (above pH 6.0) following heating. Replacement of calcium with


sodium (or potassium) by ion exchange also improves the heat stability and solubility of whey proteins (dewit, 1981; Johns and Ennis, 1981; de Rharn and Chanton, 1985).

Aggregation and precipitation of p-Lg are greatly facilitated by pH and presence of calcium ions (Elfagm and Wheelock, 1978). Calcium possibly interacts with the negatively charged carboxyl groups of the protein, reducing the net charge to zero and causing isoelectric precipitation (Zittle and DellaMonica, 1956; Zittle et al., 1957). These data indicate that whey proteins, particularly when concentrated by UF, are more susceptible to thermal denaturation and that disaccharides such as lactose and sucrose may retard thermally induced denaturation of whey proteins (dewit, 1981; Bernal and Jelen, 1985; Garrett et al., 1988). Concentration of proteins prior to heat processing may be conducive to excessive denaturation, particularly of the IgG and BSA components. Other factors can affect the rate of heat denaturation, e.g., lactose concentration reduces susceptibility to heat denaturation and low concentrations of lipids enhance the thermal stability of p-Lg. Binding of 1-2 molecules of sodium dodecyl sulfate (SDS) to p-Lg increased its denaturation temperature from 82" to 90°C (Hegg, 1980). Lipid binding by BSA stabilizes the molecule but, at high concentrations of lipid, the protein is destabilized (Hegg, 1980). Thus, lactose, citric acid, pH, ionic environment, and calcium concentration can be manipulated to minimize the heat-induced insolubilization of whey proteins.

VIII. GELATION

Whey proteins can form gels that range in properties from viscous fluid soft, smooth pastes or curds to stiff, rubbery gels. These gels vary in hardness, cohesiveness, stickiness, color, and mouthfeel (Hillier and Cheeseman, 1979; Schmidt et al., 1979; Johns, 1979; Dunkerley and Hayes, 1981; Hillier et af., 1980; Johns and Ennis, 1981; Schmidt, 1981; Kornhorst and Mangino, 1985; Zirbel, 1987). WPC gels also vary in visual appearance from firm elastic transparent gels to opalescent curdlike coagula. At low protein concentrations and low ionic strength, weak gels with gray or translucent appearances are obtained. In many cases, evaluations of whey gels are based on subjective assessment; thus, standardization of methodology and terminology is necessary.

Gelation occurs when there are a limited number of specific protein-protein interactions following heating, whereas coagula or curds form when extensive random interactions occur. Gelation, i.e., the prop- erty of forming a structural network which maintains shape, has mechani-


cal strength, viscoelasticity, and retains entrapped water with minimum syneresis, is an important functional attribute of functional proteins in many food applications. Protein solutions (1-10 g/dl), when heated above a critical temperature, undergo conformational changes and on cooling may set to viscous, soft, opaque coagula or clear viscoelastic gels depending on the type of protein, concentration, heating rate, and environmental conditions, especially pH and calcium (Mulvihill and Kinsella, 1988). Thermally induced gelation may be viewed as a two-stage sequential process; and first phase involves heat-induced conformational changes in a protein which may involve unfolding of some polypeptide segments followed by subsequent protein-protein interactions resulting in a progressive build up of a network structure when protein-protein interaction is limited, i.e., approximately two interaction sites per molecule (Bernal and Jelen, 1985). Thermal activation of protein molecules to expose interactive sites may involve only very minor changes in overall molecular conformation. Where the protein concentration is adequate and the heating rate slow, protein-protein interactions leading to a three-dimensional network capable of entraining water are favored. If aggregation is more rapid than unfolding, a precipitate may form (Ferry, 1948). For the formation of an appropriate three-dimensional network, a balance between attractive and repulsive forces is necessary. In the formation of a gel from whey proteins, limited unfolding exposes hydrophobic segments on proteins which tend to associate on recooling, facilitating bonding between contig- uous molecular groups. Networks thus formed vary considerably in structure depending on the number and relative reactivity of interactive sites, and the degree of electrostatic repulsion between the solute molecules. These are markedly affected by pH and ionic strength.

The particular changes that occur in the secondary structure of proteins and the manner in which the network builds from individual molecules affect gel texture. Small conformational changes may facilitate protein-protein association to form long beads, or molecules may completely unfold to form a random network. For example, in the case of glycinin, the globular molecules associate to form chains with cross-linking or branching. In other cases, polymerization of small aggregates or of dimers and trimers into thick filaments for the formation of ropelike arrange- ments can occur (Clark et d., 1981). In BSA gels, irregular chains or filaments of protein molecules, approximately 1-2 protein molecules thick, form junction points or branch points. These filaments form a network with a density reflecting the degree of branching and cross-linking. Clark and co-workers (1981) concluded that, for BSA with a filament thickness of 6 to 12 pm (approximately double that of the undenatured molecule), thickening was caused by unfolding of the native molecule. p-


Lg gels contain large spherical protein aggregates linked together to form a continuous branching network at both high and low ionic strength (Har- walkar and Kalab, 1985b). Some proteins, e.g., lysozyme form networks composed of straight, rodlike filaments, whereas other protein molecules seem to be assemblies of small, irregular protein clumps with a beadlike repeating structure (Clark et al., 1981).

A. GEL STRUCTURE

Proteins vary considerably in the amount of native tertiary and secondary structure which is lost prior to aggregation and gel formation. Little work has been done to quantify the extent of unfolding prior to gelation. In the case of BSA gels, there is limited unfolding and the molecules string together to form irregular chains 2 to 3 times as thick as the native molecule (Clark et al., 1981). Limited conformational changes occur during heating and gelation. Clark et al. (1981) reported an increase in p-sheet and unordered structure with a concomitant decrease in the a-helical content of BSA, which is common in fibrous protein systems, following heating. Timasheff et al. (1967) noted that aggregation of p-Lg was accompanied by an increase in P-sheet and unordered structure.

Generally, clear gels reflect the formation of uniform networks of fine filaments and there is greater linear aggregation with frequent cross-linking and/or branching processes. As network density becomes less regular, gels increase in turbidity. In the case of BSA gels, gel clarity was decreased by adding salts or decreasing pH toward the isoelectric point, i.e., minimization of net charge (Clark et al., 1981). In the case of coagula, phase separation occurs and the protein network is composed of aggregates of protein separated by regions of water.

Noncovalent protein-protein interactions occur in the formation of both reversible and irreversible gels (Table XVI). The number and type of noncovalent interactions (hydrophobic and van der Waals’ interactions, hydrogen bonding, and ionic interactions between charged amino acid side chains) vary with the protein, pH, heat treatment, and ions present. Cross-linking is essential for gel formation, which together with the solvent provides the fluidity, mechanical strength, elasticity, and flow behavior of gels (Schmidt, 1981). The degree of cross-linking can be variable, and thus provides a mechanism by which the strength of gels may be manipulated. Understanding the nature of the interactions provides the means to control the extent of cross-linking, permitting manipulation of gel properties. Some ion cross-linking may be important in calcium- induced gels (Schmidt, 1981; Mulvihill and Kinsella, 1987). In the formation of irreversibly set gels, covalent disulfide cross-linking may occur


TABLE XVI POSSIBLE FORCES INVOLVED IN THE FORMATION AND STABILIZATION OF PROTEIN

FILMS AND GELS

Energy Interactive Groups Type (kJ/mol) distance (A) involved Effects

Hydrophobic van der Waals

Hydrogen bonding

Ionic Cation

Covalent bridging

4.2-

8.4- 12.6

33.6

42-84 25.2- 50.4 336-

378

3-5 Apolar Strengthening and

2-3 NH-OC Strengthens initiating contacts

associations 0 I1

2-3 C-0:- +NH, Strengthens cross-links 2-3 Calcium Cross-linking

1-2 RSH:S-S Bridging, ordering, strengthening

(Huggins et al., 1951). Electrostatic interactions (the net result of attractive and repulsive forces) are critical in determining gel formation and gel properties. Thus, the gelation of p-Lg at pH 7.0 or above is enhanced by calcium chloride (3-6 mM) or sodium chloride (0.2-0.4 mM). Gel strength is also affected by salts but their effects on gel properties vary with anion type. This may reflect differential binding by the anions, exerting variable effects on electrostatic interactions (Mulvihill and Kinsella, 1987).

B. GELATION OF WHEY PROTEINS

Whey proteins have excellent gelling characteristics, particularly above pH 7.0. Undenatured, soluble whey proteins prepared under mild processing conditions form irreversible gels at the appropriate pH, ionic strength, and protein concentration. Heating p-Lg above pH 6.5 causes formation of gels that become clearer as pH is increased (dewit, 1984). Coagula are formed on heating p-Lg at pH values below 6.5 and concentrations greater than 7 g/dl, reflecting extensive enhanced attractive interactions. The higher gelation tendency in the region above pH 8 may reflect some disulfide cross-linking and matrix formation via thiol-disulfide interchange. Whey proteins can form clear gels or opaque gels. Clear gels tend to be more elastic and hold water more effectively. Apparently, they possess finer filaments, smaller mesh size, more even spacing of intermo-


lecular linkages, and are generally formed at higher pH values (Schmidt, 1981).

Although much information exists concerning factors affecting gel formation and properties of whey protein gels (Dunkerley and Hayes, 1981; Schmidt, 1981; Mulvihill and Kinsella, 1987), there are limited data on the kinetics of gel formation and gelling properties of the individual proteins. Dynamic shear measurements at small total strain amplitudes during heat- induced gelation of whey protein concentrates were made and the changes in the storage modulus (G’) and the loss modulus (G”) were calculated by Beveridge et al. (1984). As the gel network formed, the samples became more elastic. WPC (7 g/dl protein solution) showed a slow increase in G’ (2 x lo3 dynekm’) on heating (85°C for 10 min) compared to egg white in which G’ was 4 x lo3 dyne/cm2 after 30 min. However, on cooling, the G’ increased to 20 x lo3 dynelcm’, i.e., the elastic properties increased. It was concluded that aggregates were formed during the initial 3-10 min of heating and that these associated to form a weak viscous network on continued heating (Beveridge et al., 1984). Disulfide bonds may be involved at this stage. However, on cooling, an extensive elastic network is formed due to extensive hydrophobic, ionic interactions and hydrogen bonding. The increased elasticity reflected the increased number of cross-links formed during cooling. Reheating the gels reduced the elasticity somewhat but it significantly increased on cooling (Beveridge et al., 1984).

Little is known of the effect of additional materials, such as lipids, on the gel-forming characteristics of whey proteins. Sternberg et al. (1976) reported a decreased tendency of whey protein solutions to gel after addition of milk lipids. On heating in the presence of calcium, Yamauchi et al. (1980) observed gelation of a coconut oillwater emulsion of whey protein. Gelation did not occur when calcium was omitted. Jost and co-workers (1986) investigated the amount of lipid which could be incorporated into whey protein gels without compromising desired gel textural qualities. Generally, the firmness of heat-induced gels increased with increasing emulsion fineness and was affected by changes in the protein and oil content. The pH range within which oillwater emulsions stabilized by whey protein could get extended from pH 3.5 to 8.0 (Jost et al., 1986).

C. COMPOSITION AND GELLING PROPERTIES

The properties of whey protein gels are affected by protein concentration, time and temperature of heating, pH, ionic strength, etc. (Schmidt, 1981). The appearance of whey protein gels varies from translucent and


elastic to curdlike brittle aggregates. Weak translucent gels are obtained at lower protein concentrations (5%) and low heating temperatures (55" to 70°C). Stronger and more aggregated opaque gels which form at higher protein concentrations and higher heating temperatures (90°C) reflect a more extensive network. Translucent gels are usually obtained when protein is heated at low ionic strength (Schmidt, 1981).

Gel strength increases with the concentration and purity of the protein. Prior denaturation of the protein reduces gelation and gel strength (Lang- ley et al., 1986). Commercial samples of WPC (10 g/dl protein solution) vary tremendously in gel formation and gel properties when studied under identical conditions. The time required for gelation may range from 1 to 17 min at lOO"C, and some samples did not form a gel even after 30 min heating. This may reflect extent of prior denaturation, composition of the whey, pH, and ion concentration (Schmidt, 1981; Langley et al., 1986). Dynamic rheological techniques (Mitchell, 1980) permit the viscoelastic properties of individual whey proteins to be assessed separately. BSA was characterized as having good, p-Lg intermediate, and a-La poor thermal gelling properties under conditions of pH 6.6, 1% NaCl, and heating range from 30" to 95°C (heating rate l"C/min). The BSA gels were purely elastic, while the p-Lg gels had viscous elements (Paulsson et al., 1986). In protein mixtures, the influence of various whey proteins at equal concentrations on heat-induced gelation decreased in the order BSA, p-Lg, a-La and the gelation behavior was influenced mostly by the protein in highest concentration (Paulsson et al.. 1986). The presence of lactose and ash may impair gelation by interfering with network formation, but generally increasing the protein concentration, relative to nonprotein components, improves gelation. Thus, gels formed following dialysis of WPC are more translucent, stronger, more cohesive, less springy, and more chewy, than gels from the nondialyzed WPC.

The pH has a marked effect on gelling properties of whey proteins, depending on the particular preparation, and this effect may be related to calcium content. Gels formed at low pH (pH <6) are more coagulated and less elastic than gels formed at pH 7 to 9 (Hillier et al., 1980; Schmidt, 1981). The addition of 0.1 M phosphate enhances the rate of gelation at higher pH values, possibly due to chelation of calcium (Schmidt, 1981). Exposure of WPC to pH 9 followed by readjustment to pH 7 prior to heating facilitates the subsequent gelation of WPC. This may reflect some unfolding and/or rupture of disulfide bonds under alkaline conditions. The pH of the solution during heating markedly affects gelation reactions; thus increasing the pH from 7 to 11 reduces gel strength, particularly above pH 9. The relative strengths of gels made by heating whey protein solutions at 100°C for 15 min at pH 7, 9, 10, and 11 progressively de-


creased from 100, 57, 23, 0 (N/m*), respectively (Schmidt et al., 1979; Schmidt, 1981). Exposure to alkaline pH conditions accelerates heat-induced gelation and reduces the temperature treatment required for gelation.

Hillier and Cheeseman (1979) and Hillier et al. (1980) confirmed the effects of pH on the gelling time. As the pH of the solution was increased above 6.5, gelling time of deionized WPC (10 g/dl) increased, particularly at pH 9 and above. Below pH 6.4 and especially around pH 5 , the proteins precipitated on heating. Thus pH, by affecting protein conformation and net charge, markedly affects the rate and extent of gelation, i.e., where electrostatic repulsion is excessive a limited network is formed. The effects of pH in turn are influenced by the presence of ions. Calcium may reduce net repulsion above the isoelectric pH and facilitate hydrophobic interactions. Calcium may also serve in cross-linking adjacent anionic molecules, thus facilitating network formation (Mulvihill and Kinsella, 1988).

Because the degree of electrostatic interactions between proteins is critical in forming a three-dimensional matrix with desirable water-holding capacity, the heat-set and thermal denaturation characteristics of two acidic proteins, BSA and WPI, containing added basic protein components, e.g., clupeine (pZ = 12), hydrolyzed clupeine, or modified p-Lg (pZ = 9.9, at low ionic strength, were examined by Poole et al. (1987a). An optimum concentration ratio of 3 : 0.3% (w/v, BSA : clupeine) yielded a gel at pH 8 comparable to that formed with BSA alone at pH 5 . How- ever, using the same concentrations of hydrolyzed clupeine, a much weaker gel was formed, demonstrating the importance of size of the basic protein in determining whether cross-linking or aggregation occur. Modi- fied p-Lg with a net positive charge also enhanced gelation, but the minimum total protein concentration required for gelation was higher (4 : 0.4% w/v, BSA : p-Lg), reflecting weaker interactions with BSA possibly because of a lower pZ than clupeine. Clupeine promoter the gelation of WPI but only at a critical ratio above 5 : 0.4% (w/v, WPI : clupeine). However, the gel had a coarser texture and poorer water-holding capacity than the corresponding BSA gels. It was concluded that basic protein may promote gelation by cross-linking (partially) denatured acidic proteins via electrostatic attractive forces. DSC and spectral analyses demonstrated that basic proteins tend to destabilize acidic proteins at a pH value greater than the pZ, thus facilitating protein unfolding (Poole et al., 1987a).

The gel structure of whey proteins is affected by salt concentration (Ta- ble XVII). The action of sodium chloride can be explained in terms of a charge-shielding effect of counterions which surrounds like-charged macromolecules in solution, causing repulsion (Dickinson and Stainsby,


TABLE XVII EFFECT OF SALTS AND THIOL GROUPS ON THE STRENGTH OF

WHEY PROTEIN GELS*

Penetration Hardness Treament (dcm) (kg)

Whey protein control (WPC) 533 0.56 WPC + NaCl (0.2 M) 996 0.82 WPC + CaCI,(IOmM) 1785 0.89 WPC + cysteine (25 mM) 1980 0.69

“Data after Schmidt et nl. (1979).

1982). Thus, in the presence of sodium chloride (0.2 M), a coarse gel of large aggregates is formed compared to finer gels developed in the absence of salt (Schmidt et al., 1979). The addition of calcium (5-20 mM) tends to restore the strength of whey protein gels formed at pH values of 7 and above (Schmidt et al., 1979; Mulvihill and Kinsella, 1988). This cation reduces electrostatic repulsion and permits greater protein-protein interactions and network formation. The effect of calcium addition varies with each whey preparation, reflecting variability in composition, e.g., citrate, phosphate, calcium content. Dialyzed WPC shows greater response to calcium addition; addition of 10 mM calcium to dialyzed WPC improved gelation and gel strength (Schmidt, 1981).

The rheological properties of gels made from p-Lg are affected by addition of calcium (Table XVIII), suggesting that, in the absence of added salts, electrostatic repulsive forces, which are enhanced at high pH, pre- dominate to prevent gelation but dissipate on increasing the ionic strength (Mulvihill and Kinsella, 1987, 1988). Maximum gel strength occurs at 10 mM CaCl, in contrast to 200 mM NaCl, reflecting the greater effectiveness of calcium compared to sodium in increasing rheological parameters related to gel strength (Mulvihill and Kinsella, 1987).

In the case of gels made at pH 6, replacement of 30,67, and 100% of the calcium in UF/WPC with sodium progressively increased the hardness, springiness, and chewiness of 10% WPC gels (Johns and Ennis, 1981). Additionally, gels became more translucent with calcium replacement, and the time for gelation increased from 44 to 166 min on heating at 70°C at pH 6.7 (Johns and Ennis, 1981). Calcium may interfere with gelation by causing excessive coagulation following heating rather than enabling the protein network to form properly.


TABLE XVIII

RHEOLOGICAL PROPERTIES OF p-Lg GELS~.’

EFFECTS OF CALCIUM CHLORIDE CONCENTRATION ON THE

[CaC121 (mM) Stress (kN/m*) AMD (kN/m’)

5 10 I5 20

0.5 7.0 2.5 1.5

2.0 16.5 11.5 8.6

“Data after Mulvihill and Kinsella (1988). ”Gels measured at 20% compression. AMD, Apparent modu-

lus of deformability.

Thiol groups at reasonably low concentrations facilitate gel formation by enhancing the rate of unfolding of proteins since reduction of strained disulfide bonds permits greater conformational freedom and perhaps greater interactions between unfolded molecules (network formation). However, excessive disulfide reduction may impair network formation (Schmidt, 1981).

Disulfide bonds are important in stabilizing the structure of gels since, on addition of thiol reagents, they can be disrupted (dewit, 1981). The addition of cysteine (up to 10 mM) prior to heat treatment enhances gelling properties, whereas, at higher concentrations, gelation is impaired (McKenzie, 197 1).

The content of apolar amino acids in proteins influences the changes which occur on heating because a high molar percentage of apolar side groups can overcome the repulsive electrostatic forces arising when pH values are outside the isoelectric region (Circle et al., 1964; Shimada and Matsushita, 1981). Thus, small proteins with high molar percentages of apolar amino acids (>33%), such as p-Lg and a-La, are concentration independent in gelling systems (Shimada and Matsushita, 1980). How- ever, the disposition of apolar amino acid side chains along the polypeptide chain may be more important to the development of intermolecular interactions than the total content of these residues.

D. WHEY PROTEINS AS GELLING INGREDIENTS

WPC has been promoted as a replacement for egg white proteins in gelling applications (Melachouris, 1984). At protein concentrations of 10 g/dl, WPC forms gels when heated above 85°C for 15 to 30 min. Egg white


proteins gel at 60°C and many applications of egg white in food processing are based on this feature. Thus, when replacing egg white with whey protein, the food may have to be heated above 80°C for adequate gel formation and this may be incompatible with other functional attributes of the particular product. This is particularly true of cakes; for example, in angel food cake the extra heat may cause oven-collapse. The addition of poly- phosphates to whey protein treated with pepsin improved its gelling and whipping properties, which were comparable to those of egg white (Nakai and Li-Chan, 1985), but this preparation was found to be unsuitable as an egg white substitute in angel food cakes because of the instability of whey protein foams. The differences in thermal characteristics of egg white and whey must be recognized before advocating WPC as a suitable replacement. Thus, the temperatures required to induce gelation and the effects of the various components on the temperature of gelation are extremely important.

The gelling behavior of proteins is important in processed and reformed meat products for setting into a matrix but also because the binding (water, fat, flavors) properties are related to gelling behavior (Acton, 1972). In this respect, whey proteins have acceptable binding properties but, for optimum performance, their thermal properties should be compatible with those of the myofibrillar proteins. Thus, when functional proteins are added to a food system to impart gelling properties, it is necessary to know the required processing temperatures to obtain that functional attribute. For example, myosin gels at 6O"C, whereas whey protein concentrate may require higher temperatures, e.g., 80"C, to obtain an equivalent extent of gelation. This may or may not be compatible with the food product in question. Therefore, in seeking to substitute WPC for other possible gelling proteins and products, the conditions required for optimal gelation and development of requisite gel properties must be clearly defined and be compatible with the processing and preparation of the product to which it maybe added. Thus, in certain products, e.g., meats, WPC may not form a gel with the firmness required for slicing, whereas in other products, depending on concentration, heating temperatures, etc., the gel strength may be excessive. These concerns underline the necessity of testing whey proteins in actual food systems under commercially compatible conditions before claiming specific functional attributes.

In comparing gelling properties of whey protein preparations, it is important to standardize conditions of protein preparation, isolation, concentration, pH, temperature, time, and ionic strength. Standardized tests of all the physical properties of whey protein gels should be outlined to enable data from different laboratories to be compared validly. Using standardized testing, Melachouris (1984) demonstrated marked variability


in gelling properties and gel strength of WPC lots containing 50% protein. Variability in gelation behavior of whey preparations reflects differences in extent of protein unfolding during drying, composition and ion concentration, e.g., of calcium and lipids, all of which affect gelling rates and gel properties. The extent of denaturation of WPC produced by U F may range from 10 to 50%, but is usually within the range 20 tO 30%. Measures to minimize denaturation by controlling heat and/or the use of additives to minimize protein denaturation should be explored (Melachouris, 1984).

IX. SURFACE ACTIVITY OF WHEY PROTEINS

A. PROTEIN ADSORPTION

The surface active properties of proteins are important in a number of functional applications, e.g., foams and emulsions (Graham and Phillips, 1976a,b; Kinsella, 1981). Many proteins are surface active, that is, they adsorb at surfaces or interfaces at a rate which is initially diffusion controlled (Graham and Phillips, 1976a; MacRitchie, 1978; Phillips, 1981). Adsorption is thermodynamically favored because of the concomitant loss of some conformational and hydration energy by the protein molecules. The initial protein molecules arriving at an interface unfold to a varying extent (depending on their inherent molecular properties) and spread to cover a maximum area of the surface, forming an extended film (Graham and Phillips, 1979; Phillips, 198 1). Additional molecules may adsorb provided they can overcome the interfacial pressure barrier and the “energy” barrier (electric potential) created by the initially adsorbed molecules. Once adsorbed at an interface, protein molecules undergo spreading and rearrangement in order to achieve a state of lowest free energy. Thus, extensive rearrangement of polypeptide chains and interactions between polypeptides occur at the interface over time. Rearrangement involves the hydrophobic components of polypeptides orienting toward the nonaqueous phase while polar moieties become exposed to the aqueous phase (Phillips, 1981). The extent of unfolding and reorientation depends on the nature of the protein, molecular flexibility, and protein concentration at the interface. Adsorption gradually causes a decrease in interfacial tension which is proportional to the number of groups penetrating the interface. From the changes in the surface tension, surface pressure, rate of adsorption of the protein to the surface, area cleared per mole of protein, and work of compression during penetration of the protein into the interface, the approximate number of amino acid residues penetrating the interface can be estimated (MacRitchie, 1978; Waniska and Kinsella,


1985). High surface tension or low surface pressure indicates a high surface free energy. As protein molecules adsorb at an interface, the increase in surface pressure reflects a lower surface free energy (Joly, 1972). Gen- erally, the first layer of protein at an interface contributes the most to surface pressure development. Thus, protein preparations with small surface active components, e.g., PPs in whey preparations, rapidly decrease surface pressure, but because of their small molecular size, their presence may not be conducive to the formation of a continuous cohesive film. Furthermore, these same components facilitate foam formation but may actually impair foam stability. Rapid adsorption is important for entrap- ping air or encapsulating an oil droplet. As protein concentration increases, thicker films are formed (3-5 mg/m2) and tighter packing gives a more condensed compressed film with 3-8 mdm2 protein (Graham and Phillips, 1976a). Noncovalent interactions operate between adjacent molecules at the interface to form a cohesive film (Graham and Phillips, 1979, 1980).

Under equilibrium conditions, the surface activity of a protein is influenced by its rate of diffusion, which is in turn affected by molecular properties (e.g., size, composition, conformation), temperature, pH, ion species, and concentration. In this regard, small, randomly structured, i.e., flexible, amphipathic proteins (e.g., p-casein, PP) exhibit excellent interfacial properties in terms of surface pressure development. However, in food systems, equilibrium conditions may not prevail and generally there is a large energy input during mixindwhipping to form an interfacial film. Furthermore, continuous cohesive films that possess mechanical strength and appropriate viscoelastic properties are required in most food systems (Kinsella, 1981 ; Halling, 1981). Low-molecular-weight, randomly structured molecules do not form strong films, whereas larger protein molecules, which are moderately stable and retain a certain critical amount of tertiary structure at an interface, are generally better surface active agents because of greater intermolecular interactions (Graham and Phil- lips, 1979; Song and Damodaran, 1987).

The rate of surface pressure increase reflects the nature of the protein and its molecular “flexibility” (Graham and Phillips, 1979). As increasing amounts of protein are adsorbed, the extent of molecular rearrangement decreases and the film changes from an expanded to a compressed film. Globular proteins generally form more condensed interfacial films than extended proteins such as the caseins (Graham and Phillips, 1976a, 1979). Presumably, this is because globular proteins such as BSA and p-Lg retain a greater amount of residual secondary and perhaps tertiary structure when adsorbed at the interface, facilitating greater polypeptide entangle- ments and interactions to yield a condensed film. In an expanded film,


the extent of unravelling and loss of tertiary structure may be significantly greater for globular proteins than for extended proteins (Graham and Phil- lips, 1976a; MacRitchie, 1978). Globular proteins such as p-Lg, BSA, and a-La form condensed fdms with relatively high surface viscosity. The viscoelastic properties of BSA and p-Lg films reflect a continuous two- dimensional network, formed via extensive protein-protein interactions (Kim and Kinsella, 1985; Waniska and Kinsella, 1985).

B. PROTEIN STRUCTURE AND FILM-FORMING PROPERTIES

Different proteins behave differently at interfaces and protein adsorption at an interface is highly dependent on the nature of the protein and predominating forces, i.e., molecular size and shape, and conformational, ionic, and hydrophobic characteristics. It is essential to understand how each of these parameters affects the dynamics of the adsorption and spreading processes. In order to elucidate the contribution of molecular conformation, different proteins with varying structural characteristics have been extensively studied (Graham and Phillips, 1979, 1980; Damo- daran, 1989). p-Casein adsorbs and spreads rapidly at the interface to occupy the maximum area per molecule compared to BSA or lysozyme (Benjamins et al., 1975; Phillips, 1981). BSA rapidly diffuses to the interface where some of the molecular domains reorient, but the bulk of the tertiary structure is retained and anchors in the interface (Graham and Phillips, 1979). Ovalbumin, a compact and nonhydrophobic protein molecule, takes 0.36 sec to form a film of surface concentration 1.8 mg/m2 from a 0.03% solution (Bull, 1972). It was concluded from these results that differences in the surface behavior could be attributed to differences in conformational characteristics. However, differences in amino acid composition and disposition along polypeptide chains also exist and should not be discounted in contributing to the surface behavior of these proteins.

One approach to assess the influence of protein conformation on interfacial properties has been to study the adsorption and molecular behavior at fluid interfaces of structural intermediates of BSA, i.e., protein molecules which have been partially unfolded and permitted to refold in variable amounts of time (Song and Damodaran, 1987). The kinetic data from rates of surface pressure development for six structural intermediates of BSA, ranging from 0 to 50% a-helix, suggest that an optimum ratio of ordered to disordered structure is apparently essential to facilitate maximum changes in surface behavior (Song and Damodaran, 1987).

Because of the importance of charge in influencing the behavior of proteins in solution, i.e., during adsorption, unfolding and protein-protein


interactions at the interface, the pH and ionic strength have dramatic affects on the film-forming properties of proteins. A high net charge retards adsorption and protein-protein interactions in the film, causing the formation of expanded films with limited cohesiveness. Maximum adsorption rates occur near the isoelectric point if the protein is soluble, and strong viscous condensed films with maximum protein-protein interactions are obtained (Halling, 1981; Kinsella, 1981). Salt can enhance the rate of adsorption by minimizing electrostatic repulsion between protein molecules and facilitate molecular packing in the film (Cumper, 1953). Adsorption is increased by temperature as long as the protein remains soluble (Hal- ling, 1981).

The effects of aging on the surface rheology of interfacial films of several proteins (e.g., BSA, p-Lg, ribulose 1,5-bisphosphate carboxylase, K-

and p-caseins) have been reported and discussed (Benjamins er al., 1975; Kim and Kinsella, 1985; Waniska and Kinsella, 1985; Barbeau and Kinsella, 1986). Rates of surface pressure development, a function of protein concentration and conformation at an aidwater interface, surface viscosity, and film hysteresis increased as the protein films aged. The pH of the solutions also affected these parameters, i.e., maximum values were attained at or near the isoelectric points of the proteins. The data indicate that there is a limit to the number of polypeptide segments which can occupy the interface (MacRitchie, 1978) and significant protein-protein interactions occur between segments both above and below the plane of the interface (Graham and Phillips, 1979, 1980).

The' physical properties of the film once formed are the principal deter- minants of foam stability. Stability depends on the extent of protein-protein interactions, film thickness, mechanical strength, viscoelasticity, and on various external factors such as temperature and pH that alter the nature and magnitude of protein-protein interactions. Thicker films are usually more stable because of better rheological properties and mechanical strength. Thus, films of amphipathic bulky proteins which retain tertiary structure are usually thicker and more stable, (e.g., BSA versus p- casein) and this is reflected in more stable foams and emulsions. Such films have better viscoelastic properties and can adapt to shocks, compression, and distortion without rupture. For example, BSA forms viscous films while caseins show limited viscosity because of weak protein-protein interactions in the film (Graham and Phillips, 1979; Phillips, 1981).

The viscoelastic behavior of protein films has been explained in terms of protein-protein interactions; however, the nature of such interactions remains largely unknown. Several investigators have proposed a correlation between surface hydrophobicity and the surface activity of proteins


(Scriven and Sternling, 1960; Kato and Nakai, 1980; Townsend and Na- kai, 1983; Nakai, 1983; Voutsinas et al., 1983). It is conceivable that at or near the isoelectric pH of a protein, hydrophobic interactions are facilitated, thus enhancing hydrogen bonding and electrostatic interactions between polypeptides in the film. This premise has been supported by experiments on the effects of salts enhancing the foam stability of a protein (Barbeau and Kinsella, 1986).

The surface activity (rate of surface pressure development) of p-Lg was maximum around pH 4.9, which is slightly below the isoelectric point of 5.25 (Waniska and Kinsella, 1985). The highest rates of adsorption and rearrangement are observed when proteins possess limited net charge (Graham and Phillips, 1979), reflecting minimum electrostatic repulsion between polypeptides at the interface (MacRitchie, 1978). Surface viscosity, surface elasticity, and dilatational moduli are measurements of the cohesiveness, rigidity, compressibility, and extensibility of films (Graham and Phillips, 1976a,b). All are relevant to foam stabilization and indicate the yield strength, elasticity, and flexibility of the film and determine its capacity to withstand the normal shocks and stresses encountered in foams. Films with a yield point and viscosity can dissipate sheardshocks and the elasticity helps restore the film. Excessive viscosity results in brittle films (MacRitchie, 1978).

C. WHEY PROTEIN FILMS

Whey proteins possess good surface active properties. Jackson and Pallansch (1961) reported that the relative rates of adsorption of whey proteins occurring at an interface were in the order BSA > a-La >> p- Lg. Partial unfolding of p-Lg improved its rate of adsorption. Native p- Lg, a-La, and BSA showed rather similar aidwater interfacial adsorption behavior. Heating of a-La and/or p-Lg markedly accelerated the rate of surface pressure development, particularly during the initial 3- to 5-min adsorption period (Jackson and Pallansch, 1961). After 10 min, the surface pressures at surface concentrations of 2 m g h ’ were 12 dynekm for native and 16 dynekm for heat-treated p-Lg. The surface pressure for native a-La was approximately 16 dynekm and 21 dyne/cm for heat- treated a-La, while the value for heated-treated BSA was approximately 15 dynekm. The initial development of surface pressure was closely related to the diffusion coefficient during the first minutes of measurement (Jackson and Pallansch, 1961).

Tornberg reported that p-Lg adsorbed more rapidly than BSA and observed that the ability of whey proteins to form an interfacial film was enhanced by 0.2 M salt. The rate of diffusion and adsorption of whey


protein was greatly decreased below concentrations of 1 mg/dl (Tornberg 1978a,b). Shimizu and co-workers (1981) reported that whey proteins form films containing 2-3 mg tightly polymerized protein per mz of film surface. The amount of protein in the interfacial film was 2.0, 7.6, 3.0, and 2.7 mg/mz at pH 3, 5, 7, and 9, respectively. Analyses of the film material indicated that a-La, BSA, and p-Lg were preferentially adsorbed at pH 3, 5, and 7-9, respectively.

Waniska and Kinsella (1985) demonstrated that the rapid adsorption of p-Lg was accelerated at pH 5.3, close to the isoelectric point, although true steady-state “equilibrium” was not attained until after 360 min. The maximum surface pressure reflected both the adsorption of the protein and the subsequent rearrangement of the adsorbed protein molecules in the film. Maximum surface viscosity occurred in the pH range 5-6 and decreased by 40% at pH 7.0. These values correspond to the pH at which foams of p-Lg show maximum strength. In the case of BSA, surface pressure development, surface viscosity, surface yield stress, and film elasticity all showed maximum values in the pH range 5-6. Significantly, maximum foam stability was observed in the same pH region, indicating a relationship between film and foaming properties (Kim and Kinsella, 1985).

D. FILM-FORMING PROPERTIES OF GLYCOSYLATED P-LACTOGLOBULIN

The balance of forces operating between molecules at the interface is subject to protein-protein and protein-solvent interactions, and disruption of these interactive forces by altering protein structure and conformation may either enhance or diminish protein film-forming properties. Protein hydrophilicity , as well as protein hydrophobicity, net charge, and osmotic and steric effects, contribute to protein film formation since the hydrophilic segments of proteins resist penetration of the interfacial film as this would require dehydration of the tightly bound water groups, a thermodynamically unfavorable process (Cumper and Alexander, 195 1 ; Phillips, 1977; Waniska and Kinsella, 1987).

The cumulative effects of glycosylation on P-Lg were an enhanced molecular weight, reduced net charge, increased relative viscosity, and a significant reduction in the amount of a-helical content. These parameters directly reflected changes in the size, shape, and conformation of the de- rivatized protein molecules. Because of apparent enhanced hydrophilic interactions between the modified proteins in water, a reduction in ionic and hydrophobic interactions resulted in destabilization of the native


structure of p-Lg (Waniska and Kinsella, 1987; Kinsella and Whitehead, 1988).

The added glycosyl moieties on p-Lg enhanced hydrogen bonding between neighboring protein molecules and solvent and alteration of net charge on the molecule caused changes in the nature and magnitude of intermolecular forces between protein molecules in the film, i.e., reduction in sensitivity of the protein to pH because of diminished elctrostatic forces (Kuntz and Kauzmann, 1974; Waniska and Kinsella, 1987). Changes in the hydrodynamic volume of modified proteins apparently contributed to the reduced rate of diffusion to the interface, thereby slow- ing the rate of surface adsorption. Loss of conformational energy upon glycosylation (loss of some secondary structure) resulted in a decreased gain in free energy of the modified proteins upon adsorption at the interface. The combination of these factors reduced the extent of protein interactions, i.e., hydrogen bonding and electrostatic interactions, which resulted in weakened films in foams and emulsions (Kinsella and Whitehead, 1988).

X. FOAMS

A typical foam is composed of millions of bubbles each encapsulated by a protein film and separated by thin water-filled canals (lamella). Foam bubbles tend to assume polyhedral shapes and water is held in the space between adjacent bubbles by capillarity, by binding to exposed polar residues of the protein, and to some extent by negative pressures in the Pla- teau border regions. Maintenance of the lamella is essential for foam stability because contact between adjacent bubbles results in disproportionation, coalescence, and collapse of the foam. Separation of adjacent bubbles is aided by electrostatic repulsion between the adjacent films, by steric hindrance (which retards contact), and by retention of the lamellar water column. This is affected by its viscosity (more viscous fluids are held more effectively), and to some degree by the negative pressure at Plateau borders (Halling, 1981 ; Kinsella, 1981). Foam instability is enhanced by fluid drainage from the lamella due to gravity and rupture of the film resulting from shocks, etc. Drainage can be slowed either by increasing viscosity or by using film materials with polar water-binding groups which possess high surface viscosity. Such features as high yield values and surface viscoelasticity reflect strong cohesion between the protein molecules in the film and also reflect the properties of the component proteins, i.e., their surface activity, molecular flexibility, and ability


to interact with neighboring molecules. Proteins that form cohesive films with high surface viscoelasticity generally form more stable foams (Gra- ham and Phillips, 1976a; Halling, 1981).

The foaming capacity of proteins is related to the rate of decrease in surface tension and rate of film formation, while foam stability very much depends on the nature and strength of the film, reflecting the extent of protein-protein interactions within the film matrix per se (Table XIX). Thus, while flexible proteins like p-casein can reduce surface tension very rapidly and facilitate a large volume increase in a short time, the foam is relatively unstable because of low resistance to shear, reflecting limited interactions. Graham and Phillips (1976a) and Kim and Kinsella ( 1985) have elucidated relationships between film-forming behavior and foaming properties of BSA and p-casein. p-Casein is very surface active and rapidly forms foams, but because of limited protein-protein interactions, the foams collapse easily. In contrast, globular proteins such as BSA, which retain considerable tertiary structure at the interface, form stable foams because of more extensive intermolecular network formation.

A. PROTEIN STRUCTURE AND FOAM STABILITY

The capacity of proteins to form foams is important but the ability to form viscous foams that are stable is required in food applications. Struc- tural features which favor rapid foam formation, i.e., low molecular weight, amphipathic flexible molecules, may not be conducive to the formation of stable foams. Upon formation of a protein-encapsulated bubble, the component proteins should interact extensively via hydrogen bonding, and electrostatic and hydrophobic interactions (and perhaps disulfide bonds) to form a strong viscoelastic, continuous, cohesive, poly-

TABLE XIX SOME FACTORS AFFECTING STABILITY OF PROTEIN-BASED FOAMS

Enhance stability Reduce stability

Increased viscosity of aqueous phase Protein concentration and film thickness Film mechanical strength and yield stress Surface viscoelasticity Gibbs-Marangoni effect Film net charge Heterogeneous proteins with residual

tertiary structure

Drainage (gravitational) Disproportionation Mechanical shockdvibrations Capillary pressureldrainage Permeable film Surface active lipids Temperature Overw hipping


meric, impervious film that retains the air and provides a strong structural matrix for the foam. Several forces act to destabilize a foam but drainage of the lamellar water is a major cause leading to foam collapse. This can be minimized by maintaining capillary hydrostatic pressure, increasing viscosity, and preventing approach and contact of the films of adjacent bubbles; an event resulting in eventual coalescence and rupture of the bubbles. During drainage, van der Waals’ attractive forces between adjacent films increase as the distance apart decreases. These are counter- acted by repulsive forces acting between protein groups, i.e., electrostatic forces, steric hindrance, volume restriction, and osmotic effects between protein moieties. However, over time, bubbles coalesce by disproportionation, films become thin and rupture, and fluid is lost via drainage, resulting in eventual collapse of foams (MacRitchie, 1978; Halling, 1981; Phillips, 1981). The rates of these consecutive events are greatly affected by the properties and composition of the protein films. A heterogeneous population of proteins with a range of properties, for example, the proteins of egg white, form more stable films. Soluble proteins which can orient and interact to form thicker, more viscous films generally create the best foams. It has been reported that acidic-basic protein mixtures generally display improved foaming properties as a result of enhanced electrostatic interactions between protein molecules at the bubble surface (Poole et af., 19876). However, the differences in pls of the acidic and basic proteins must be sufficiently large so that at intermediate pHs, interaction is strong enough to yield good foaming properties. The ability of basic proteins, e.g., lysozyme and clupeine with PI> 9.0, to enhance the foaming power of acidic proteins (e.g., p-Lg BSA) is attributed to not only net charge effects but also to the molecular size and conformation of the complex (Poole et af., 1987b). The addition of salts improves foaming by modifying net charge (masking of net repulsive forces), enhancing adsorption, and perhaps preventing excessive denaturation during foam formation (Halling, 1981; Kinsella, 1981).

For optimal foam formation a protein should rapidly diffuse to the newly created interface and possess sufficient segmental molecular flexibility to spread at the interface, reduce interfacial tension, facilitate surface expansion, and encapsulate the nascent air bubble with a protein membrane. The protein, in adjusting to new thermodynamic conditions, undergoes some conformational changes: the hydrophobic segments or loops occupy the apolar air phase, while the polar and/or charged groups occupy the aqueous phase, and the major bulky tertiary structure occu- pies the interface (Graham and Phillips, 1976a; Phillips, 198 I). Ideally, segments of the protein in the aqueous phase should be charged to repel the adjacent film and entrap water, thereby retarding drainage. Simultane-


ously, there should be extensive protein-protein interactions (ionic, hydrogen bonding, van der Waals’ and hydrophobic interactions) to form a continuous, three-dimensional cohesive film that is impermeable to air and possesses mechanical strength, viscoelasticity, and extensibility in order to stabilize the film against shocks, gravity, and rupture. The component protein molecules in the film (or in the aqueous solution) should possess sufficient mobility to occupy thin or weakened segments of the film. Input of energy by whipping is required to expand and crate new interface and perhaps aid in spreading the protein at the interface.

B. WHEY PROTEIN FOAMS

Whey proteins are reasonably good foamindwhipping proteins but commercial WPC preparations vary immensely in whipping properties because of variability in extent of denaturation of the proteins, high ash content, the presence of lipids and possibly PPs (devilbiss et al., 1974; Richert et al., 1974; Richert, 1979; Phillips et al., 1987). The term “foaming properties” can be misleading, as researchers have reported good foams from whey preparations when the reported overruns may range from 300 to 1500%. Egg white is the premier ingredient in protein-stabilized foams; under normal conditions it forms foams with a 10-fold increase in volume on whipping for 5 min and the foam is stable for 30 min. Most importantly, egg white foam can withstand the addition of other ingredients, e.g., sucrose, and it sets to a rigid, permanent structure on heating. Unfortunately, while whey protein concentrates form good foams, they are not very tolerant of other components and in applications involving heating, the functional performance of whey protein foams is poor.

DeVilbiss et al. (1974) studied the overrun stabilities of whey protein foams from 11 different WPC samples containing from 29 to 88% protein and observed remarkable differences in foaming and drainage that ap- peared to be independent of protein concentration, denaturation, and pH of the respective dispersion. A WPC solution (20% total solids) formed an angel food cake foam similar to that of egg white (devilbiss et al., 1974). However, when the batters were baked at 191”C, the WPC foam collapsed after 15 min of heating. By increasing WPC to 25% total solids, cake volume was retained; however, the texture was crumbly and the cake was filled with large holes, which reflects an unstable foam. In an angel cake, the foam must be capable of incorporating the other ingredients without detriment to the foaming properties and baking. In contrast to egg white, which attained maximum stability after 5 min, WPC required 10 min of whipping time to attain maximum stability. The presence


of fat in excess of 0.5 to 0.9% significantly depressed foaming performance. The angel food cake test provides a practical method for evaluating WPC in an actual application, and such tests may become an integral method of evaluating model foams (Harper, 1984).

In the application of WPC in foam batters, the addition of 44% sucrose to WPC increased their apparent viscosities from 8 to 34 and 9 to 79 centi- poise, respectively, for 14 and 20% WPC total solids (devilbiss et al., 1974). The addition of sucrose also enhanced foam density from 0.16 to 0.2 g/ml, but the foam was too light to hold the other flour ingredients and the batter collapsed on mixing. Increasing the solids concentration reduced the rate of drainage of lamellar water, i.e., at 5 and 15% total solids, the times required for 3 ml to drain were 6 and 15 min, respectively. At around 15% total solids, egg white foam shows superior water retention properties. The addition of sucrose to WPC dispersions containing 20% total solids shows superior water retention compared with egg white, suggesting that drainage is not a major problem with WPC foams containing added sucrose (devilbiss et al., 1974). On whipping of WPC or egg white, there is a progressive increase in the amount of protein denaturation and an enhanced stability of the foam. Heating WPC (10 g/ dl protein) at 55°C for 30,60, and 90 min at pH 4.7 increased the insoluble proteins from 10 to 15 to 2096, respectively. There was a progressive increase in the stability of foams made from WPC containing 10 to 18% denatured protein (deVilbiss et al., 1974).

Richert et al. (1974) studied the effects of heat, pH, calcium, redox potential, and sodium lauryl sulfate on the foaming properties of WPC. Heating WPC dispersions above 70°C for 30 min caused protein aggregation, particularly in the pH range 4 to 5 and this impaired foaming behavior. WPC dispersions heated above 70°C required longer whipping times and produced diminished foams with varying stability; overruns were reduced from approximately 1200 to 700%, the foams were very viscous, dense, and resembled whipped cream, while drainage was markedly reduced. Heating, pH, redox potential, and calcium level all had inconsis- tent effects on whipping time, overrun, drainage, and viscosity of whey protein foams. Higher temperatures of heating and increased reducing conditions reduced the foam volume. Compared to unheated controls, heating at 50", 60", or 65°C for 30 min increased the whipping time from 9 to 13 min, enhanced the overrun, and increased the stability of WPC foams. Heating WPC dispersions from 65" to 70°C improved foaming, whereas higher temperatures impaired foaming properties (Table XX). Maximum apparent stability was observed at approximately pH 7 and 70°C. This has been explained as a balance between the disaggregation effect of pH and the tendency toward aggregation at higher temperatures.


TABLE XX EFFECT OF HEAT TREATMENT ON FOAMING

PROPERTIES OF WHEY PROTEIN CONCENTRATEa

Heating Denatured Overrun Stability (“C/30 min) protein (%) (%I (min)

Unheated 10 800 4 65 20 1500 14 70 15 1500 - 75 23 1350 - 80 38 1150 - 85 60 800 -

“Data after Richert ef al. (1974).

As heating temperature was increased from 65” to 85”C, the denatured protein content increased from 40 to 80%, while the maximum overrun decreased from 1500 to 800%. Increasing the pH from 5 to 7 improved foam stability, and this effect was enhanced at higher temperatures, indicating that “partial” unfolding of whey proteins enhances foaming properties (Richert er al., 1974).

Spray-dried WPC powders prepared from cheddar cheese whey and casein whey, containing 49 to 63 and 76 to 80% protein, respectively, were evaluated for whipping and foaming properties (Haggett, 1976). Dis- persions of 10% protein preparations, adjusted to pH 6.0 or 8.5, were whipped using a typical electric mixer for 6 min. Generally, higher foam volumes, i.e., 1000 versus 1450% overrun, were observed at pH 6.0 and 8.5, respectively (Haggett, 1976). The presence of sucrose in the mix significantly depressed foaming. Heat treatments up to 55°C for 5 to 10 min enhanced whipping, particularly at pH 6.0, and also improved stability. Whey pasteurization enhanced the foaming properties of the treated proteins. Heating WPC containing antifoaming lipids may cause the formation of lipoprotein complexes which precipitate out of the foaming mix and facilitate foam formation (Haggett, 1976).

C. FACTORS AFFECTING THE FOAMING PROPERTIES OF WHEY PROTEINS

The whipping properties of WPC are influenced by the source, method of manufacture, pasteurization, extent of clarification (removal of precipitated materials and lipids formed during pasteurization), methods used


for concentrating the protein (ion exchange, diafiltration, gel chromatography), and conditions of drying. The amount of fat, calcium, and other components present in WPC preparations markedly affected foaming properties (Cooney, 1974; deVilbiss et al., 1974; Richert et al., 1974; Hag- gett, 1976; Richert, 1979; Phillips et al., 1987).

The foaming properties of WPC generally increased with increasing solids content, with an optimum observed around 10 g/dl protein. This has been attributed to the viscosity effect and increased protein concentration. Solids content ranging from 2.5 to 35% and protein from 1.5 to 17.5% yielded overruns from 300 to 1400% without any apparent consistency and foam stability was highly variable. Generally, foam stability increased at higher solids though exceptions have been observed (devilbiss et al., 1974; Richert et al., 1974; Richert, 1979).

The effects of pH are significant, but wide variations have been noted depending on the composition of the whey foam, the salt concentration, and the presence of glycomacropeptides in renneted wheys. Maximum foam volumes in the pH range 5-6 and maximum stabilities have been reported around the isoelectric pH, presumably because of enhanced protein-protein interactions in the film (Cooney, 1974). Richert (1979) has noted the difficulty in concluding anything about the affects of pH on foaming other than it is an important variable which must be controlled. Whey protein concentrates (73% protein) that displayed 90% solubility and were prepared from renneted casein by ultrafiltration and spray drying had excellent foaming properties (Morr, 1985). Overrun was much better at pH 4.5 than at pH 9, while stability was better at pH 9.

Ions, by affecting protein conformation and solubility, influence film formation and hence foaming properties. Some investigators have reported that calcium chloride decreased the overrun and firmness of WPC foams, whereas others reported these properties as improved. Calcium is more effective than equimolar concentrations of sodium in decreasing foam stability and this has been attributed to a decrease in the thickness of the electrical double layer, thereby facilitating coalescence of protein- coated air bubbles (Cooney, 1974). Foam stability decreased linearly with the square root of ionic strength and maximum overrun occurred at 0.05 M NaCl. The effects varied with ion species and concentration; the progressive replacement of calcium with sodium reduced foaming capacity but apparently had little effect on foam stability (Johns and Ennis, 1981).

The addition of sucrose or soluble starch to WPC solutions before whipping decreased overrun (Haggett, 1976). The effects of sucrose on protein solutions are attributed to the increase in viscosity of the solution, but it is also conceivable that sucrose, by increasing the stability of the protein, minimized unfolding at the interface, thereby decreasing foam-


ing. Phillips clearly showed that sucrose improved the heat stability of p- Lg and whey protein foams (Phillips, 1988).

The presence of small amounts of fat in whey protein preparations causes foam instability, particularly if the lipids are composed of mono- glycerides and polar lipids. These may cause desorption and also weaken the protein film. Centrifugation of WPC solutions to reduce lipid content resulted in a marked increase in foam overrun (Cooney, 1974). The addition of phospholipids to WPC solutions increased overrun but decreased stability (Cooney, 1974). Heating WPC solutions reduced the negative impact of the lipid materials, conceivably by enhancing binding to the proteins. The addition of basic proteins (e.g., clupeine) to acidic proteins (e.g., BSA, WPI, WPC) dramatically enhanced the lipid tolerance in protein foaming systems (Poole et al., 1986). However, the foaming power is highly dependent on both the type of acidic protein and the type of lipid incorporated; BSA was more effective than either WPI or WPC. Lecithin, which can disrupt the film more effectively than other types of lipids, can only be tolerated at low levels compared with corn oil and butterfat (Poole et al., 1986). The addition of clupeine markedly improved the foaming performance of whey protein and egg albumen contaminated by lipids, while also withstanding the addition of sugar, and such mixtures could produce novel aerated foods (Poole et al., 1986).

Generally, mild heat treatment induces the partial unfolding of whey proteins, thereby reducing whipping times, increasing overrun, and enhancing the stability of WPC foams. However, several investigators have reported contrary results (devilbiss et al., 1974; Richert et al., 1974; Richert, 1979). The efficacy of heating is influenced by the pH of the protein solution; mild heating (below 70°C) is preferred at acidic pH, e.g., pH 5.0, whereas higher temperatures (above 80°C) are preferred around neutral pH (Richert et al., 1974). Whey protein preparations contain varying quantities of PPs which are surface active and, at concentrations of 1-2%, they can be potent defoaming agents (Volpe and Zabik, 1975). However, it has been shown that the pp was not the foam depressant in whey preparations but a high-molecular weight (> lO0,OOO) lipoprotein component may be responsible (Phillips, 1988).

Because of the confusing state of the literature, it is recommended that foaming properties be evaluated for each particularly designed food system. Generally, the literature indicates that whey protein foams do not possess the same properties as egg white foams and whey proteins cannot presently be used interchangeably without prior modification.

There is a need for standardized methods and criteria in assessing the whipping properties of whey proteins. Furthermore, data relating whipping behavior in model systems to actual food systems is questionable. The approaches of Harper (1984) and deWit (1984) in using appropriate


food systems warrant evaluation. A method has recently been described for the evaluation of foams which offers both practicality and standardization (Phillips et al., 1987).

XI. EMULSIONS

Emulsions are heterogeneous systems consisting of one or more phases dispersed in a continuous phase. Stabilization of an emulsion system is achieved by amphiphilic surface active agents possessing an affinity for both phases. The major function of emulsifiers is to reduce the interfacial energy and facilitate dispersion of the discontinuous phase. In protein- stabilized emulsions, the role of the protein is to form an interfacial membrane rapidly around the oil droplet to prevent coalescence, flocculation, creaming, and oiling-off. Different criteria are required in a fluid compared to a viscous emulsion: in salad dressing, the rheological and viscoelastic properties of the film formed are critical, while in sausage-type products, thermal stability of the film and the ability to set to age1 on cooling are important properties of the component proteins (Kinsella, 1984b).

Protein must be soluble to be an effective emulsifier (Halling, 1981; Kinsella, 1982). The ability of a protein molecule to unfold at the interface, exposing hydrophilic and hydrophobic residues toward their preferred aqueous and nonaqueous environments on either side of the Gibbs’ surface is critical (Dickinson, 1986). The capacity of a protein to produce an emulsion of large interfacial area correlates strongly with its ability to lower the interfacial tension at the oil/water interface. Factors which influence the conformation of protein, i.e., those that facilitate the unfolding of protein at the oil/water interface subsequent to adsorption, may improve the emulsifying properties of whey protein (Hayes et al., 1979; Shimizu et al., 1981).

A. EMULSIFYING PROPERTIES OF WHEY PROTEINS

The adsorption of whey proteins onto the surface of a fat globule is selective and influenced by pH, presence of salts, protein concentration, and temperature (Yamauchi et al., 1980). The creaming stability of whey protein-coconut oil dispersions was minimal at pH 5 , while viscosity and adsorption of protein were maximum at pH 5 , suggesting that emulsion stability is dependent on the electrostatic nature of the proteins (Yamau- chi et al., 1980). However, the effective hydrophobicity, i.e., the abun- dance of apolar groups exposed at the molecular surface, may also be an important factor in protein adsorption because, initially, hydrophobic interactions are predominant between the protein and oil.


Whey proteins can be effective emulsifying agents (Graham and Phil- lips, 1976b; Tornberg and Lundh, 1978; Morr, 1981; Kinsella, 1983) and display average emulsifying properties (dewit, 1981 ; Reimerdes and Lor- enzen, 1983; dewit, 1984). The emulsifying properties of whey proteins may be improved by partial unfolding, especially during the process of emulsion formation, e.g., homogenization in food systems (Tornberg and Hermansson, 1977).

Slack et af. (1986) assessed the emulsifying properties of oil/water emulsions stabilized with either p-Lg-enriched or a-La-enriched WPC samples. Similarities in the data for emulsifying capacities and stabilities between WPC and p-Lg-enriched samples suggest that the origin and processing of whey have little effect on their emulsifying ability. The results also indicate that p-Lg-enriched samples are adequate emulsifying agents while emulsions made with a-La-enriched samples display average emulsion capacity but poor stability (Slack et al., 1986).

The emulsifying activities (EA) of reduced, urea-denatured, and acylated BSA over the pH range 2-10 were determined in order to assess the importance of protein structure and charge on emulsifying properties (Waniska et al., 1981). Reduction of disulfide bonds permitted the BSA to unfold into a more expanded conformation. This resulted in a decreased EA compared with the native protein, suggesting that native BSA, with a greater degree of tertiary structure, formed a stronger and more cohesive film. Furthermore, the EA of reduced BSA was more sensitive to pH changes compared with native BSA, indicating greater response to electrostatic repulsions, particularly at the higher pH values. Complete disruption of the tertiary and secondary structure of BSA by urea eliminated its EA (Waniska et al., 1981).

Shimizu et al. (1981) observed that emulsions of coconut oil stabilized with whey proteins contained three times more protein associated with the interfacial material at pH 5 than at pH 7. At pH 9, p-Lg was selectively adsorbed and was the predominant protein in the isolated interfacial material. At pH 3 the amount of a-La associated with the membrane had progressively increased. Apparently, pH-dependent conformational changes affected adsorption and emulsifying properties, i.e., alkaline- induced molecular expansion of p-Lg causes the protein to become adsorbed more readily, while in the acidic pH range a-La may lose the stabilizing effect of bound calcium, permitting facile adsorption and spreading.

B. MOLECULAR FLEXIBILITY, SURFACE HYDROPHOBICITY, AND EMULSIFYING PROPERTIES

Ample evidence exists which indicates that the ease of protein unfolding and the accessibility of hydrophobic residues at an interface are


closely related (Kato and Nakai, 1980; Kato et al., 1981; Morr, 1981; Voutsinas et al., 1983). The number and disposition of apolar groups contribute to the effective hydrophobicity of proteins by promoting a greater affinity of the protein for the oil phase, which facilitates reorientation of polypeptide segments. Shimizu et al. (1985) investigated the relationship between protein hydrophobicity and emulsifying properties of p-Lg at different pH values. Although the surface hydrophobicity of p-Lg changed on lowering the pH, no significant difference in the secondary structure of the protein between pH 3 and 7 was observed by circular dichroism (CD) spectral analysis. Results from sedimentation velocity analysis, surface tension measurements, and urea and guanidine hydrochloride denaturation experiments strongly indicated that p-Lg has a relatively rigid conformation at pH 3 and resists surface denaturation (Shimizu et al., 1985). This structural feature may explain the reduced protein adsorption and low emulsifying activity observed at pH 3.

Changes in the emulsifying properties of several food proteins, e.g., p- Lg and BSA, were monitored during heat denaturation and the results were correlated with changes in surface hydrophobicity, which were measured with the fluorescent probe cis-parinaric acid (Kato et d., 1983). The emulsifying activity and emulsion stability of all proteins examined displayed a linear correlation with surface hydrophobicity, although protein structure, measured by CD spectroscopy, was altered significantly during heat denaturation, particularly for BSA and p-Lg (Kato et al., 1983). The emulsifying activity of p-Lg and BSA decreased, along with a reduction in the surface hydrophobicity, in proportion with the amount of heat-induced denaturation. This may be explained by the fact that both native BSA and p-Lg are hydrophobic proteins and the significant structural changes observed at the thermal transition points triggered conformational changes which reduced the accessibility of apolar residues to hydrophobic probes.

Kato et ul. (1985) have asserted that the functional properties of proteins, particularly surface active properties, cannot be attributed to surface hydrophobicity alone. For example, a-La shows average emulsifying and foaming properties but has a low surface hydrophobicity (Kato et al., 1985). A possible relationship between protein flexibility and surface behavior has been pointed out by several investigators (Shimizu et al., 1981; Townsend and Nakai, 1983; Kato et al., 1985, 1986). The presump- tion that flexible protein molecules are more susceptible to surface denaturation (and thus to molecular rearrangements) at interfaces than rigid protein molecules has been demonstrated by proteolysis of adsorbed proteins (Kato et d., 1985, 1986). BSA and p-Lg are susceptible to protease digestion and this correlates with a high emulsifying activity index compared with lysozyme and ovalbumin, which were resistant to protease


digestion and displayed inferior emulsifying properties (Kato et al., 1985). Since protein unfolding is known to increase protease susceptibility (Pri- valov, 1979), it is probable that ovalbumin and lysozyme retain more folded conformations at the interface because of their more compact conformations since they form less cohesive films. Thus, molecular flexibility along with surface hydrophobicity may be considered important structural factors governing the surface properties of proteins.

C. FACTORS AFFECTING EMULSIFYING PROPERTIES

Compositional factors such as lipid, ash, and sulfhydryl content also contribute to the emulsifying properties of whey protein concentrates. Peltonen-Shalaby and Mangino (1986) have proposed using the sulfhydryl content of WPC samples to predict the performance of various WPC samples in aerated emulsion systems.

Whey proteins had good emulsifying properties in fluid emulsions but were less effective than isolated p-Lg, which was inferior to casein, in ease of emulsion formation (Pearce and Kinsella, 1978). Heat-denatured WPC in solutions of sodium phosphate displayed good emulsifying properties at pH 6.4 and maximum stability at pH 7.0 (Mutilangi and Kilara, 1985). The emulsifying capacity of whey proteins is greatly affected by the extent of denaturation and loss of solubility and this may account for some of the variations observed in the emulsifying properties of different whey preparations. The emulsifying systems used in many studies which rely on a high energy input may partly overcome this problem by enhanced physical spreading of the protein at the interface during homogenization. The various factors which affect the stability of emulsions made with milk proteins have recently been reviewed by Leman and Kinsella ( 1989).

XII. LIGAND BINDING BY WHEY PROTEINS

A. LIPID BINDING

The interaction between ionic surfactants such as sodium dodecyl sulfate and whey proteins is pH dependent (Brown, 1984). Apparently, the initial interaction (in the pH range 5.5 to 7.0) is electrostatic, followed by hydrophobic interactions between the lipid hydrocarbon chain and the protein (Brown, 1984). This is consistent with the fact that, at pH 5 , a greater amount of whey protein remains with emulsion droplets that have been subjected to low centrifugal forces than at pH 7, where only one-


third of the protein remains associated with the emulsion (Yamauchi et al., 1980). Binding of ionic surfactants to p-Lg at high molar ratios of surfactant to protein (S/P = 1600) increased the -helical content to twice that observed when the protein is in buffer alone (Mattice et al., 1976) and perhaps this stabilizes the protein against thermal denaturation (Hegg, 1980).

Both p-Lg and BSA can bind a variety of apolar molecules, especially fatty acids and amphipathic lipids (McMeekin et al., 1949). Binding of the detergent n-octyl benzene-p-sulfonate (OBS) to p-Lg is concentration dependent: at low OBS concentrations, the p-Lg dimer bound two to three molecules of detergent (intrinsic dissociation constant of 1.59 x lop5 M) and binding was attributed to hydrophobic interactions between the protein and the apolar tail region of the OBS molecule (Hill and Briggs, 1956). At higher detergent concentrations, a stoichiometry of 1 mol of detergenvmol of protein monomer for native, S-carboxymethy- lated-, alkylated-, and carboxypeptidase A-hydrolyzed p-Lg was observed and the binding interaction involved the charged head group of the OBS molecule with basic amino acid residues of p-Lg (Seibles, 1969).

Recently, studies using gel filtration chromatography demonstrated that p-Lg can bind p-nitrophenyl phosphate @-NPP), an analog of retinol, with a stoichiometry of 1 mol ligand per 18,360-Da monomer (Farrell et al., 1987). Fluorescence intensity of the protein was quenched upon binding p-NPP, and the CD spectra (260 to 300 nm region) implicated tryptophan and phenylalanine residues in the binding of the ligand (Farrell et al., 1987). These results coupled with the K D values obtained from gel filtration clearly indicated the formation of a complex between p-Lg and p-NPP. The KD for the p-Lg-p-NPP complex is independent of pH in the range 4.0 to 7.5. In addition, a complex of p-Lg and dodecyl sulfate bound retinol in an manner analogous to the native protein (Fugate and Song, 1980). Robillard and Wishnia (1972) estimated that the apolar binding site of p-Lg may be limited in size to single ring aromatics. The tryptophan and phenylalanine residues which are purportedly involved in binding of p-NPP (or retinol) have been located at or near the p-barrel structure recently elucidated by crystallographic studies (Sawyer et al., 1985 ; Papiz et al., 1986; Monaco et al., 1987). This structure has been identified as an ideal structural motif for the transport of small apolar molecules such as retinol (Sawyer, 1987). Retinol forms the tightest binding complex with p-Lg (Table XXI) and the strong structural homology between retinol- binding protein (RBP) and p-Lg points to a possible evolutionary and functional relatedness (Sawyer et al., 1985; Papiz et al.. 1986). However, further experimental evidence is required in order to establish the biological role of retinol binding and transport for p-Lg (Sawyer, 1987).


TABLE XXI COMPARISON OF THE DISSOCIATION CONSTANTS ( K D ) OBTAINED FOR

BINDING OF NONPOLAR COMPOUNDS BY P-LACTOGLOBULIN A

Ligand KD ( P ‘ w Reference

Toluene 2200 Robillard and Wishnia (1972) Pyridoxal phosphate 320 Farrell ef al. (1987) p-Nitrophenyl phosphate 31 Farrell el a / . (1987) Retinol 0.020 Fugate and Song (1980)

B. FLAVOR BINDING

Because overall flavor, as perceived by the consumer, is important in determining the acceptability of many foods, interactions of flavors with proteins is of practical interest. A problem rarely mentioned is the off- flavor associated with whey proteins. The components responsible for off-flavor development have not been quantitatively identified, but may include some volatile fatty acids, carbonyls from oxidized lipids (e.g., phospholipids, lactic acid/esters), and amino acid products. These conceivably are bound to whey proteins such as BSA and p-Lg, both of which have a high affinity for apolar ligands (O’Neill and Kinsella, 1987a).

The binding of flavors and off-flavors to food components, especially proteins, is a challenging problem as food technologists attempt to fabri- cate and flavor new foods using protein ingredients. For example, the flavor characteristic of meats consists of at least 400 flavors and, to manufacture meat analogs with the appropriate flavor, correct concentrations, binding characteristics (i.e., relative binding affinities, release rates, and partitioning of individual flavor components between food components), and, particularly, partitioning between the complex food medium and the appropriate oral chemoreceptors need to be determined. The consequences of flavor binding to the food industry are actually twofold because, in addition to off-flavor development, loss of desirable flavor in the formulated food may also occur (Mills and Solms, 1984). Further- more, though functionally acceptable, many protein preparations possess undesirable flavors, and release of these reversibly bound off-flavor compounds hinders their more widespread use in foods (Kinsella and Damo- daran, 1981).

The ability to either mask undesirable flavor(s) or simulate the desired food flavor is significantly influenced by the flavor-binding capacity of the chosen protein. The structure of some proteins may enhance or dimin-


ish their capacity to adsorb added flavors and variable amounts must be added to obtain the desired flavor impact. Binding of flavor compounds by protein results in the suppression of their primary flavoring impact. Therefore, techniques for controlling adsorption of specific flavors must be sought and used in fabricated foods. The excessive binding of flavors by proteins, the uneven retention of flavors during processing treatments and storage, and/or the preferential release (or retention) of some components of a flavor blend during mastication, are problems confronting the manufacturer of fabricated foods. Because the perceived flavor is ulti- mately most important in determining food acceptability, the phenomenon of flavor binding and release is extremely significant.

Information concerning the interaction of selected flavor compounds has previously been reviewed (Beyeler and Solms, 1974; Franzen and Kinsella, 1974; Damodaran and Kinsella, 1980; Kinsella and Damodaran, 1981; Kinsella, 1989). For the fabrication of food, knowledge of the affinity of food components for flavor compounds and the differences in binding characteristics of different food components are critically important. Quantitative data are needed to determine the binding of flavors to proteins in order to compare the binding affinities of different proteins, and to determine if there are differences between the extent of binding of components in a flavor blend. Furthermore, the rate and extent of release of flavor compounds from proteins during ingestion and mastication are important. However, little information is available in this highly relevant area.

The binding of saturated aldehydes and methyl ketones (e.g., heptanol and nonanone) to unprocessed whey protein has been studied, with emphasis on the effects of lactose, salt, and residual fat, in order to understand off-flavor development in commercial products (Mills and Solms, 1984).The lactose (up to 82 g/lOO g protein) and salt (0.5 M) contents of all samples tested had little effect on the binding of heptanol; however, the effect was more marked with nonanone. Very little difference in the binding of heptanal was observed when the fat content of the whey powder was reduced from 4.82 gAO0 g protein to 0.91 g/lOO g protein, whereas the binding of nonanone decreased by 50% with the same decrease in fat content (Mills and Solms, 1984).

The effect of pH and temperature on the binding of flavor compounds is of particular interest since many different conditions of pH and temperature are employed during the industrial isolation of whey proteins (Mar- shall, 1982). At pH 6.89 and 25"C, the binding of heptanal was greater than the binding of nonanone, while at pH 4.66, the opposite effect was observed (Mills and Solms, 1984). As temperature was increased from 25" to 50°C. the amount of heptanal irreversibly bound to whey protein


increased to the extent that, at the highest total heptanal concentration, only 10% of the heptanal bound at 50°C was released, while binding of nonanone was completely reversible at both pH values and temperatures (Mills and Solms, 1984). Thus, the binding of some classes of compounds to whey protein may be achieved by careful choice of pH, temperature, and processing conditions to minimize the level of bound off-flavor compounds.

The predictable effects of various flavors bound by proteins are also complicated by the highly variable, intrinsic properties of the protein components in a food system, i.e., conformation, state of the protein (native/denatured), surface area and topography, and presence of other factors, such as lipids and additional nonspecific interactions between the flavor compounds and proteins (Franzen and Kinsella, 1974; Damodaran and Kinsella, 1980). In order to optimize flavoring of food proteins and to develop practical methods for the removal of off-flavors, the mecha- nisms and thermodynamics of flavor binding need to be understood. Stud- ies to elucidate the characteristics of flavor-protein interactions have been attempted by employing the classical approach for protein-ligand associations (Damodaran and Kinsella, 1980).

For a protein (P) having a number of equal and independent binding sites, the interaction between the ligand, i.e., flavor molecule (L), and the protein may be represented by the equation.

P + nL = PL,

Based on this model, the interaction between flavor molecules and protein can be represented thermodynamically by the Scatchard equation:

- - V/[L] = nK - VK

where f? is the number of moles ligand bound/mole protein, [L] is free ligand concentration, n is total number of binding sites, and K is the intrinsic binding constant. A plot of f?/[L] versus f?gives a straight line with a slope of - K and an intercept equal to nK. This equation assumes no protein-protein interactions at higher concentrations and may only apply to single polypeptide chains, e.g., BSA or p-Lg, such that, at a given free ligand concentration, the molal ratio of binding is the same irrespective of protein concentration (Kinsella and Damodaran, 198 1). When analyzing binding, it is important to measure accurately the amount of ligand that is actually associated with the protein. Using a liquid-liquid partition equilibrium method, the kinetics and thermodynamics of flavor binding to BSA and p-Lg have been investigated. The protein in solution was


equilibrated with the flavor ligand and the amount of flavor bound to BSA or p-Lg was determined by gas chromatography (Damodaran and Kinsella, 1980; O'Neill and Kinsella, 1987a).

Differences in the slopes of the binding curves of 2-heptanone and 2- nonanone indicated differences in the affinity of these components for the binding sites in BSA. The initial number of binding sites (approximately 6 to 7) was similar for both compounds. Increasing the chain length of the ligand by two methylene groups increased the binding energy sixfold, indicating that hydrophobic interactions were dominant in the binding of these apolar ligands to BSA. The curvilinear relationships of the binding isotherms were positive, possibly reflecting unfolding of the protein molecule at higher ligand concentrations and resulting in the exposure of nonspecific binding sites which are unavailable in the native protein. Such structural changes were monitored from changes in the UV absorption and fluorescence emission spectra of the protein-ligand complex. Thus binding of these ligands at low concentrations stabilized the BSA molecule initially, but above this concentration the molecule began to unfold (Damodaran and Kinsella, 1980). The presence of certain types of salts affected flavor binding. Ligand binding affinity progressively increased with anion concentration (SO2; > C1- > Br-), in the order of the lyo- tropic series of anions. This is the order in which these anions stabilize protein structure via enhancement of hydrophobic interactions (Damo- daran and Kinsella, 1981a).

C. LIGAND BINDING BY P-LACTOGLOBULIN

p-Lg and RBP display a strong sequence homology with respect to several amino acid locations that have been demonstrated to be critical for the maintenance of structure and retinol binding (Pervaiz and Brew, 1985). X-Ray crystallographic analyses reveal striking conformational similarities between p-Lg and RBP, most notably in a cross-hatched, eight-stranded 9-barrel (the core of which is lined with apolar side chains) (Newcomer et al., 1984; Papiz et al., 1986). This particular structural feature has been identified in several extracellular transport molecules and is ideally suited for binding small apolar molecules (Sawyer, 1987). Two molecules of retinol are bound per dimer of p-Lg with a dissociation constant of 2 x lo-" M. The binding is primarily hydrophobic in nature because it is pH independent, and is unaffected by the presence of urea (8 M) or SDS (Fugate and Song, 1980). The TrpI9 residue (located at the bottom of the p-barrel) apparently is the binding site for the p-ionone moiety of retinol in RBP and is highly conserved in p-Lgs from several species sequenced to date (Newcomer et al., 1984; Pervaiz and Brew,


1985). Although the binding of retinol by p-Lg could reflect a general affinity for small apolar molecules, the relationship between p-Lg and RBP infers that binding is a significant and specific phenomenon that suggests a specific biological function for p-Lg (Pervaiz and Brew, 1985). p-Lg is remarkably stable at pH values below 3.0 (Kella and Kinsella, 1988), an attribute that enables the protein molecule to remain intact under the prevailing acidic conditions of the stomach (Reddy et al., 1988). Thus, it is possible that a biological role of p-Lg may be vitamin A transport in the bovine neonate via p-Lg-specific receptors in the small intestine (Papiz et al., 1986).

p-Lg readily binds carbonyls, e.g., alkanones and methyl ketones (O’Neill, 1986), and the double reciprocal plots of 2-heptanone, 2-octanone, and 2-nonanone binding to native p-Lg B indicate that there is one binding site per p-Lg monomer. The free energies of association are compared with those of BSA (Table XXII) (Damodaran and Kinsella, 1981b; O’Neill and Kinsella, 1987a). The binding affinity of alkanones for p-Lg increases with chain length and for each additional methylene group there is a corresponding change in free energy of -3.36 kJ/mol. The effect of chain length on the free energy of association suggests that the protein-ligand interaction is primarily hydrophobic in nature. The binding constants for the interactions of alkanones with p-Lg are higher than those obtained for either BSA or soy protein (Damodaran and Kinsella 1980, 1981b; O’Neill and Kinsella, 1987b). A binding constant of 930 M-’ for p-Lg was obtained for heptanone, which is approximately 2.5 times that of soy protein (O’Neill and Kinsella, 1987b).

Urea at increasing concentrations caused unfolding of p-Lg and pro-

TABLE XXII THERMODYNAMIC (AG, kl/mol) AND BINDING CONSTANTS ( K , M-’) FOR

BOVINE SERUM ALBUMIN AND P-LACTOGLOBULIN”

Ligand

2-Heptanone 2-Octanone’ 2-Nonanone

Protein‘ K AG K AG K A

BSA 270 - 13.8 (810) (- 16.6) 1800 - 18.5 P-Lg 150 - 12.5 480 - 15.4 2440 - 19.4

“Data from O’Neill and Kinsella (1987a) and Damodaran and Kinsella (1980). ”Constants in parentheses are calculated values. ‘Protein concentration was 0.974% at 25°C in 1 M NaCI.


gressively decreased the binding affinity of alkanones, although the number of binding sites remained unchanged (O’Neill and Kinsella, 1987a). Heat treatment of p-Lg at 75°C caused an increase in the number of binding sites for alkanones but the binding affinities of the protein decreased because of heat-induced conformational changes (O’Neill and Kinsella, 1988). Similar results were observed when the free carboxyl groups of p- Lg were esterified, possibly reflecting a tendency of the modified protein to form hydrophobically associated aggregates (O’Neill and Kinsella, 1988).

Thus, the nature and extent of the interactions between flavors and proteins are altered by heat treatment, and proteins may bind more or less of a given flavor compound, depending on the intensity of heat treatment. Because heat-denatured p-Lg forms aggregates with an increased number of binding sites, the strength of the binding is reduced; hence, substitution of heat-treated p-Lg for native p-Lg in a whey-based flavored food might result in increased binding of that flavor than allowed for in the original flavor formulation, resulting in a stronger or unbalanced perceived flavor.

D. INTERACTIONS OF FLAVORS WITH WHEY PROTEINS

a-La also binds aldehydes and methyl ketones (Franzen and Kinsella, 1974; Jasinski and Kilara, 1985), although the binding capacity is lower than that of p-Lg. Interestingly, whey protein preparations (88% protein) exhibit a very high flavor-binding capacity that apparently exceeds the sum of the binding capacities of the component proteins (Jasinski and Kilara, 1985). This may reflect unfolding and/or denaturation of the proteins, which increases the number of nonspecific binding sites (Franzen and Kinsella, 1974; O’Neill and Kinsella, 1988), and also the presence of lactose, which avidly binds flavors (Nickerson er al., 1976).

In considering the binding of flavors to proteins, the association constant is an index of the tendency of that compound to partition to the protein and bind. For sensory perception of the flavor, it must be released from the protein and bind to the chemoreceptor complex; if the binding association is very high, the release rate is low and there is minimal avail- ability of flavor for perception. The rate of association which reflects the diffusion rate constant may be on the order of 10” to 10”. If the association constants are around lo8, then the equilibrium of the on/off reaction rates means that a sizable concentration of the flavor is in the unbound form and available for detection. Thus, the association constant may be a useful index of the efficacy with which flavors are released from food components.

It is relatively easy to formulate a blend of flavors in free solution to


provide a desirable sensory impact. However, if a similar concentration is blended in the presence of proteins or other components which bind specific flavors to differing extents, the perceived flavor may not be the same as the original blend. This is a major challenge in formulating food flavors for fabricated and processed foods. Because the difference between desirable and undesirable flavor impact is often one of concentration, knowledge of the flavor binding behavior of food components is critical in determining the acceptability of foods.

XIII. MODIFICATION OF WHEY PROTEINS

The importance of functional properties for the proper utilization of whey proteins underscores the need to develop methods allowing manipulation of certain properties in order to suit specific applications. Modifi- cation of proteins to alter native chemical and physical properties has been reviewed (Kinsella, 1976; Feeney and Whitaker, 1977; Kinsella and Shetty, 1979; Kinsella, 1982; Richardson and Kester, 1984; Richardson, 1985; Kinsella and Whitehead, 1987). Whey protein modification can be accomplished by enzymatic, chemical, physical, or genetic methods (Feeney and Whitaker, 1977; Fujimaki et af., 1977; Kinsella and Shetty, 1979; Fox et al., 1982; Richardson and Kester, 1984; Richardson, 1985; Kinsella and Whitehead, 1988).

A. ENZYMATIC MODIFICATION

Protein modification via enzymes generally involves limited proteolysis to yield a mixture of polypeptides. The use of enzymatic modification methods has the advantage of milder reaction conditions and the potential for stereochemical specificity (Adler-Nissen, 1986; Dickinson and Stain- sby, 1987). Apparently, there is an optimum degree of hydrolysis beyond which any improvements gained in functional behavior are lost (Mc- Nairey, 1984) and there are different degrees of hydrolysis for certain properties, e.g., emulsification and foaming, but the reason for this is un- clear. Interpretation of the physicochemical data of partial hydrolysates is complicated by the general lack of information concerning distributions of fragment sizes and peptide composition (Dickinson and Stainsby, 1987).

Enzymes may be used to form intermolecular cross-links, or attach or remove specific functional groups to the protein. Treatment with proteases reduces molecular size and may enhance the hydrophobic/hydrophilic balance, either by generating extra terminal amino and carboxyl


groups or by attaching hydrophobic substituent groups, such as L-leucine N-alkyl esters via the plastein reaction (Yamashito et al., 1979; Arai et al., 1986) to enhance the surface active properties of proteins. Partial proteolysis may facilitate unfolding of polypeptides and thereby enhance certain functional properties, e.g. solubility and also increase the heteroge- neity of the protein species (peptide molecular weight distribution), which may enhance foaming properties. Deamidation has been reported to improve many functional properties, particularly solubility, of proteins (Matsudomi et al., 1982). Enzymatic deamidation of glutaminyl and asparaginyl residues in proteins offers advantages over mild acid treatment because the latter treatment results in denaturation of protein and also cleavage of peptide bonds (Kato et al., 1987).

Limited proteolysis has been used to improve the foaming properties of whey protein preparations (Kuehler and Stine, 1974; Horiuchi et al., 1978), but because of inadequate protein-protein interactions in the protein films, proteolysis reduces foam stability. Partial proteolysis of whey protein concentrates with trypsin greatly improved thermal stability and improved emulsifying properties (Hidalgo and Gamper, 1977).

During proteolysis, there is a tendency to form bitter-tasting peptides (Kilara, 1985), although this is less of a problem with whey proteins than with caseins (Richardson and Kester, 1984). However, enzymes have been utilized in plastein reactions to reduce bitter peptides produced by limited proteolysis (Noguchi et al., 1975; Eriksen and Fagerson, 1976). Overall, proteolytic treatment may provide a practical approach for increasing the use of whey proteins in beverages because of improvement in solubility and stability in acid pH and to heating. (Dickinson and Stain- sby, 1987).

Enzymes have been used to add functional groups to proteins, e.g., attaching of phosphoryl groups using protein kinase (Bingham, 1976). In addition, transglutaminase has been used to form new intermolecular cross-links, thereby modifying the viscosity, surface activity, and gelling properties of whey proteins (Nio et al., 1986a,b). Tanimoto and Kinsella (1987) demonstrated that cross-linking of p-Lg using transglutaminase increased viscosity and greatly improved the heat stability of the cross- linked protein compared with native p-Lg.

B. CHEMICAL MODIFICATION

A limited amount of work has been conducted on the chemical modification of whey proteins. A number of the functional side chain groups in a protein, particularly the €-amino group of lysyl residues, can be modified to create protein products with remarkably different chemical and

418 J. E. KINSELLA AND D. M. WHlTEHEAD

physical characteristics. For example, whey proteins with a number of reactive polar ester or amino groups can be chemically modified to improve their surface activity (Feeney and Whitaker, 1977; Richardson, 1985; Arai et al., 1986). Methods to modi€y whey proteins chemically include acylation, phosphorylation, amidation, esterification, reductive alkylation, and thiolation (Richardson and Kester, 1984).

1 . Acylation

Acylation of protein r-amino groups with acid anhydrides is a common method of protein modification. Acetylation eliminates a positive group which reduces the electrostatic attraction between charged groups and reduces the tendency toward gelation (Richardson and Kester, 1984). Succinylation enhances hydration and solubility, improves thermal stability and surface active properties, and generally results in unfolding of the protein molecule as extent of modification increases (Waniska et al., 1981; Shetty and Kinsella, 1982). This is reflected by a marked increase in intrinsic viscosity, an indicator of molecular shape and size, because of the increase in electrostatic repulsive forces, i.e., increase in net negative charge (Shetty and Kinsella, 1982). Presumably, the higher net negative charge of succinylated proteins (at a given pH) should impart enhanced charge repulsive forces between protein encapsulated air or oil droplets at interfaces, thus favoring more stable foams or emulsions (Kinsella, 1976, 1982). Succinylated BSA (>90% lysyl residues modified) exhibited improved emulsifying activity, above pH 5 , compared to native BSA (Wani- ska et al., 1981), reflecting a more flexible conformation that facilitates diffusion to the interface and rearrangement of polypeptide segments within the interfacial film (Phillips, 1981). However, acetylation of BSA had little effect on protein solubility and resulted in a reduced emulsifying activity between pH 4 and 7 (Waniska et al., 1981).

Studies on foams generated from succinylated p-Lg suggest that ionic forces exert considerable influence on molecular interactions in the surface film (L. Phillips, 1988). This is shown by a progressive increase in surface pressure at 25% succinylation of the protein with a concomitant reduction in foam stability of about 3-fold. Although more protein was initially available at the interface as a result of relaxing of protein conformation, critical protein-protein interactions, i.e. , ionic, hydrophobic, and possibly steric, were diminished to an extent that inhibited stable film formation.

Succinylation has been used to enhance the functional behavior of heat- denatured whey proteins. Succinylation of heat-coagulated WPC improved several properties, e.g., solubility and emulsifying power, but did


not improve whippability. The modified proteins absorbed 12-fold and 2- fold the molecular weight in water and fat, respectively, and displayed excellent emulsifying properties (Thompson and Reyes, 1980). This product was an effective replacement for sodium caseinate in coffee whiteners, egg yolk in salad dressings, and NDM in meat patties, ice cream, and instant pudding (Thompson and Reyes, 1980; Thompson et al., 1982).

2 . Phosphorylation

Phosphate groups can be covalently attached to free €-amino groups on proteins with phosphorus oxychloride to increase the net negative charge (Woo et al., 1982; Woo and Richardson, 1983; Matheis and Whitaker, 1984). Chemical phosphorylation of proteins may also involve derivatization of the hydroxyl group of serine, the imidazole nitrogen of histidine, and other free amino groups. Furthermore, it may cross-link proteins to varying degrees via phosphate bridges or isopeptide linkages (Woo et al., 1982). Phosphorylation generally enhances several physical properties of proteins, such as solubility (by increasing net hydration of protein chemical groups), electrostatic repulsive forces, water absorption, water-holding capacity, and viscosity, all of which can be exploited for use in commercial food products.

The lysyl residues of p-Lg were phosphorylated with phosphorus oxychloride, incorporating approximately 13. mol phosphate per mole of protein and resulted in loss of the native protein conformation (Woo et al., 1982). Phosphorylated p-Lg readily gelled without heating, via calcium cross-linking, to yield a fluid, yogurt-like gel (Woo and Richardson, 1983). Modified p-Lg also displayed markedly improved emulsion stability (creaming properties), presumably because of enhanced electrostatic repulsive forces between emulsion droplets.

3. Amidation and Esterification

Generally, proteins possess a net negative charge around pH 7 and this charge can be effectively reduced by derivatization of the carboxyl group of the aspartic and glutamic acid. residues. Amidation is accomplished via a carbodiimide-mediated condensation of ammonium ions with carboxy- lic groups forming asparagine and glutamine, respectively. Esterification is achieved using an acidified alcohol medium (Mattarella et al., 1983). Amidation of 78% and esterification of 83% of-the carboxyl groups of p- Lg increased its isoelectric pH to 10 and 9.8, respectively. Intermediate levels of esterification yielded proteins with a range of isoelectric points (Mattarella et al., 1983; Mattarella and Richardson, 1983; Halpin and


Richardson, 1985). Modification increased the random configuration of p- Lg. These positively charged derivatives may form complexes with negatively charged protein components, in the neutral pH range, because of strong electrostatic interactions. This may improve emulsifying and foaming properties since the increased association of modified whey proteins with other proteins during film formation should increase the viscosity of films, resulting in enhanced foam and emulsion stability (Poole et al., 1987b). Richardson and Kester (1984) reported that ethyl-esterified p-Lg absorbed to the interface four times more rapidly than the nonmodified protein. Further research on the properties of amidated/esterified whey proteins is warranted.

4. Thiolation

The presence of thiol and disulfide groups in proteins markedly affects their functional behavior and provides an excellent approach for modification via disulfide reduction, thiol oxidation, and thiol-disulfide interchange reactions (Cup0 and Pace, 1983; Kella et al., 1986). Alteration of molecular conformation, protein flexibility, and intramolecular cross- linking to enhance solubility, adjust intermolecular interactions, and improve viscosity and network formation necessary in gelation is possible. Free thiol (-SH) groups are involved in many important reactions of proteins required in gelation, thermostability, viscosity, and complexation with other proteins. Reduction of p-Lg with mercaptoethanol or dithio- threitol enhanced its aggregation tendencies and increased its flavor-binding capacity (O’Neill and Kinsella, 1988).

Thiolation of proteins is generally accomplished using N-acetylhomo- cysteinethiolactone (N-AHTL) and S-acetylmercaptosuccinic anhydride (S-AMSA) (Feeney and Whitaker, 1977; Richardson and Kester, 1984). Thiolation with S-AMSA involves alkylation of the amino groups so that the -SH function is protected in the form of an acetylthiol group. The acetyl group is subequently removed by nucleophilic displacement with hydroxylamine (Richardson and Kester, 1984). The reaction with N-AHTL under alkaline conditions involves an imidazole-catalyzed alkylation of the amino groups of the protein followed by opening of the thio- lactone ring with exposure of a new -SH group (Richardson and Kester, 1984). 0-Lg has been successfully thiolated using both of these reagents, which acylated the e-amino groups and reduced the isoelectric pH of de- rivatized p-Lg compared to the native protein. Intermolecular disulfide cross-linking occurred on oxidation of thiolated p-Lg and resulted in high- molecular-weight polymers that apparently possessed enhanced resistance to heat, increased viscosity, improved foaming power, and gelation

PROTEINS IN WHEY 42 I

properties (Richardson and Kester, 1984). In the presence of calcium ions, these polymers formed strong, transparent, heat-stable gels.

5. Reductive Alkylution

Methylation or ethylation of amino groups can be accomplished via a reaction using formaldehyde and a strong reducing agent, such as sodium borohydride or cyanoborohydride. Reductive methylation causes minor changes in the conformation of proteins. This is a useful way of labeling proteins with radioactive derivatives, such as [ ''C]formaldehyde. In this way, any changes occurring during processing may be easily monitored in order to study factors affecting protein-protein interactions, surface accumulation of p-Lg, etc. (Richardson and Kester, 1984).

6. Glycosylution

Is it possible to alter the size, conformation, and physicochemical character of proteins with high-molecular-weight derivatives, e.g., single sugar groups, using relatively mild conditions (Waniska and Kinsella, 1984b). Covalent attachment of carbohydrate residues of varying sizes, e.g., mal-

TABLE XXl l l DIFFERENCES I N COMPOSITION BETWEEN HUMAN A N D

BOVINE MILK"

Human milk Bovine milk

gldl 76 ddl 96

Total proteins Caseins Whey proteins

a-Lactdlbumin P-Lactoglobulin Lactoferrin Serum albumin Lysozyme k A IgG IgM Others Lactose Minerals

0.89 0.25 0.64 0.25

0.17 0.05 0.05 0 . 10 0.003 0.002 0.07 7.4 0.2

-

100 35 65 17

17 6 6

I I

-

- 8

3.30 2,60 0.70 0.12 0.12 Trace 0.03 Trace 0.003 0.06 0.003 0.15 4.8 0.8

I 00 79 21 2.5 9.0

I .0

3.0 -

- 4.5

"Data after Hambraeus (1984).


tosyl and p-cyclodextrinyl, to the €-amino groups of p-Lg altered molecular size, conformation, and several physicochemical properties, including surface hydrophobicity (Waniska, 1982; Kinsella and Whitehead, 1988). Many surface active properties, including the foaming characteristics, of p-Lg were improved as a result of alterations in the molecular nature of the protein induced by the introduction of sugar moieties, i.e., net charge, hydrophobicity, and hydrophilicity (Waniska and Kinsella, 1984a, 1987; Kinsella and Whitehead, 1987). Synthetic glycoproteins of p-Lg have been reported to display high solubility at low ionic strength and improved heat stability compared to the native protein (Kitabatake et al., 1985).

XIV. NUTRITIONAL ASPECTS OF WHEY PROTEINS

Infant formula provides a significant potential market for refined whey proteins. Ideally, in preparing commercial formulations the end-product must simulate as close as possible the composition of human milk, particularly with regard to protein content. The disparities in the caseidwhey protein ratios and lactose content between human and bovine milks also require adjustment.

In human milk, casein (2.5 g/liter) accounts for only 20% of the total nitrogen. Whey proteins represent 70% of human milk protein and consist mostly of a-La, lactoferrin, lysozyme, y-globulins, and serum albumin (Table XXIII) (Hambreus, 1984). Trace amounts of p-Lg may be present. Lactoferrin is an iron-binding protein which can be involved in supplying iron to the child by facilitating intestinal absorption, in addition to having an apparent bacteriostatic effect and possibly stimulating development of the gastric mucosa (Reiter, 1985). Lysozyme is relatively abundant in human milk and also exerts an antimicrobial function in the intestinal tract (Wharton, 1980; Hambreus, 1984). Lysozyme, like lactoferrin, is relatively resistant to proteolytic digestion. These proteins are present in trace levels in bovine milk. Human milk also contains significant amounts of immunoglobulins (IgG fraction) compared to bovine milk.

In the manufacture of infant formula, bovine milk is fortified with additional whey to correspond with the ratio of casein to whey protein (20 : 80) present in human milk as closely as possible. The whey is demineralized by ion exchange, electrodialysis, and/or UF/DF to adjust the concentration of minerals, lactose, and NPN materials in the diluent whey (Mar- shall, 1982; Morr, 1982; Jost e? al., 1987). However, protein compositional differences still exist because bovine milk contains p-Lg, which accounts for approximately 60% of total whey protein whereas human


milk contains higher levels of a-La, lactoferrin, lysozyme, and immunoglobulins and lacks P-Lg. It has been suggested that addition of bovine colostrum may “boost” immunoglobulin concentrations in infant and baby foods.

There is concern about limited pepsin digestibility of p-Lg and the aller- genic reaction frequently elicited by bovine p-Lg when present in sufficient quantities; consequently, there is interest in reducing and/or elimi- nating it from infant formula. Thus, methods such as gel filtration, selective precipitation, and enzymatic hydrolysis have been pursued to reduce or remove p-Lg from whey.

The addition of ferric chloride (7 mM) to whey at pH 4.5 selectively precipitates p-Lg (Kuwata et al., 1985). However, this method also results in removal of the immunoglobulins from the precipitate. If the whey is adjusted to pH 3 in the presence of ferric chloride (4 mM), then all proteins except p-Lg remain in solution. Excess iron is then removed by ion exchange or U F to yield a whey protein preparation devoid of p-Lg. Selective heat precipitation by pH adjustment is also an alternative because, between pH 2.6 and 3.0, most whey proteins, except p-Lg, are thermally coagulated. The selective thermal precipitation technique of Pearce (1983) for separation of p-Lg from other whey proteins may be useful in this regard.

During enzymatic hydrolysis, cleavage of the polypeptide chains de- stroys existing epitopes on native protein, thereby rendering the protein nonantigenic. Treatment of whey proteins with proteases reduces the an- tigenicity of both p-Lg and a-La by three orders of magnitude (Pahud et al., 1985; Jost et al., 1987).

For specialized dietary uses, there is a growing need for hydrolyzed proteins with a balanced essential amino acid composition. In cases where injury or disease prevents digestion and/or absorption of ingested protein, intravenous feeding of complete whey protein hydrolysates may be of critical nutritional value (Manson, 1980).

XV. SUMMARY AND CONCLUSIONS

There is abundant information concerning the functional behavior of whey proteins in model systems. The data on functional properties reported by different researchers, however, reveal wide discrepancies in values. For example, in the case of comparable whey preparations, apparent solubilities may range from 10 to 100%; strength of gels from 0.3 to >10 N, foam overruns from 250 to 1500%, and foam stabilities from 0.5 to 30 min. Many of the data are of limited value in assessing the true


functional characteristics of different preparations, treatments, or processing effects. Reports to date are useful in indicating the relative behavior of different proteins; however, the data do not always predict the performance of such proteins in actual food systems. This reflects the fact that in foods, extensive interactions with other components may occur, resulting in modified behavior of the proteins. Harper, (1984) has advo- cated the testing of these various preparations in simulated food systems which should validly relate the behavior to performance in commercial systems. Emphasis on standardization of specific protocols, with regard to order of addition in ingredients, temperature, pH control, and amount of energy input during mixing, homogenization, emulsification, etc. de- serves serious consideration. While this approach is justifiable in terms of providing valuable data to commercial users, it does not minimize the importance of examining these proteins in model systems where the physicochemical basis of each functional attribute can be described in molecular terms (Kinsella, 1987). Such information is necessary to expedite appropriate methods of processing in order to control compositional variability, extent of denatauration, and possible protein modification. In addition, rapid, reliable tests for routine quality assurance that can provide practical information concerning functional applications would be of great value.

Whey protein preparations vary immensely in functional behavior and are presently relegated to limited use as functional ingredients in the food industry. This need not be the case since conventional and new technologies permit rigorous control of production protocols, e.g., careful control of heat treatments can result in the production of whey protein preparations with consistent, reliable functional properties (dewit, 1981, 1984; Harper, 1984; Morr, 1985). As the market for functional proteins contin- ues to expand, the whey industry must seek the means to refine whey protein products; determine useful functional properties; develop standardized manufacturing protocols; demonstrate the effectiveness of whey as a functional ingredient; promote, and then market, whey on the basis of performance at competitive cost.

RESEARCH NEEDS

Because of the enormous resources of whey available worldwide, there is ample justification in continuing research for its improvement in effective use for the food industry. This is underscored by the urgent need to develop standard methodology for evaluating the functional properties of whey protein concentrates and for correlating these properties with specific commercial uses of the protein, which may encourage effective utili-


zation of whey proteins. Standard methods should emphasize the use of model systems which closely approximate end-use applications. Exam- ples of research needs include

Examination of the functional properties of whey proteins in relation to their composition, as affected by lipids, salts, and proteose-peptone content. Development of methods applicable to commercial practice for elimi- nating lipids from whey protein concentrates. Evaluation of the functional properties of whey protein fractions and proteins isolated by different methods. Elucidation of relationships between protein structure and specific functional properties, with emphasis on the effects of heat treatments. Investigation of processes for production and modification of whey proteins designed for specific functional applications. Development of practical methods for concentration (including, freeze concentration and drying) and isolation of highly functional products. Examination of the usefulness of whey proteins for blending with and/or complementing other food proteins. Determination of the effects of modification by physical, enzymatic, chemical, or genetic methods on functional properties. Determination of physicochemical attributes required in proteins for optimum performance in specific functions.

ACKNOWLEDGMENTS

This review was supported in part by grants from the National Dairy Promotion Board and the Wisconsin Milk Marketing Board.

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