Ch3-V1-Functionality of Bovine Whey Proteins and Their Enzymatic Hydrolysates with Special Reference to Colostral Whey Proteins

Functionality of Bovine Whey Proteins and Their Enzymatic Hydrolysates with Special Reference to Colostral Whey Proteins

H. Korhonen, P. Rantamäki, M. Kaunismäki and T. Rokka

Agriultural Research Centre of Finland, Food Research Institute, FIN-31600 Jokioinen, Finland

Introduction

Figure1. Major constituents of bovine milk and

colostrum (%) (Senft and Rappen, 1968).

The functional properties of individual whey proteins, whey protein concentrates (WPC), whey protein isolates (WPI) and their enzymatic hydrolysates have been reviewed extensively in a number of scientific articles and textbooks over the last 25 years (Morr et al.,1973; Kinsella, 1976; de Wit, 1989; Kinsella and Whitehead, 1989; Mulvihill and Fox, 1989; Konrad, 1990; Dybing and Smith, 1991; Morr and Ha, 1993; Jost, 1993; Giese, 1994; Panyam and Kilara, 1996). A majority of these reviews focuses on the proteins present in cheese whey. On the other hand, the functional properties of whey proteins derived from bovine colostrum have been researched only marginally (de Wit et al., 1988; Lindström et al., 1994). Bovine colostrum differs from normal milk in many respects, e.g., in the content and composition of proteins (Senft and Rappen, 1968),(Fig.1). A major protein class in colostrum are represented by immunoglobulins (Ig) (Butler, 1994). The well-documented biological role of Ig’s is to provide a new-born calf with passive immunity (Nocek et al., 1984; Butler, 1994; Nousiainen et al., 1994). In this review, the functional characteristics of ordinary whey proteins are discussed. Further, the functionality of the major colostral whey proteins as well as their concentrates and hydrolysates will be discussed mainly by reference to unpublished results of the present authors.

Functional properties of whey proteins

The rapid development of membrane filtration techniques in the 1970's provided new possibilities for the large scale manufacturing of whey proteins. Since then, more than one hundred thousand tons per year of whey protein concentrates (WPC) and isolates (WPI) with various properties have been produced as food ingredients used in both conventional foods as well as various dietetic preparations (Riedel, 1994; Cunningham, 1995; Valeur, 1997). Recently, the functional and biological properties of individual whey proteins have become a focus of commercial interest as potential ingredients of so-called functional or health-promoting foods (Mulvihill, 1992; Mann 1996; Horton 1995). Pilot and industrial-scale technological methods have been developed for isolation of several individual whey proteins in purified form, such as -lactalbumin (a-la) and -lactoglobulin (b-lg), lactoperoxidase, lactoferrin, glycomacropeptide and immunoglobulins (Yoshida and Xiuyun, 1991; Yoshida and Ye, 1991; Burling, 1994; Fukumoto et al., 1994a, Mitchell et al., 1994; Outinen et al., 1995; Outinen et al., 1996; Konrad and Lieske 1997). The whey proteins differ widely in their functional properties. (Mulvihill and Fox, 1989; Paulsson, 1990).

Generally, the most important functional properties of whey proteins are considered to be

· solubility · gelation properties

· dispersibility · water holding capacity

· acid stability · adhesion

· emulsification properties · film formation

· foaming properties · organoleptic properties

· viscosity · binding properties

(Mulvihill and Fox, 1987; de Wit et al., 1988; Giese, 1994; Lawson, 1994).

The basic element which determines the functionality of protein is the molecular structure.Other important factors affecting the functional properties of whey proteins are the origin of whey, season-dependent variations of its components and the processing steps and conditions involved, e.g., heat treatments, pH, ionic strength, and presence of minerals (Paulsson, 1990; Zhu and Damodaran, 1994a,b; Haque and Sharma, 1997; Ju et al., 1997). In addition, effective factors for functionality are the presence of lipids, e.g., phospholipids and antagonistic or synergistic effects between whey constituents (Gezimati et al., 1996; Lieske and Konrad, 1996a), followed finally by the interaction of proteins with other ingredients in the food products (Lawson,1994). At a pH of about 4.2 and at 55-65 C, a-la undergoes isoelectric precipitation due to dissociation of calcium ions and hydrophobic interactions (Bramaud et al., 1995). Further, other minor whey proteins precipitate under these conditions, while the b-lg remains soluble and can be separated, concentrated by membrane techniques and finally dried. The product has superior gelling and water-binding properties in many foodstuffs. The purified a-lactalbumin is more suitable for infant formulas than WPC’s since human milk does not contain b-lg (Walstra and Jenness, 1984).

a-lactalbumin has good emulsifying properties, but its gelation ability is poor. Instead, b-lactoglobulin has excellent gelling and foaming properties (Rojas et al. 1997). The functionality of lactoferrin (LF) and lactoperoxidase (LP) in various food systems has not been studied, though their antimicrobial effects in vitro and in vivo are well documented (IDF, 1991; Hambraeus and Lönnerdahl, 1994; Stadhouders and Beumer, 1994; Tomita, 1994; de Wit and Hooydonk, 1996). BSA and IgG fractions have not, as of yet, shown any special functional ability (p. 4), but studies of their functional properties are very limited. The soluble glycomacropeptide (CMP) which is derived from k-casein by the action of chymosin, also belongs to very scarcely known components of whey. Langley and Green (1989) have regarded gelation properties of glycomacropeptides very poor. Marshall (1991) determined the foaming ability of CMP and ranked it between egg white and WPC.

Functional properties of colostral whey proteins

In bovine colostrum, the protein content is 3-4 times higher ( up to 15 grams per litre vs. 3 - 4 grams per litre) than in normal milk. This is primarily attributed to high concentration of whey proteins, as shown in Table 1. Among colostral whey proteins, the Ig’s represent up to 75 % of total protein nitrogen in the first milking as compared to about 10 % in normal milk (Korhonen, 1977; Fox 1989).The content of Ig’s varies individually, ranging from 20 to over 100 g / l (Korhonen, 1977; Stott et al., 1981; Nousiainen et al., 1994). After post partum, the content of Ig’s decreases below one gram per litre over the course of one week (Korhonen, 1977). Also, the content of LF, a-la and b-lg is higher in colostrum than in milk. Likewise, differences are found in the content of minor proteins and enzymes. Ig-enriched preparations have been produced for the market in many countries as calf milk replacers (Mee and Mehra, 1995). It has been suggested that infant formulas could be fortified with colostral Ig’s and LF (Goldman, 1989; Seung and Jae, 1995). Also, preparations containing specific colostral Ig’s (antibodies) may in the future find applications in the prevention and treatment of human microbial diseases (Ruiz, 1994). Still, the possibilities to use colostral WPCs for human consumption await for further research. Since bovine colostrum also contains other biologically active compounds such as growth-promoting factors and essential nutrients (Yamauchi, 1992; Fox and Flynn, 1992), research in this field seems highly promising.

The Ig’s are a heterogeneous group of proteins with a variable high molecular weight. Three classes of Ig’s have been identified in bovine milk: IgG, IgA and IgM (Butler,1994). The IgG is represented in colostrum by two sub-classes, IgG1 and IgG2. IgG1is the principal immunoglobulin in respect to the passive immunization of calves. IgA, which occurs mainly in its secretory form SIgA, is more abundant in colostrum than in normal milk, and IgM is an important agglutinating antibody in blood (Butler, 1994). In milk or whey, even 80% of the immunoglobulins consists of IgG1. IgG molecules have a relatively larger content of basic amino acids than the other whey proteins, and, therefore, also their isoelectric point is higher (pI = 6.3-7.0) (Eigel et al., 1984). They are composed of four polypeptide chains joined by disulphide bridges and show a higher denaturation (unfolding) temperature than -la and -lg. In the presence of other whey proteins, however, the Ig’s are very thermolabile, which may be related to the activity of thiol groups of -lg and BSA. As a result of modern technology, it has been possible to overcome thermolability by means of membrane sterilization (Fukumoto et al., 1994b).

In the earlier literature it has been documented that Ig’s have interesting technological properties such as the cold agglutination of fat globules in milk (Mulder and Walstra, 1974) and binding of fatty solutes and bacteria in desalted acidified whey (de Wit et al., 1978).

De Wit (1988) studied the functional properties of immunoglobulin G (IgG) fraction obtained by ultrafiltration, diafiltration and gel filtration of sweet whey, comparing them to those of -la, -lg and BSA. The solubility of Ig G fraction at pH 6.5 was comparable to those of beta-la, -lg and BSA, but at pH 4.6 the solubility of Ig G was lowered to 91.5%, which indicates partial denaturation of protein. Foamability of Ig G and BSA were only about 25% compared to -lg. This was estimated to be due to the presence of fatty components which impaired the foaming properties. Also, the emulsifying behaviour of IgG and BSA was poor. Both IgG and BSA fractions formed gels, the strength of which were 38% and 15% of that of -lg, respectively. IgG, BSA and b-lg contain reactive thiol groups as initiators of gelation - unlike -la which did not form any gel in this study. Lindström et al. (1994) studied the effect of heat treatment on microfiltered bovine colostral Ig fraction. According to their results, the Ig’s were the most heat-stable among the whey proteins. A thermally induced unfolding was irreversible and not pH-dependent. The presence of NaCl in phosphate buffer stabilized immunoglobulins against aggregation. More research in this area is required in order to understand how profoundly various processing techniques affect the functional properties of whey proteins.

Enzymatic modification of whey proteins

In the pursuit to improve the functional properties as well as the nutritional qualities of whey proteins, various chemical and physical modifications have been applied. The classical means is thermal modification which can be combined with changes in pH. Many chemical modifications have been studied, such as

· acylation

· esterification

· amidation

· reductive alkylation

· lipophilization

· glycosylation

· phosphorylation

· arylsulphonation

· alkylsulphonation

· amino acid attachment (Nakai and Li-Chan, 1989; Schwenke, 1997)

Chemical modification may, however, produce impaired in vitro metabolization of whey proteins (Lieske and Konrad 1994) which, in turn, could have toxic physiological effects. The end product may also contain unreacted chemicals and products of unwanted side reactions.

Although enzymes catalyze many modifications of proteins, e.g., glycosylation, hydroxylation and methylation (Watanabe and Arai, 1982), probably the most examined method of modification of whey proteins at present is based on enzymatic hydrolysis (Panyam and Kilara, 1996). Enzymatically modified proteins have long been available in many conventional foods such as ripened cheese and fermented soy protein products. Pure protein hydrolysates have been shown to have valuable dietetic properties and high nutritional value (Frokjaer, 1994). Therefore, they can be applied in hospital diets, special diets for elderly people and sport products as well, the last of which would appear to represent a growing market world-wide (Blenford, 1994; Frokjaer, 1994; Schmidl et al., 1994).

The most commonly used enzymes in the production of whey protein hydrolysates are pepsin, trypsin and chymotrypsin, which are also human digestive enzymes. Also, plant originated papain and some bacterial and fungal proteases have been used in many studies (Schmidt and Poll, 1991; Lahl and Braun, 1994; Althouse et al. 1995, Panyam and Kilara, 1996). The ability of enzymes to hydrolyze whey proteins is highly variable. Trypsin is capable of hydrolyzing native b-lg (Schmidt and Poll, 1991), but chymotrypsin is not (Reddy et al., 1988). Lieske and Konrad (1996a) demonstrated that BSA and b-lg were hydrolyzed by papain. Instead, a-la was resistant. However, a-la was hydrolyzed completely at acidic pH when calcium binding was absent (Lieske and Konrad, 1996b). According to Schmidt and van Markwijk (1993) a-la was hydrolyzed by pepsin but pepsin was not capable of hydrolyzing native b-lg. Native -lg is rapidly hydrolysed by papain and there is a difference between genetic variants A and B of b-lg; the A variant is hydrolyzed more rapidly than B. Enzymatic hydrolysis of -lg at an elevated hydrostatic pressure has become more attractive in cases where the hydrolysis is slow or the enzyme is not capable of hydrolyzing -lg under ambient conditions (Stapelfeldt et al. 1996). Hydrolysis of IgG by papain yields three peptides: two so-called 'fragment binding' ((Fab)2) peptides from the N-terminal segments with antibody activity, and one 'fragment crystallizable' (Fc) peptide with no antibody activity (Fox, 1989). Pepsin cleaves the heavy chains on the C-terminal side of the disulphide linking the two heavy chains (Fox, 1989). It is known that IgA of the secretory type (SIgA) is more stable in enzymic, e.g., pepsin degradation than Ig’s without a secretory component (Kanamaru et al., 1993). Using pepsin, it is possible to eliminate IgG1 without marked degradation of IgM and SIgA, and so attain the product which is better suitable for infant formulas (Kanamaru et al. 1993).

Functional properties of whey protein hydrolysates

Enzymatic modification of whey proteins by controlled proteolysis can alter their functional properties over a wide pH range and other processing conditions. The hydrolysis of peptide bonds can increase the number of charged groups and hydrophobicity, decrease molecular weight, and modify molecular configuration (Phillips and Beuchat, 1981). Changes in functional properties are greatly dependent on the degree of hydrolysis. The most common changes in functionality are an increase in solubility and a decrease in viscosity. When the degree of hydrolysis is high, hydrolysates often tolerate strong heating without precipitating, and solubility is high even at pH 3.5 - 4.0. Hydrolysates also have far lower viscosity than intact proteins. The difference is especially striking in solutions with a high protein concentration. Other effects are altered gelation properties, enhanced thermal stability, increased emulsifying and foaming abilities and decreased emulsion and foam stabilities (Nakai and Li-Chan, 1989; Panyam and Kilara, 1996; Lieske and Konrad 1996a; Otte et al., 1996). Haque (1993) has described the profound effect of hydrolytic amphipathic peptides on the functionality of milk proteins. A potential future method is to separate peptides by molecular filtration in accordance with their molecular weight (Maubois and Ollivier, 1997; Tossavainen et al. 1997).

Functional properties of peptides derived from milk proteins

Functional properties of individual peptides obtained from partial enzymatic hydrolysis of milk proteins are still inadequately known. Some literature is available on the properties of peptides derived from caseinates (Shimizu et al., 1984; Lee et al., 1987; Panyam and Kilara, 1996), but the properties of peptides derived from whey proteins are known almost only as peptide fractions consisting of a certain range of molecular weights and isolated by selective membrane filtration.

Turgeon et al. (1992) identified two highly functional peptides from -lg, -lg 21-40 and -lg 41-60. The peptides were responsible for the improvement of the emulsifying properties of whey proteins upon tryptic hydrolysis. These two peptides were found in the mixture of a polypeptide fraction, obtained by UF-concentration, of the total hydrolysate using a 1 kDa membrane. A study of the interfacial behaviour of fractions containing -lg 21-40 and -lg 41-60 showed increased rates of adsorption at oil/water interface by a factor of up to 200 compared to other peptidic fractions. Hydrolysates containing these highly functional peptides were used successfully as replacements for eggs in mayonnaise-like emulsions and pancakes, and as a water-holding agent in processed meats. Barbeau et al. (1995) showed that the -lg 21-40 peptide can stabilize -lg against heat denaturation by increasing both its denaturation temperature and enthalpy. The exact nature of the peptide - protein interaction responsible for this phenomenon is not known. Partial proteolysis of LF by pepsin yields peptides which have a higher antimicrobial activity (Tomita, 1994; Dionysius and Milne, 1997).

It has long been known that peptides and amino acids can produce many types of taste sensations (Eriksen and Fagerson, 1976). A problem in the use of proteolysis in improving functionality and nutritional value has been the formation of bitter peptides, which are formed, e.g., from -lg as well as an aftertaste from the original protein sources. Ney (1971) postulated that bitter peptides have an average hydrophobicity greater than 1400 kcal/mole, irrespective of the amino acid sequence of the peptides, and that proteins with a high average hydrophobicity have a tendency to yield bitter peptides on hydrolysis. Bitterness is generally related to the hydrophobicity of the amino acids in the peptides. Because hydrolysis of milk protein causes frequently bitter peptides, the problem has aroused plenty of research in this area. As a solution, peptides with a high content of hydrophobic amino acids have been filtered by means of active coal (Murray and Baker, 1952; Helbig et al., 1980). Other efficient adsorbants are phenolic formaldehyde resin (Roland et al., 1978), glass fibre (Helbig et al., 1980), and hexyl-sepharose (Lalasidis, 1978). Plastein reaction has aroused interest due to the fact that this phenomenon is able to debitter protein hydrolysates (Fujimaki et al., 1970; Yamashita et al., 1970). In addition to selective separation other methods used are masking and enzymatic treatment (Pedersen, 1994). Recently this problem has been - at least in some cases - overcome by selecting suitable hydrolysis conditions concerning pH, temperature and substrate-enzyme ratio (Castro et al. 1996).

Apart from functional properties, enzymatic proteolysis of whey proteins is likely to modify their biological properties. Depending on the final use of the resulting hydrolysate, the changes in the molecular structure and amino acid sequence of peptides as formed may appear physiologically beneficial in many ways. A number of studies have shown that the allerginicity of whey proteins, in particular that of b-lg, can be reduced substantially if the whey proteins are hydrolyzed with pepsin or trypsin or using combinations of proteolytic enzymes (Ena et al. 1995). Heat treament prior to hydrolysis increases the DH value, further reducing allergicinity (Asselin et al., 1989; Van Beresteijn et al., 1994). Whey protein hydrolysates have, therefore, found increasing use in infant formulas and dietetic products. In recent years, a lot of scientific interest has been focused on physiologically active peptides deriving from milk upon hydrolysis. Both caseins and whey proteins have been found to pose as precursors of bioactive peptides, which may exert various functions,such as opioid, immunomodulating, antimicrobial, antioxidative and antihypertensive (Antila et al., 1991; Meisel and Schlimme, 1996). The functionality of bioactive peptides in different food systems is little known as yet, but it will be of great importance if these peptides are to be applied in different food products.

Functional properties of colostral whey protein concentrates and their hydrolysates

In this section, results of a study carried out by the authors of this chapter will be described. In the study concerned, the functional properties of two bovine colostral whey protein concentrates (A and B) and their hydrolysates were compared with those of a commercial whey protein concentrate (C) and its hydrolysate.

For preparation of WPC’s, pooled colostrum of three milkings (from 3^rd to 5^th) from six cows was defatted and renneted by chymosin. Whey was lactose-hydrolyzed with lactase and thereafter treated as follows: A: ultrafiltration (cut-off 9000), microfiltration (0.45 m) followed by lyophilization, B: filtration (1.2 m), column chromatography (sulphonate polystyrene resin), reverse osmosis, microfiltration and lyophilization. The commercial whey protein concentrate C (Valio Ltd, Finland) was manufactured from pasteurized cheese whey using ultrafiltration, diafiltration and spray-drying.

The protein content of the products was: A 38.1%, B 58.2% and C 74.0%. The fat content of the products was: A 0.6%, B 0.7% and C 6.5%. The composition of major protein fractions, in mg/g, was as follows: A: IgG 121, -lg 140, -la 34; B: IgG 160, -lg 299, -la 84 and C: IgG 43, b-lg 370, a-la 83.

Differences found in the concentration of total protein and major protein components were considered to be mainly due to different manufacturing processes. The high content of A and B as compared to C originate from the Ig-rich colostral whey. The WPC’s were hydrolyzed by pepsin for three hours at pH 4.4. The degree of hydrolysis after 3 h was: A 15.2%, B 15.9% and C 19.4%.

The solubility at pH 6.7 of all products was better than 93%, while at 4.6 the solubility of C was 89.7% as compared to 96.7% for A and 95.9% for C, respectively. The lower solubility of C at pH 4.6 was likely to be due to a higher degree of denaturation caused by the pasteurization of whey and spray-drying of the end product.

As to foaming properties, the stability of foam and the overrun of A and B significantly exceeded those of C. The overrun of B was equivalent to that of whole egg diluted to 3% protein content (Fig.2). In the protein concentrations studied (1.5% and 3.0%), no foam formation was observed with C. This was probably due to the ten times higher fat content of C as compared to A and B. Hydrolysis increased the overrun of B by 43%, but did not affect the overrun of A and C (Table 2). After hydrolysis, C showed only a slight foaming ability which was much weaker than that of A and B. The foam stability of hydrolysate A decreased during 3 hours' hydrolysis by 43%, and in the case of hydrolysate B, by 30%, respectively. The hydrolysate C did not show any foam stability.

The specific emulsifying capacity (SEC) of all products was equal over a wide protein concentration range (0.015 - 0.250%). In lower protein concentrations, C showed the highest SEC. Hydrolysis affected SEC less than other measured functional properties (Fig. 3). The highest increase of SEC was observed in the hydrolysate C after three hours of hydrolysis (Table 2).

Maximum gel strength (0.8 - 1.2 N) was observed at pH 6.0 - 8.0 for both A and B and 7.5 - 8.0 for C, respectively. The hydrolysis significantly increased the gel strength of A and decreased that of B and C’s (Fig. 4)(Table 2).

Conclusions

The functional properties of protein concentrates and isolates as derived from cheese whey can be improved significantly by different enzymatic modifications. The application of these products as ingredients in different food systems is ,therefore, likely to be expanded. Purified whey proteins have specific functional and biological properties which will probably be exploited more in the future in the development of specialty products with distinct nutritional, health-promoting and textural properties. Colostral whey protein concentrates are comparable and even partially superior to WPC’s manufactured from cheese whey with regard to functional properties. In addition, colostral WPC’s may find specific applications in different industries, since they may naturally contain high amounts biologically active compounds.

Figure 1. Major constituents of bovine milk and colostrum (%) (Senft and Rappen, 1968).

Figure 2. Foaming properties of whey protein concentrates A, B and C, whole egg and egg white determined as overrun (%). 3.0% whey protein solutions were used. Whole egg and egg white were diluted with distilled water to 3.0% protein solution.

Figure 3. Specific emulsifying capacity (SEC) of hydrolysates of whey protein concentrates A, B and C. SEC was measured after 1 and 3 hours’ pepsin hydrolysis.

Figure 4 . Gel strength of 10% hydrolysates of whey protein concentrates A, B and C at pH 6.5. Gel strength was measured after 1 and 3 hours’ pepsin hydrolysis.

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