The whey proteins in milk: Thermal denaturation, physical interactions, and effects on the functional properties of milk

C H A P T E R

9 The whey proteins in milk: Thermal denaturation, physical interactions, and effects on the functional properties of milk Skelte G. Anema Fonterra Research and Development Centre, Palmerston North, New Zealand

Introduction Milk is produced in the mammary gland of female mammals and is intended for the feeding of the neonate from birth to weaning. Milk is a highly nutritious, readily digested food that is rich in protein, minerals, and energy in an aqueous solution. It also provides the neonate with many other essential compounds such as protective agents, hormones, and growth factors. Milk is a highly perishable fluid and was intended by nature to be consumed soon after production. Humans have used milk and dairy-derived foods to supplement their diet for centuries, and dairy products are still a major food source. Because of the commercial and nutritional significance of dairy products, manufacturing processes to preserve the food value of milk long after its initial production have been developed. Over the last century, modern dairy milk processing has been transformed from an art into a science. Traditional products such as cheeses and yogurts combine century-old knowledge with modern science, technology, and processing techniques. In contrast, more recently developed products (such as spray-dried milk products, milk protein concentrates, micellar casein, and whey protein concentrates) have been based on modern technologies of the time. The majority of the milk processed is of bovine (cow) origin; however, significant quantities of buffalo, goat, and sheep milk are also manufactured into dairy products (Fox, 2003). In milk, the lactose, some of the mineral components, and the native whey proteins are in true molecular solution. However, the casein and most of the calcium and phosphate are

Milk Proteins https://doi.org/10.1016/B978-0-12-815251-5.00009-8

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found in large macromolecular assemblies called casein micelles. The colloidal suspension of casein micelles in milk serum is a remarkably stable food protein system. Milk can be subjected to high temperatures and pressures, high shear, and variations in concentration without appreciable damage to the casein micelle system. Even the extreme action of drying milk to a powder does not significantly alter the milk system, as milk powders can be reconstituted to produce liquid milks that have many properties similar to those of the milk from which they were derived (Singh and Newstead, 1992; Kelly et al., 2003; Nieuwenhuijse and van Boekel, 2003; O’Connell and Fox, 2003).

The casein micelle To understand and rationalize any changes to the properties and stability of milk, it is necessary to have some knowledge of the casein micelle structure. Despite extensive research efforts, the detailed structure and assembly of the casein micelle has not been unequivocally established. Several models have been proposed over the years, and these models have been progressively updated or modified as more information on the casein micelle has become available (Fig. 9.1; Schmidt, 1982; Walstra, 1990; Holt, 1992; Holt and Horne, 1996; Horne, 1998; Walstra, 1999; Dalgleish, 2011; Huppertz et al., 2017). Although there is some agreement over some aspects of the various models, there is still considerable debate over the detailed structure of the casein micelle, as evidenced from the numerous recent papers and reviews devoted to this subject (Farrell et al., 2006; Qi, 2007, 2009; Dalgleish, 2011; Trejo et al., 2011; Dalgleish and Corredig, 2012; de Kruif et al., 2012; Holt et al., 2013; Horne, 2017; Huppertz et al., 2017; Lucey and Horne, 2018; also see Chapter 6). Evidence from early electron microscopy and light scattering studies suggested that the casein micelle was assembled from smaller subunits, and as a consequence, submicelle models of the casein micelle structure were developed. In the later iterations of these submicelle models, the casein proteins were hydrophobically aggregated to form the submicelle units, and these submicelle units were linked by colloidal calcium phosphate (CCP) via serine phosphate clusters at the submicelle surfaces to form the casein micelle. The distribution of κ-casein between submicelles was heterogeneous, and submicelles with high levels of κ-casein were located at the micelle surface, whereas those with low levels of κ-casein were in the interior, thus giving a surface location to κ-casein that was consistent with experiment (Fig. 9.1A; Schmidt, 1982; Walstra, 1990). Subsequent experimental evidence did not support the existence of submicelles, and therefore, the validity of the submicelle model of the casein micelles was questioned (Holt, 1992; Holt and Horne, 1996; Horne, 1998; Walstra, 1999; Horne, 2002, 2006). In particular, there was evidence to show that the CCP was uniformly distributed through the casein micelle, which precluded submicelles being linked by CCP to form the micelle. In addition, it was considered to be unlikely that there would be heterogeneous populations of casein submicelles with different levels of κ-casein or that assembly into casein micelles via calcium phosphate would occur only after the casein submicelles had been formed. In addition, electron micrograph images of casein micelles using modern techniques did not display the internal substructure expected for casein submicelles, and it was considered

(A)

(B)

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(D)

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FIG. 9.1 Recent models of the casein micelle. (A) Original “hairy” submicelle model of the casein micelle. (B) Modified hairy submicelle model of the casein micelle. (C) Nanocluster model of the casein micelle. (D) Dualbinding model of the casein micelle. (E) Dalgleish model of the casein micelle. (F) Huppertz model of the casein micelle (Huppertz et al., 2017). (A) Reproduced with permission from Walstra, P., Jenness, R., 1984. Dairy chemistry and physics. In: Dairy Chemistry and Physics. John Wiley and Sons, New York and Walstra, P., 1990. On the stability of casein micelles. J. Dairy Sci. 73, 1965–1979. Copyright (1984) John Wiley & Sons. (B) Reproduced with permission from Walstra, P., 1999. Casein sub-micelles: do they exist? Int. Dairy J. 9, 189–192. Copyright (1999) Elsevier. (C) Reproduced with permission from Holt, C. (1992). Structure and stability of bovine casein micelles. Adv. Protein Chem. 43, 63–151. Copyright (1992) Elsevier. (D) Reproduced with permission from Horne, D.S., 1998. Casein interactions: casting light on the black boxes, the structure in dairy products. Int. Dairy J. 8, 171–177. Copyright (1998) Elsevier. (E) Reproduced with permission from Dalgleish, D.G., 2011. On the structural models of bovine casein micelles-review and possible improvements. Soft Matter, 7, 2265–2272. Copyright (2011) Royal Society of Chemistry. (F) Used with permission.

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that the appearance of submicelles in earlier micrographs was an artifact of the early preparation techniques for electron microscopy (McMahon and McManus, 1998; Horne, 2006). In an attempt to reconcile this new evidence, the submicelle model of the casein micelle was refined to change the role of CCP from that of linking the submicelles to a charge-neutralizing agent to allow for a uniform distribution of CCP, and the submicelles were now linked together via hydrophobic interactions (Fig. 9.1B; Walstra, 1999). However, new models for the casein micelle that did not rely on the formation of submicelles were developed (Holt, 1992; Holt and Horne, 1996; Horne, 1998; de Kruif and Holt, 2003; Dalgleish, 2011). Nonsubmicelle models included the nanocluster model (Fig. 9.1C; Holt, 1992; Holt and Horne, 1996; de Kruif and Holt, 2003) and the dual-binding model (Fig. 9.1D; Horne, 1998). There has been some convergence of these two models as the combined results of detailed experiments on micelle structure using small angle x-ray/ neutron scattering and static light scattering were consistent with calculation only if weak interactions (hydrophobic interactions, hydrogen bonding, ion pairing, etc.) were incorporated into the nanocluster model (de Kruif et al., 2012). The dual-binding model proposed by Horne (1998) describes the types of interactions involved in the assembly of casein micelles and demonstrates that micelles with a consistent arrangement of casein proteins and CCP can be achieved. However, this dual-binding model does not give a detailed description of the surface or the internal structure of the casein micelle. A recent model of the casein micelle proposed by Dalgleish (2011) and Dalgleish and Corredig (2012) (Fig. 9.1E) has a relatively sparse hairy layer of κ-casein on the surface, dense enough to stabilize against approach by other casein micelles or other large colloidal particles but sufficiently diffuse to allow denatured whey proteins to interact with the para-κ-casein region of κ-casein or to allow β-casein to dissociate from the micelles on cooling and to reassociate with the micelles on subsequent warming. In addition, this model attempts to reconcile the structural arrangement of the interior of the micelles with the high hydration of the micelle by giving a specific role to β-casein. It is proposed that some of the β-casein acts as a surfactant in stabilizing the hydrated internal channels of the micelle. This β-casein may be loosely bound to the micelles so that it is able to dissociate and reassociate during cold and warm temperature cycling. Very recently, Duerasch et al. (2018) presented a casein micelle model with a similar structure to that proposed by Dalgleish (2011) and Dalgleish and Corredig (2012); however, they suggest that “αS2-casein must be the major compound of calcium phosphate nanoclusters forming the inner structure of casein micelles” (Duerasch et al., 2018). It is highly unlikely that αS2-casein has greater importance than αS1-casein in casein micelle structure as, in bovine milk, αS1-casein is more dominant than αS2-casein, and αS1-casein can bind high levels of calcium phosphate (Swaisgood, 1982, 1992, 1993). In addition, the low level of αS2-casein in bovine milk would require this protein to bind large amounts of calcium phosphate if it was the major casein of the calcium phosphate nanoclusters. The fact that bovine milk and caprine milk may have vastly different αS1-casein and αS2-casein levels yet similar sized casein micelles (Richardson et al., 1974; Anema and Stanley, 1998; Raynal and Remeuf, 1998; Tziboula and Horne, 1999) suggests that αS1-casein and αS2-casein play similar roles in casein micelle structure and, in particular, similar roles in stabilizing the colloidal calcium phosphate nanoclusters.

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Another very recent model of the casein micelle has a structure that is composed of nonspherical subparticles, referred to as “primary casein particles” (PCPs) rather than submicelles (Fig. 9.1F; Huppertz et al., 2017). In many respects, this model is similar to that described by Schmidt (1982) and Walstra (1990), with the core of the micelle being composed of PCPs depleted in κ-casein (and enriched in αS2-casein) and the surface of the micelle being composed of PCPs enriched in κ-casein (and depleted in αS2-casein). The PCPs are cross-linked into a three-dimensional structure by the CCP through the phosphoserine centers on the αS1-casein, αS2-casein, and β-casein. The primary differences between the model by Huppertz et al. (2017) and the Schmidt/Walstra models (Schmidt, 1982; Walstra, 1990) are that the PCPs are nonspherical whereas the submicelles in the earlier models were spherical and that the PCPs are somewhat smaller, containing about 9–11 casein molecules, whereas the submicelles were considered to contain about 15–25 casein molecules. In a review on submicelle models of the casein micelle, Walstra (1999) indicated that experimental evidence was strongly against the concept of submicelles being linked via calcium phosphate bridges; however, this concept is being reintroduced in this latest casein micelle model. From the regular emergence of new casein micelle models and the continuing debate on casein protein interactions with respect to casein micelles (e.g., Carver et al., 2017; Horne and Lucey, 2017), it is clear that there is still not universal agreement on the casein micelle structure. There are proponents of submicelle models, and convincing arguments based on a structural biology perspective have been presented (Farrell et al., 2006; Qi, 2007). Even within the groups that support the models without submicelles, there are diverse views on the structural arrangements and the relative importance of different types of bonding (Horne, 2006; McMahon and Oommen, 2008; Dalgleish, 2011; Dalgleish and Corredig, 2012; de Kruif et al., 2012; Carver et al., 2017; Horne and Lucey, 2017). Although various models of the casein micelles have been proposed, they have largely been derived from the same pool of research data and are therefore different depictions or interpretations of similar information. As a consequence, many of the salient features of the structure, assembly, and stability of the different models are similar (Schmidt, 1982; Walstra, 1990; Holt and Horne, 1996; Horne, 1998; Walstra, 1999; Dalgleish, 2011; Dalgleish and Corredig, 2012; de Kruif et al., 2012; Huppertz et al., 2017). Hydrophobic interactions and CCP are important in maintaining micelle integrity. Therefore, micelle integrity can be modified or destroyed by disruption to hydrophobic interactions or by the dissolution of the CCP. In all recent models, κ-casein has a preferential surface location, with the C-terminal region protruding from the surface layer as a flexible hair. These models of the casein micelles, with the surface layers of κ-casein and an internal structure maintained by hydrophobic interactions and CCP, have been used to explain micelle stability and the destabilization by the enzymes in rennet, by acidification, or by the addition of alcohol (Walstra, 1990; Holt and Horne, 1996; Horne, 1998, 2003). However, less studied and less well understood are the mechanisms responsible for the changes occurring to the casein micelles during the heating of milk, in particular the interactions with the denatured whey proteins, the heat-induced, pH-dependent dissociation of the casein (especially κ-casein) from the micelles, and the eventual heat-induced coagulation of the casein micelles.

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The heat treatment of milk The effect of heat on the milk system is an important consideration in dairy chemistry as a heat treatment is involved in the manufacture of almost all milk products. The heat treatment may range from thermization (about 65°C for 15 s) to sterilization (about 120°C for 10–20 min) or ultrahigh-temperature (UHT) treatment (typically 138–142°C for several seconds). As the thermal history of milk influences its behavior in subsequent applications, the effects of heat on milk have been the subject of intensive, if somewhat intermittent, research, and many reviews and books on the subject are available (Singh and Creamer, 1992; International-DairyFederation, 1995, 1996; O’Connell and Fox, 2003; Singh, 2004). When milk is heated, a number of competitive and often interdependent reactions occur, and the importance of each reaction is determined by the heating conditions and by factors such as milk composition or concentration. When considering the protein components of milk, reactions of particular importance are whey protein denaturation, the interactions of denatured whey proteins with other proteins (including those of the casein micelles), and casein micelle dissociation. These three reaction processes can markedly modify the physicochemical properties of milk and may play a major role in determining the stability of milk and the functional performance of heated milk products.

Whey protein denaturation The whey proteins are typical globular proteins with well-defined secondary and tertiary structures. They (especially α-lactalbumin and β-lactoglobulin) retain their native conformations only within relatively limited temperature ranges. Exposing the whey proteins to extremes of temperature results in the denaturation and aggregation of the proteins; this process can be expressed using the simple reaction scheme as shown in Eq. (9.1): ðPN Þn ⇆nPN

(9.1a)

PN ⇆PU

(9.1b)

PU + A ! ðP  AÞ

(9.1c)

For protein species where the native protein is in the form of noncovalently linked oligomers (such as dimeric β-lactoglobulin), the first step in the denaturation process is the reversible dissociation of the oligomer into monomeric species (Eq. 9.1a). The monomeric protein can then unfold, disrupting the native conformation (Eq. 9.1b). In principle, this unfolding step is reversible; however, in complex mixtures such as milk, the unfolding process is accompanied by the exposure of reactive amino side chain groups, which allows irreversible aggregation reactions to occur. The unfolded whey protein can undergo aggregation reactions with other (unfolded) whey proteins, with aggregates, or with the casein micelles (represented by A in Eq. 9.1c). At a fundamental level, protein denaturation is often defined as any noncovalent change to the secondary or tertiary structure of the protein molecule (Eq. 9.1b). From this denatured state, the protein can revert to its native state (refold) or interact with other components in the system (aggregate). Under this definition, α-lactalbumin is generally regarded as one

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of the most heat-labile whey proteins, whereas β-lactoglobulin is one of the most heat-stable whey proteins (Ruegg et al., 1977). However, for the dairy industry, it is the irreversible aggregation processes that largely determine the functional properties of dairy products. Hence, it is common practice to define whey protein denaturation as the formation of irreversibly denatured and aggregated whey proteins (Sanderson, 1970a; Singh and Newstead, 1992; Kelly et al., 2003), and therefore, this encompasses only the irreversible process shown in Eq. (9.1c). Unless otherwise stated, the irreversible denaturation process is the definition used in this chapter. Using this definition for the denaturation of the whey proteins in milk, the immunoglobulins are the most heat labile, and α-lactalbumin is the most heat stable of the whey proteins, with β-lactoglobulin and bovine serum albumin being intermediate (Larson and Rolleri, 1955). In general, significant denaturation of the major whey proteins, α-lactalbumin and β-lactoglobulin, occurs only on heating milk at temperatures above about 70°C. Assessment of the denaturation of whey proteins in milk A considerable amount of research has been directed toward determining and understanding the denaturation processes of the major whey proteins when milk is heated. In early studies, the casein and denatured whey proteins were precipitated by the adjustment of the pH to the isoelectric point of the casein (about pH 4.6). The supernatants from the unheated and heated milks were analyzed for protein nitrogen, which gave estimates of initial native whey protein levels and levels after heat treatment (Rowland, 1933). However, a rapid method for determining the native whey protein levels was required for assessing milk powders for suitability in applications in the bakery industry (Harland and Ashworth, 1947) and also for categorizing milk powders based on the heat treatments received during the powder manufacturing process (Sanderson, 1970b,c). From these requirements, the whey protein nitrogen index (WPNI) method was developed. In the WPNI method, the casein and the denatured whey proteins were precipitated and separated from the native whey proteins by saturation of the milk with common salt, and the supernatant containing the native whey proteins was analyzed for protein content. This was originally achieved by dilution and pH adjustment of the supernatant to produce a turbid solution, with the turbidity proportional to the level of native whey protein present in the milk (Harland and Ashworth, 1947; Kuramoto et al., 1959; Leighton, 1962). However, the WPNI method displayed considerable variability in the degree of turbidity developed for samples with similar levels of whey protein denaturation. To overcome this, Sanderson (1970a) combined a dye-binding method for determining the total protein content of milk with the original WPNI method, thus giving a more accurate and reliable method for determining the WPNI. The original WPNI method or one of its variants is still the industry standard for determining the native whey protein levels of milk powder products and is still widely used to classify milk powders according to the heat treatment received (Singh and Newstead, 1992; Kelly et al., 2003). However, a recent report indicates that Fourier transform near-infrared spectroscopy may have potential as a rapid method for the determination of the WPNI of milk powders in the dry state, eliminating the necessity for reconstitution, precipitation, and filtration (Patel et al., 2007).

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Although the WPNI method can give an estimate of the level of whey protein denaturation, research into the denaturation and interactions of the individual whey proteins requires more accurate separation and analysis procedures. There are numerous quantitative methods for separating and determining the level of the individual whey proteins in milk, and these can be used directly or can be adapted to determine the level of denaturation after defined heat treatments. Methods that have been used include polyacrylamide gel electrophoresis (PAGE; e.g., Hillier and Lyster, 1979; Dannenberg and Kessler, 1988b; Kessler and Beyer, 1991; Anema and McKenna, 1996), capillary electrophoresis (e.g., Fairise and Cayot, 1998; Butikofer et al., 2006), differential scanning calorimetry (e.g., Ruegg et al., 1977; Manji and Kakuda, 1987), highperformance liquid chromatography (HPLC; e.g., Kessler and Beyer, 1991), and various immuno-based assays (e.g., Lyster, 1970). In recent years, miniaturized and/or rapid automated methods have been adapted for determining whey protein denaturation and protein compositions, such as PAGE techniques on microfluidic chips (Wu et al., 2008; Anema, 2009; Costa et al., 2014; Gubic et al., 2016) or optical biosensor-based assays (Dupont and MullerRenaud, 2006; Indyk, 2009). In general, good correlations have been observed when the various methods for determining whey protein denaturation have been compared (Manji and Kakuda, 1987; Kessler and Beyer, 1991; Anema and Lloyd, 1999; Patel et al., 2007; Anema, 2009; Indyk, 2009). These methods are more time consuming than the traditional WPNI methods and therefore cannot be used for the routine analysis and classification of milk products. However, they have higher accuracy and reproducibility and can be used to determine the denaturation behavior of the individual whey proteins. In addition, variations of the techniques or coupling to additional detection devices can provide further information on the interactions of the denatured whey proteins with other components in the milk (e.g., Lowe et al., 2004; Patel et al., 2006, 2007; Donato and Guyomarc’h, 2009). Kinetic evaluation and modeling of whey protein denaturation Early studies showed that the denaturation of whey proteins was a kinetic phenomenon and therefore dependent on both the temperature and the duration of the heat treatment (Rowland, 1933; Harland and Ashworth, 1945). Although these early studies considered the whey protein components as a single entity, it was noted that the denaturation process did not follow a simple exponential law and was not a first-order (unimolecular) process. In addition, there was a change in temperature dependence above about 80°C, which was probably the first indication of the complex nature of the irreversible denaturation of the whey proteins in milk (Rowland, 1933). Although early studies on the effect of temperature and heating time on the denaturation of the individual whey proteins had been performed (e.g., Harland and Ashworth, 1945; Gough and Jenness, 1962), it was the kinetic study of Lyster (1970) over a wide temperature range (68–155°C) that conclusively demonstrated the complexity of the denaturation process of the whey proteins. Lyster (1970) found that the denaturation of α-lactalbumin appeared to follow first-order kinetics and that the denaturation of β-lactoglobulin was second order. Arrhenius plots for both α-lactalbumin and β-lactoglobulin indicated that the irreversible denaturation reaction was not a simple process, as a change in temperature dependence was observed at about 80–90°C for both α-lactalbumin and β-lactoglobulin (Fig. 9.2). The rate

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FIG. 9.2 Effect of milk concentration on the Arrhenius plot for the thermal denaturation of β-lactoglobulin (A) and α-lactalbumin (B) over the temperature range 75–100°C. , 9.6% total solid (TS) milk; ●, 19.2% TS milk; □, 28.8% TS milk; and , 38.4% TS milk. (A) Reproduced with permission from Anema, S.G., 2000. Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of β-lactoglobulin. J. Agric. Food Chem. 48, 4168–4175. Copyright (2000) American Chemical Society. (B) Reproduced with permission from Anema, S.G., 2001. Kinetics of the irreversible thermal denaturation and disulfide aggregation of α-lactalbumin in milk samples of various concentrations. J. Food Sci. 66, 2–9. Copyright (2001) Blackwell Publishing.

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constants increased more rapidly with an increase in temperature in the low-temperature ranges than at higher temperatures. Further studies confirmed the complexity of the denaturation process and provided relationships between compositional aspects and the rate of denaturation (Lyster, 1970; Hillier and Lyster, 1979; Manji and Kakuda, 1986). The kinetic and thermodynamic studies of Dannenberg and Kessler (1988b) provided insights into the possible mechanisms responsible for the complex temperature dependences of the denaturations of α-lactalbumin and β-lactoglobulin. Dannenberg and Kessler (1988b) found, in milk, that the denaturation of β-lactoglobulin had an order of about 1.5, which is now generally accepted, and that the denaturation of α-lactalbumin was pseudo first order. From thermodynamic evaluations of the denaturation reactions of β-lactoglobulin and α-lactalbumin in the two temperature ranges (i.e., at temperatures above and below the marked change in temperature dependence for the denaturation reactions; Fig. 9.2), information on the possible rate-determining steps in the denaturation reactions was obtained. At temperatures below about 90°C for β-lactoglobulin and 80°C for α-lactalbumin, the high values for the activation energies and enthalpies indicated that a large number of bonds were disrupted, and the positive activation entropies indicated a lower state of order of the reaction

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products. These kinetic and thermodynamic parameters were interpreted as indicating that the unfolding (reversible denaturation) of the whey proteins was the rate-determining step in the lower-temperature ranges. At higher temperatures, above 80°C for α-lactalbumin and above 90°C for β-lactoglobulin, the considerably lower activation energies and enthalpies were typical of chemical reactions, and the negative activation entropies indicated a higher state of order. These parameters suggested that chemical (aggregation) reactions were the rate-determining step in the higher temperature ranges. Subsequent studies have supported these interpretations in skim milk and whole milk under industrial processing conditions (Anema and McKenna, 1996; Oldfield et al., 1998a). In a recent study, kinetic and thermodynamic evaluations were conducted by treating the whey proteins (α-lactalbumin and β-lactoglobulin) in milk as a single “total whey protein” entity (Anema, 2017b). When considered as total whey protein, the denaturation followed second-order reaction kinetics, with the temperature dependence changing at about 85°C. As with the denaturation of the individual proteins, the thermodynamic parameters of total whey protein denaturation were consistent with unfolding reactions as the rate-determining step at temperatures below 85°C and aggregation reactions as the rate-determining step at higher temperatures. From the kinetic evaluation, equations that could predict the denaturation levels of the total whey proteins after any defined heat treatment were developed, and these predicted denaturation levels were highly correlated with the experimental WPNI of the milk samples (Anema, 2017b). When considering compositional changes of the milk, the denaturation reactions of both β-lactoglobulin and α-lactalbumin are enhanced when the pH of the milk is increased from the natural pH and are retarded when the pH is decreased (Law and Leaver, 2000). The denaturation of β-lactoglobulin was retarded when all components in the milk were concentrated, although the effect was less pronounced as the temperature was increased (Fig. 9.2A; Anema, 2000). In contrast, the denaturation of α-lactalbumin was hardly affected by the milk concentration, with similar rates of denaturation at all milk concentrations regardless of the heating temperature (Fig. 9.2B; Anema, 2001). The seemingly contrasting effects of milk concentration on the denaturation of α-lactalbumin and β-lactoglobulin have been explained through detailed studies on the effect of the concentrations of the individual components of milk on the denaturation reactions. Increasing the protein concentration of milk while maintaining essentially constant concentrations of nonprotein soluble components increased the rate of denaturation of both α-lactalbumin and β-lactoglobulin (Fig. 9.3; Law and Leaver, 1997; Anema et al., 2006), with a similar effect at all temperatures (Anema et al., 2006). Increasing the concentration of nonprotein-soluble components while maintaining constant protein concentrations retarded the denaturation of both β-lactoglobulin and α-lactalbumin; however, the effects on these two proteins were somewhat different (Fig. 9.3; Anema et al., 2006). For β-lactoglobulin, increasing the nonprotein-soluble components caused a substantial retardation of denaturation in the lower-temperature range, and this effect became less pronounced at higher temperatures. In contrast, the effect of increasing the nonprotein-soluble components on α-lactalbumin denaturation was less pronounced than for β-lactoglobulin denaturation and was similar at all temperatures investigated (Fig. 9.3). The increase in lactose concentration, the major component of the nonprotein-soluble

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FIG. 9.3 Comparison of the effects of the concentrations of (●, ) protein, ( , □) nonprotein-soluble components, (▲, 4) lactose, and (⧫, ◇) TS on the rate constants for the denaturation of (A) β-lactoglobulin (A) and (B) α-lactalbumin at 80°C (filled symbols) and 95°C (open symbols). Reproduced with permission from Anema, S.G., Lee, S.K., Klostermeyer, H., 2006. Effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible thermal denaturation of β-lactoglobulin and α-lactalbumin in skim milk. J. Agric. Food Chem. 54, 7339–7348. Copyright (2006) American Chemical Society.

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components, explained much of the effect of increasing nonprotein-soluble components; however, clearly, other compositional factors such as pH and mineral components also have an effect (Fig. 9.3; Anema et al., 2006). From these results, it was possible to explain the effects of milk concentration on the denaturation of β-lactoglobulin and α-lactalbumin. For α-lactalbumin, on increasing the total solid (TS) concentration of the milk (both protein- and nonprotein-soluble components), the retardation of the reaction rate by increasing the nonprotein-soluble components concentration was almost exactly offset by the increase in the denaturation rate on increasing the protein concentration. As this effect was similar at all temperatures, increasing the TS of the milk appeared to have no effect on the rate of denaturation of α-lactalbumin (Figs. 9.2B and 9.3B; Anema, 2001; Anema et al., 2006). For β-lactoglobulin, the retardation of the rate of denaturation on increasing the concentration of the nonprotein soluble components was not completely offset by the increasing rate of denaturation on increasing the protein concentration; therefore β-lactoglobulin denaturation was retarded by increasing the TS concentration of the milk. However, the nonprotein-soluble components were less effective in retarding the denaturation of β-lactoglobulin at higher temperatures, and as a consequence, the increase in TS concentration appeared to have a smaller effect on the denaturation of β-lactoglobulin at the higher temperatures and particularly

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above about 90°C (Figs. 9.2A and 9.3A; Anema, 2000; Anema et al., 2006). The effects of the nonprotein-soluble components concentration or the lactose concentration on the denaturation of β-lactoglobulin and α-lactalbumin have been discussed in terms of the preferential hydration theory (Anema, 2000; Anema et al., 2006).

Interactions between denatured whey proteins and κ-casein/casein micelles An understanding of the denaturation reactions of the whey proteins provides information on the initial steps of a complex series of aggregation reactions that can occur when milk is heated. This aggregation process can involve other milk protein components and may involve numerous reaction pathways or interaction processes. Although the reactions of the denatured whey proteins with other milk protein components are important, these types of reactions are considerably more difficult to measure than the irreversible denaturation processes, particularly in a complex mixture of components such as found in (skim) milk. Interactions between denatured whey proteins and κ-casein in model systems One of the major reactions of interest is the interaction between the denatured whey proteins and the casein micelles, particularly interactions of denatured β-lactoglobulin with κ-casein at the micelle surface. Early studies on model systems indicated that there was an interaction between β-lactoglobulin and κ-casein when these components were heated together (Zittle et al., 1962; Long et al., 1963; Sawyer et al., 1963). These conclusions were drawn from electrophoretic studies, which showed that the discrete bands assigned to κ-casein and β-lactoglobulin observed in unheated solutions produced species of intermediate mobility when the solutions were mixed and heated together. Sedimentation velocity experiments also confirmed complex formation, as the β-lactoglobulin-κ-casein complex formed on heating had markedly higher sedimentation coefficients than did the individual proteins when heated separately (Zittle et al., 1962). Once interactions between κ-casein and denatured β-lactoglobulin had been confirmed, subsequent investigations in heated model systems were aimed at determining the types of bonds involved in complex formation, the stoichiometry of the complexes formed, and the involvement of other whey proteins (particularly α-lactalbumin) in the complexes. It was shown that reducing agents dissociated the heat-induced complexes and that thiolblocking agents prevented the formation of the complexes (Sawyer et al., 1963). These results supported earlier suggestions that the free thiol group of β-lactoglobulin was involved in the interactions (Trautman and Swanson, 1958; Zittle et al., 1962), and it was suggested that intermolecular disulfide bonds were formed between κ-casein and denatured β-lactoglobulin (Sawyer et al., 1963). This has been corroborated by numerous subsequent studies (e.g., Grindrod and Nickerson, 1967; Purkayastha et al., 1967; Sawyer, 1969; Tessier et al., 1969). Some studies indicated that the heat-induced self-aggregation of β-lactoglobulin was limited when κ-casein was present, which suggested that κ-casein formed complexes with intermediate species of aggregated β-lactoglobulin (Sawyer, 1969; McKenzie et al., 1971). In contrast, other studies indicated that the aggregation of β-lactoglobulin was not a prerequisite for its interaction with κ-casein (Euber and Brunner, 1982). The reason for these apparently

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conflicting observations may have been resolved through the detailed study of Cho et al. (2003), in which many of the possible pathways involved in the aggregation of β-lactoglobulin with κ-casein in heated model systems were elucidated. Cho et al. (2003) proposed that, when mixtures of β-lactoglobulin and κ-casein were heated, the free thiol of β-lactoglobulin was exposed and this initiated a series of thiol-disulfide exchange reactions of β-lactoglobulin with other denatured β-lactoglobulin molecules or with κ-casein. The products formed ranged from 1:1 β-lactoglobulin-κ-casein complexes to large heterogeneous aggregates, and the product mix was dependent on the ratio of κ-casein to β-lactoglobulin. The aggregate species were held together by either or both disulfide bonds and hydrophobic interactions. Although there have been some indications of interactions between α-lactalbumin and κ-casein on heating (Shalabi and Wheelock, 1976; Doi et al., 1983), others have reported that interactions between these proteins do not occur (Baer et al., 1976; Elfagm and Wheelock, 1978). It is now generally believed that interactions between α-lactalbumin and κ-casein will occur only if β-lactoglobulin (or another whey protein with a free thiol) is present during heating, and this may require the initial formation of a β-lactoglobulin-α-lactalbumin complex, which subsequently interacts with κ-casein (Baer et al., 1976; Elfagm and Wheelock, 1978). A recent study has shown the importance of thiol-disulfide interactions in the irreversible denaturation and aggregation of α-lactalbumin in milk and whey protein solutions (Nguyen et al., 2018). The irreversible thermal denaturation of α-lactalbumin was promoted when low levels of cysteine (or other thiol-bearing reagents) were added to milk, with significant denaturation occurring at temperatures as low as 61°C. At higher temperatures (above about 70°C), the thermal denaturation of β-lactoglobulin was also enhanced on addition of these thiol reagents. It was proposed that, at temperatures between 61 and 70°C, the added cysteine initiated thiol-disulfide interchange reactions with the disulfide bonds of the unfolded α-lactalbumin, thus promoting irreversible aggregation reactions. At temperatures above 70°C, the cysteine, along with the free thiol of unfolded β-lactoglobulin, initiated thiol-disulfide interchange reactions with the disulfide bonds of both α-lactalbumin and β-lactoglobulin, thus promoting the irreversible aggregation of both these proteins (Nguyen et al., 2013; Nguyen et al., 2018). The addition of thiol reagents to milk prior to heating markedly increased the level of α-lactalbumin interacting with the casein micelles; however, only a small increase in β-lactoglobulin interacting with the micelles was observed (Nguyen et al., 2013). There is considerable evidence to show that disulfide bonds are involved in the aggregates formed between the denatured whey proteins and κ-casein; however, there are reports that suggest that noncovalent bonding may be important in these interactions, particularly in the early stages of heating and at lower heating temperatures (Sawyer, 1969; Haque et al., 1987; Haque and Kinsella, 1988; Hill, 1989). Other studies have shown that, although a substantial part of the denatured whey proteins in heated milk are involved in disulfide-bonded aggregates, there is a significant proportion that can be recovered as monomeric protein under dissociating but nonreducing conditions, indicating that noncovalent interactions are also involved (Oldfield et al., 1998b; Anema, 2000). As Cho et al. (2003) have suggested, it is likely that both hydrophobic and disulfide interactions are important in the early stages of aggregate formation, with the interaction mechanism being dependent on the composition of the system and the conditions of heating.

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Interactions between denatured whey proteins and κ-casein/casein micelles in milk systems Most of the early studies examining the heat-induced interactions between denatured whey proteins and κ-casein were in model systems using purified proteins in buffer solutions. Milk is considerably more complex, with numerous protein species that could potentially interact on heating. A number of the milk proteins have free thiol groups and/or disulfide bonds. Although β-lactoglobulin is the major whey protein component, denatured α-lactalbumin and bovine serum albumin can also be involved in thiol-disulfide exchange reactions and therefore can be incorporated in the aggregated products. For the caseins, as both κ-casein and αS2-casein have disulfide bonds, both could participate in thiol-disulfide exchange reactions with denatured β-lactoglobulin or other denatured thiol-bearing whey proteins. As a consequence of this complexity, there are numerous potential thiol-disulfide interaction pathways, as well as noncovalent interactions, and therefore, the separation and analysis of the reaction products can be difficult. Studies on the interactions between the proteins in heated milk suggest that, despite the complexity of the system, the reactions between β-lactoglobulin and κ-casein may be similar to those occurring in the model systems. In early electrophoretic studies on heated milk, it was noted that the bands corresponding to β-lactoglobulin disappeared, along with a reduction in the intensity of the bands corresponding to casein. This was accompanied by the formation of bands corresponding to new (heterogeneous) components (Slatter and van Winkle, 1952; Tobias et al., 1952). When thiol-blocking agents were added, the band pattern was comparable with that of the original skim milk, indicating that thiol-disulfide exchange reactions were involved in the interaction mechanisms (Trautman and Swanson, 1958). Subsequent studies confirmed that an interaction between denatured β-lactoglobulin and κ-casein on the casein micelles occurred on heating milk, although, as expected, the other denatured whey proteins were also involved in the interactions (Snoeren and van der Spek, 1977; Elfagm and Wheelock, 1978; Smits and van Brouwershaven, 1980; Noh et al., 1989b; Noh et al., 1989a; Corredig and Dalgleish, 1996a,b, 1999; Oldfield et al., 1998b). Unlike κ-casein, αS2-casein does not readily interact with denatured whey proteins when milk is heated, although some interactions in UHT milks have been reported (Snoeren and van der Spek, 1977; Patel et al., 2006). This low reactivity may be due to the location of αS2-casein in the interior of the casein micelles, which makes it less accessible for interaction, whereas κ-casein is located at the casein micelle surface and is therefore more accessible for interaction (Walstra, 1990; Horne, 1998; Huppertz et al., 2017). Interestingly, in pressuretreated skim milk, disulfide-bonded aggregates between αS2-casein and the denatured whey proteins are observed, suggesting that the disulfide bonds of αS2-casein may become accessible to thiol groups of the denatured whey proteins when the casein micelle structure is disrupted under pressure (Patel et al., 2006). The degree of interaction of the denatured whey proteins with the casein micelles is dependent on many variables including the time, temperature, and rate of heating; the milk and individual protein concentrations; the milk pH; and the concentration of the milk salts (Smits and van Brouwershaven, 1980; Corredig and Dalgleish, 1996a,b; Oldfield et al., 2000; Anema and Li, 2003b; Oldfield et al., 2005). For example, when the temperature of milk is gradually increased above 70°C, as in indirect heating systems, most of the denatured

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β-lactoglobulin and α-lactalbumin associates with the casein micelles, presumably as disulfidebonded complexes with κ-casein at the micelle surface (Smits and van Brouwershaven, 1980; Corredig and Dalgleish, 1996b). In contrast, when milk is heated rapidly, as in direct heating systems, only about half of the denatured β-lactoglobulin and α-lactalbumin associates with the casein micelles, with the rest remaining in the milk serum (Singh and Creamer, 1991a; Corredig and Dalgleish, 1996a; Oldfield et al., 1998b). Corredig and Dalgleish (1999) suggested that, on heating milk, α-lactalbumin and β-lactoglobulin initially aggregate in the serum phase at a ratio that is dependent on the initial individual whey protein concentrations. These complexes subsequently associate with κ-casein at the casein micelle surface on prolonged heating. However, Oldfield et al. (1998b) proposed that, under rapid heating rates, β-lactoglobulin forms aggregates in the serum before interacting with the casein micelles and this limits the level of association with the casein micelles, whereas at slower heating rates, monomers or smaller aggregates of β-lactoglobulin may interact with the micelles, and this may allow greater association with the casein micelles. The pH of the milk at heating is important in determining the level of interaction between the denatured whey proteins and the casein micelles. When milk is heated at high temperatures (about 140°C), the heat coagulation time/pH profiles of most milks show increasing heat stability with increasing pH to a maximum at about pH 6.7, followed by decreasing stability to a minimum at about pH 6.9, and increasing stability again as the pH is increased further (Rose, 1961). Considerable research has been undertaken over decades in an attempt to explain this unusual pH-dependent heat stability of milk, and numerous factors are known to influence the heat stability behavior. Many review papers on the heat stability of milk are available (Singh and Creamer, 1992; International-Dairy-Federation, 1995; O’Connell and Fox, 2003; Singh, 2004). The results from the studies on the heat stability of milk have influenced the direction of the future research on the effects of heat on milk and, in particular, on the interactions between denatured whey proteins and κ-casein/casein micelles. Therefore, it is appropriate to briefly review aspects of the pH dependence of heat stability that are relevant to understanding the interactions between denatured whey proteins and κ-casein/casein micelles. Electron microscopic studies showed that, when milk was heated at high temperatures (90–140°C) for long times (30 min) at pHs below 6.7, the denatured whey proteins complexed on to the micelle surfaces as filamentous appendages. However, when the milk was heated at higher pH, the denatured whey proteins were found in the serum phase as aggregated complexes (Creamer et al., 1978; Creamer and Matheson, 1980). These were the first indications that the pH at which the milk is heated may influence the interactions between the denatured whey proteins and the casein micelles. Kudo (1980) showed that the amount of nonsedimentable protein in milk heated at pH 6.5 was lower than that in unheated milk; however, the level of nonsedimentable protein increased with the pH at heating, so, at above pH 6.7, the level was markedly higher than in the unheated milk and increased with increasing pH. Kudo (1980) concluded that the denatured whey proteins cosedimented with the casein micelles at low pH (about pH 6.5), whereas most of the denatured whey proteins along with some casein (particularly κ-casein) was released from the casein micelles at pHs above 6.8. It was also proposed that the transition from

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whey protein-coated casein micelles to protein-depleted forms with changing pH at heating could explain the pH dependence of the heat stability of milk at high temperatures. Singh and Fox (1985a,b 1986, 1987a,b,c), in a series of extensive studies, showed that the dissociation of κ-casein-rich protein on heating was dependent on the pH at heating. At pHs below about 6.8, little dissociation of micellar κ-casein occurred, whereas at higher pH, particularly above pH 6.9, high levels of κ-casein dissociated from the micelles, with the level increasing proportionally with increased pH. The whey proteins, particularly β-lactoglobulin, played an important role in the heat-induced, pH-dependent dissociation of κ-casein (Singh and Fox, 1987a,b), as did mineral components such as calcium and phosphate (Singh and Fox, 1987c). The results from these studies have been used to develop detailed mechanisms for the pH-dependent heat stability of milk, including concentrated milk systems (Singh and Creamer, 1992; O’Connell and Fox, 2003; Singh, 2004). Initially, it was reported that the dissociation of κ-casein from the casein micelles occurred only when milk at high pH (above about pH 6.8) was heated at high temperatures, particularly 90°C or above (Singh and Fox, 1985a). However, subsequent studies demonstrated that, at these pH values, the dissociation of κ-casein occurred as soon as the temperature was raised above ambient, with the level of dissociated κ-casein increasing proportionally with temperature up to 90°C. In these studies, the dissociation of αS-casein (αS1-casein and αS2-casein combined) and β-casein showed unusual temperature dependence. Increasing levels of these caseins dissociated as the temperature was increased up to about 70°C, with the levels then decreasing again at higher temperatures (Fig. 9.4; Anema and Klostermeyer, 1997; Anema, 1998). The unusual temperature dependence of αS-casein and β-casein dissociation was a consequence of the whey proteins, particularly β-lactoglobulin. When whey protein-depleted milk was heated, the levels of αS-casein and β-casein dissociating from the casein micelles increased with increasing temperature up to 90°C. When compared with heating standard milk, this indicated that higher levels of αS-casein and β-casein dissociated from the micelles in the whey protein-depleted milks at temperatures above about 70°C (Anema and Li, 2000). It was postulated that all the caseins dissociated from the micelles on heating. On subsequent cooling, the dissociated κ-casein stabilized the dissociated αS-casein and β-casein as small serum-phase aggregates if the heating temperature was below about 70°C. However, above about 70°C, κ-casein associated with denatured whey proteins. It was already known that the complex formed between κ-casein and denatured β-lactoglobulin was less effective than uncomplexed κ-casein at stabilizing αS-casein and β-casein in the presence of calcium ions (Zittle et al., 1962); therefore, this interaction may have prevented κ-casein from stabilizing the other caseins, and they either reassociated with the casein micelles or formed larger aggregates on subsequent cooling (Anema and Li, 2000). Early studies on the effect of the pH at heating on the interaction of denatured whey proteins with the casein micelles tended to use relatively large pH steps. In a model milk system containing casein micelles and β-lactoglobulin, about 80% of the denatured β-lactoglobulin associated with the casein micelles when the milk was heated at pH 5.8 or pH 6.3, whereas only about 20% associated with the casein micelles at pH 6.8 or pH 7.1 (Smits and van Brouwershaven, 1980). The studies on the heat-induced, pH-dependent dissociation of κ-casein from the casein micelles showed that this dissociation was accompanied by increases in the levels of

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FIG. 9.4 Effect of temperature and pH on the level of protein in the supernatants obtained from 10% TS reconstituted skim milk samples heated for 30 min: (A) κ-casein, (B) αs-casein, and (C) β-casein. , pH 6.3; ●, pH 6.5; □, pH 6.7; , pH 6.9; and 4, pH 7.1. Reproduced with permission from Anema, S.G., Klostermeyer, H., 1997. Heat-induced, pH-dependent dissociation of casein micelles on heating reconstituted skim milk at temperatures below 100°C. J. Agric. Food Chem. 45, 1108–1115. Copyright (1997) American Chemical Society.

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denatured whey proteins remaining in the serum (Singh and Creamer, 1991b), and this was confirmed by Anema and Klostermeyer (1997) and Oldfield et al. (2000), who reported that 80%–90% of the denatured whey proteins associated with the casein micelles when milk was heated at pH below 6.7, whereas only about 20% associated with the casein micelles at pH above 6.8. Corredig and Dalgleish (1996b) measured the ratio of β-lactoglobulin or

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α-lactalbumin to κ-casein in the colloidal phase obtained from heated milk adjusted to pH 5.8, 6.2, or 6.8. Although the denatured whey proteins interacted with the casein micelles at a faster rate at lower pH and at higher temperatures, the ratios of denatured whey proteins to κ-casein on the casein micelles were not markedly different under the different heating conditions. Further studies demonstrated the extreme importance of pH in the association of denatured whey proteins (α-lactalbumin and β-lactoglobulin) with the casein micelles when milk was heated above 70°C, particularly at pH 6.7 or below, where differences in association behavior could be measured at pH differences as small as 0.05 pH units (Anema and Li, 2003a,b; Vasbinder and de Kruif, 2003). From these studies, it was shown that, on heating milk at 90°C for 30 min, about 80% of the denatured whey protein associated with the casein micelles at pH 6.5 and that this level of association decreased linearly as the pH at heating was increased, so that only about 30% was associated at pH 6.7. At higher pH (above pH 6.7), very low levels of denatured whey proteins associated with the casein micelles on heating the milk (Fig. 9.5). Although the heat-induced, pH-dependent dissociation of κ-casein from the casein micelles could explain the low levels of denatured whey proteins interacting with the casein micelles at pH above 6.8, it had been reported that very little κ-casein dissociated from the casein micelles at pH below 6.8 (Singh and Fox, 1985a; Nieuwenhuijse et al., 1991; Singh, 2004). Therefore, it was initially unknown why small shifts in pH between 6.5 and 6.7 affected the association of denatured whey proteins with the casein micelles when milk was heated. The level of κ-casein in the serum phase was low; therefore, it was initially believed that κ-casein was not involved in this partition of the whey proteins between the serum and colloidal phases (Oldfield et al., 1998b; Anema and Li, 2003b; Vasbinder and de Kruif, 2003).

FIG. 9.5 Level of whey proteins associated with the casein micelles/nonsedimentable whey proteins in skim milk samples that were heated at 90°C for various times. The pH values of the milk samples prior to heating were ●, pH 6.5; , pH 6.55; ., pH 6.6; 4, pH 6.65; , pH 6.7; □, pH 6.9; and ⧫, pH 7.1. Reproduced with permission from Anema, S.G., Lee, S.K., Lowe, E.K., Klostermeyer, H., 2004a. Rheological properties of acid gels prepared from heated pH-adjusted skim milk. J. Agric. Food Chem. 52, 337–343. Copyright (2004) American Chemical Society.

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However, subsequent studies showed that the heat-induced dissociation of κ-casein was pH dependent from pH 6.5 to pH 7.1, with a linear increase in serum-phase κ-casein as the pH was increased throughout the pH range from 6.5 to 7.1 (Fig. 9.6A), and that the level of serum-phase κ-casein was correlated with the level of serum-phase denatured whey protein (Fig. 9.6B; Anema, 2007). At the natural pH, about 30%–40% of the κ-casein dissociated on heating the milk at 90°C for 20–30 min (Fig. 9.6A; Anema, 2007), and recent studies showed that, when milk at its natural pH was heated under commercial direct or indirect UHT processing conditions (approximately 140°C/3 s), high levels of κ-casein (up to 60%) dissociated from the casein micelles. Under these UHT heating conditions, most of the denatured whey proteins remained in the serum phase (Anema, 2017a; Gaur et al., 2018). The differences in the level of dissociated κ-casein between the earlier studies and the later studies may be related to the conditions of centrifugation. Early studies used very high centrifugal forces (about 50,000  g), which may have masked the effects at the lower pH, especially under

FIG. 9.6 (A) Effect of the pH at heating on the level of nonsedimentable κ-casein in milk. ●, serum-phase κ-casein in unheated milk; , serum-phase κ-casein in milk heated at 90°C for 20 min; ., serum-phase κ-casein in milk heated at 90°C for 25 min; 4, and serum-phase κ-casein in milk heated at 90°C for 30 min. (B) Relationship between the serum-phase denatured whey protein and the level of serum-phase κ-casein for the heated milk samples. ●, milk heated at 90°C for 20 min; , milk heated at 90° C for 25 min; and ., milk heated at 90°C for 30 min. Reproduced with permission from Anema, S.G., 2007. Role of κ-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk. J. Agric. Food Chem. 55, 3635–3642. Copyright (2007) American Chemical Society.

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conditions where the particles are less hydrated and more readily deposited. Later studies used lower centrifugal forces (about 25,000  g), which allowed smaller dissociated particles to be observed (Parker et al., 2005; Rodriguez del Angel and Dalgleish, 2006; Anema, 2007). Although the level of κ-casein in the serum phase at pH below 6.7 was relatively low (less than about 30% of the total κ-casein), the ratio of denatured whey protein to κ-casein was high and relatively constant (about 2.5 whey protein molecules to each monomeric κ-casein molecule) for the serum-phase proteins at all pH values. In contrast, the ratio of denatured whey protein to κ-casein was only about 1:1 for the whey protein associated with the casein micelles at pH 6.5, and this decreased to about 0.5:1 at pH 7.1 (Anema, 2007). Intensive studies on the soluble whey protein-κ-casein complexes formed when milk was heated at the natural pH also showed that κ-casein was intimately involved in the serum-phase aggregates and that a high ratio of denatured whey proteins to κ-casein was observed (Guyomarc’h et al., 2003). Electron micrographs of the serum-phase whey protein-κ-casein aggregates indicated that these particles were roughly spherical with a relatively uniform size of about 20–50 nm (Parker et al., 2005; Rodriguez del Angel and Dalgleish, 2006). A similar pH-dependent dissociation of κ-casein and the formation of serum-phase denatured whey proteins was observed when concentrated milk was heated at different pH values (Anema, 1998, 2008a). For milk heated at 80°C/30 min, at each milk concentration, the level of nonsedimentable whey protein increased markedly with increasing pH, and at any given pH, the level of nonsedimentable whey protein increased as the milk concentration increased. The level of κ-casein dissociating from the casein micelles when the milk was heated (80°C/30 min) increased markedly with increasing pH at each milk concentration and with increasing milk concentration at any given pH (Anema, 2008a). When milk samples of different concentrations from 10% to 25% TS and at pHs from 6.3 to 7.1 were heated at temperatures of 20–120°C for 10 min, the pH-dependent dissociation of κ-casein from the casein micelles occurred as soon as the milk was heated at temperatures above ambient conditions. At any given pH and milk concentration, the level of κ-casein dissociating from the casein micelles increased with increasing temperature. Similarly, at any given pH and temperature, the level of κ-casein dissociating from the casein micelles increased with increasing milk concentration (Anema, 1998). When the whey protein level in skim milk was increased by up to 1% by the addition of whey protein concentrate/isolate, increased levels of κ-casein were dissociated from the casein micelles, and increased levels of denatured whey proteins remained in the serum phase when the milk was heated (80°C/30 min) at its natural pH. In pH-adjusted milk, at pH 6.5, low levels of κ-casein dissociated from the casein micelles and low levels of denatured whey proteins remained in the serum phase after heating the milk. The levels of κ-casein and denatured whey protein in the serum phase increased as the pH of the milk increased and, at any given pH, higher levels of κ-casein and denatured whey proteins were in the serum phase for the milk with 0.75% added whey protein (Anema, 2018a). There is still some debate over the sequence of events in the interaction reactions between the denatured whey proteins and κ-casein. Some reports suggest that κ-casein dissociates from the micelles early in the heating process and that the denatured whey proteins subsequently interact with the κ-casein either in the serum phase or on the micelles, with a preferential serum-phase reaction (Anema and Li, 2000; Anema, 2007, 2008b). This proposal was supported by the observation that the dissociation of κ-casein is a rapid process and that significant dissociation of κ-casein can occur at temperatures below those at which the

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denaturation of whey proteins occurs (Anema and Klostermeyer, 1997). In addition, significant dissociation of κ-casein occurs in systems that have been depleted of whey proteins (Anema and Li, 2000). The higher ratio of denatured whey protein to κ-casein for the serum-phase protein aggregates regardless of the pH at heating or the level of dissociated κ-casein may also suggest a preferential serum-phase reaction between the denatured whey proteins and κ-casein (Anema, 2007). However, other reports suggest that, on heating milk, the denatured whey proteins first interact with the casein micelles and that the whey protein-κ-casein complexes subsequently dissociate from the casein micelles (Parker et al., 2005; Donato and Dalgleish, 2006; Donato et al., 2007b; Donato and Guyomarc’h, 2009). This proposal was supported by the observation that the addition of sodium caseinate to milk did not increase the level of serum-phase complexes between the denatured whey proteins and κ-casein, which was interpreted as indicating that the complexes between the denatured whey proteins and κ-casein were formed on the casein micelle surface regardless of the pH at heating (Parker et al., 2005). Milks with added κ-casein were analyzed by size exclusion chromatography (Donato et al., 2007b). The difference profiles between the sera from unheated milk and heated milk with added κ-casein produced a negative peak in the region of the native whey proteins and a positive peak in the region of the whey protein-κ-casein aggregates. As the difference spectra were the same as for milks without added κ-casein, this was also interpreted as indicating that the added κ-casein was not involved in the formation of the serum-phase aggregates and therefore that the denatured whey proteins interacted only with micelle-bound κ-casein, with the whey protein-κ-casein complexes subsequently dissociating from the casein micelles (Donato et al., 2007b; Donato and Guyomarc’h, 2009). The partial hydrolysis of κ-casein by chymosin prevented the dissociation of unhydrolyzed κ-casein from the casein micelles. This in turn prevented the formation of serum-phase whey protein-κ-casein complexes and therefore increased the level of denatured whey proteins associating with the casein micelles (Renan et al., 2007). This observation was also used as evidence to support the initial interaction of denatured whey proteins with the casein micelles, with the subsequent dissociation of whey protein-κ-casein complexes, as it was suggested that the unhydrolyzed κ-casein should still be able to dissociate and interact with serum-phase denatured whey proteins if this was the preferential reaction pathway (Renan et al., 2007). However, this proposal did not take into account the polymeric nature of κ-casein (Holland et al., 2008), and therefore, the partial hydrolysis of κ-casein would substantially increase the hydrophobicity of the polymer even when it contained some unhydrolyzed κ-casein. This increased hydrophobicity may account for the reduced dissociation of κ-casein from the micelles and the increased interaction of denatured whey proteins with the casein micelles (Donato and Guyomarc’h, 2009). It was also suggested that two mechanisms occur depending on the pH at heating. This was based on observations that the protein composition of the serum phase appeared to vary markedly depending on whether the milk was heated at a pH above or below the natural pH of the milk (Donato and Dalgleish, 2006). However, other studies did not display a marked difference in composition of the serum-phase proteins with pH (Anema, 2007). A detailed study that was specifically aimed at elucidating the sequence of events occurring when denatured whey proteins interacted with κ-casein in heated milks was conducted (Anema, 2008b). It was shown that κ-casein could dissociate from the casein micelles at temperatures that were below those at which the whey proteins denatured (Fig. 9.7A). When

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FIG. 9.7 (A) Level of serum-phase whey protein (filled symbols) and κ-casein (open symbols) in milk samples heated for 15 min at different temperatures. (B) Level of denatured (filled symbols) and micelle-bound (open symbols) whey proteins in milk samples heated at 90°C for different times. (C) Level of serum-phase κ-casein in milk samples heated at 90°C for different times. The skim milks were adjusted to (, ●), pH 6.5; (4, ▲), pH 6.7; and (□, ), pH 6.9 before heating. Reproduced with permission from Anema, S.G., 2008b. On heating milk, the dissociation of κ-casein from the casein micelles can precede interactions with the denatured whey proteins. J. Dairy Res. 75, 415–421. Copyright (2008) Cambridge University Press.

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heated at temperatures at which the whey proteins could denature, it was found that κ-casein dissociated from the casein micelles in the early stages of heating and before significant levels of whey proteins were denatured. In addition, the maximum level of serum-phase κ-casein was obtained when less than half the whey proteins were denatured, and once this maximum

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level of κ-casein was dissociated, any additional denatured whey proteins formed on prolonged heating were found predominantly in the serum phase (Fig. 9.7B), indicating a preferential interaction of the denatured whey proteins with the serum-phase whey protein-κ-casein complexes. When κ-casein was added to the milk prior to heating, the denatured whey proteins preferentially interacted with the added serum-phase κ-casein, regardless of the pH at heating (Table 9.1). Taken together, these results provide unequivocal evidence that κ-casein dissociation from the micelles can precede the interaction of denatured whey proteins with the κ-casein and that denatured whey proteins will preferentially interact with serum-phase κ-casein (Anema, 2008b). The pH-dependent changes in the association of the denatured whey proteins with the casein micelles, and the dissociation of κ-casein from the micelles, can have an effect on some of the physical properties of the milk. A marked increase in casein micelle size was observed when high levels of denatured whey protein were associated with the colloidal phase, as is observed on heating milk at pH 6.5. This change in size was less pronounced as the pH at heating the milk was increased to pH 6.7, and a decrease in casein micelle size was observed when significant levels of κ-casein were dissociated from the colloidal phase, as is observed on heating milk at pH values above 6.7 (Fig. 9.8A; Anema and Li, 2003a,b). Similar changes in viscosity (Fig. 9.8B) and turbidity (Fig. 9.8C) with the pH at heating were also observed (Anema et al., 2004c).

TABLE 9.1 Serum-phase denatured whey proteins from skim milk with different levels of added κ-casein that were adjusted to pH 6.5, 6.7, and 6.9 before heating at 90°C for 15 min; the numbers represent the average and standard deviation of triplicate measurements Added κ-casein (%)

pH at heating

Serum-phase denatured whey protein (% of total)

0

6.5

35  1a

0.1

6.5

67  2b

0.2

6.5

84  3c

0

6.7

71  2a

0.1

6.7

78  3b

0.2

6.7

92  3c

0

6.9

86  3a

0.1

6.9

91  3a,b

0.2

6.9

95  3b

Data at a given pH with the same letters are not significantly different from each other at P < .05. (Reproduced with permission from Anema, S.G., 2008b. On heating milk, the dissociation of κ-casein from the casein micelles can precede interactions with the denatured whey proteins. J. Dairy Res. 75, 415–421. Copyright (2008) Cambridge University Press.)

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9. The whey proteins in milk

FIG. 9.8 Effects of the pH at heating on the changes in (A) the size of the casein micelles, (B) the viscosity of the milk, and (C) the turbidity of the milk. The milk samples were heated at 90°C for various times, and the pH values of the milk samples prior to heating were ●, pH 6.5; , pH 6.55; ., pH 6.6; 4, pH 6.65; □, pH 6.7; □, pH 6.9; and ⧫, pH 7.1. Some of the particle size and viscosity results were reproduced with permission from Anema, S.G., Lowe, E.K., Li, Y., 2004c. Effect of pH on the viscosity of heated reconstituted skim milk. Int. Dairy J. 14, 541–548. Copyright (2004) Elsevier.

The difficulty in interpreting these changes in size, viscosity, and turbidity is determining whether the association of the denatured whey proteins with the casein micelles is directly responsible for the change in size/volume of the casein micelles by increasing the diameters of the individual particles as the proteins interact or whether there is some associated phenomenon, such as aggregation of the casein micelles, that is related to the level of whey

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protein or κ-casein that is in the serum phase or associated with the casein micelles. The strong relationship between the level of whey protein associating with the colloidal phase and the size/volume of the casein micelles, the observation that the size change plateaus on prolonged heating, and the relationships between the protein composition of the micelles and size, viscosity, and turbidity seem to suggest that the size changes are a direct consequence of the distribution of protein between the colloidal and serum phases, rather than an associated aggregation reaction (Anema and Li, 2003a,b; Anema et al., 2004c). Determination of the specific disulfide bonds formed between κ-casein and β-lactoglobulin Although many types of bonding may be involved in the early stages of interactions between the denatured whey proteins and κ-casein, there is clear evidence that disulfide bonds are involved in complex formation when model systems and milk are heated. Studies have focused on determining the specific thiol groups of κ-casein and, in particular, β-lactoglobulin that are involved in the disulfide bonding between these two protein species when they are heated in model systems or milk (Henry et al., 2002; Creamer et al., 2004; Livney and Dalgleish, 2004; Lowe et al., 2004). Understanding the specific disulfide bonds involved in the interaction process may provide useful insights into the mechanisms for the denaturation and subsequent aggregation reactions of the whey proteins in milk. Native β-lactoglobulin has two disulfide bonds and one free thiol group at Cys121 (Qin et al., 1999), whereas κ-casein is found as a heterogeneous polymeric protein that is cross-linked in a random manner by disulfide bonding via the two Cys groups in the monomer protein (Rasmussen et al., 1999; Holland et al., 2008). It was believed that the formation of disulfide bonds between the denatured β-lactoglobulin and other milk proteins, including κ-casein, during heating first involved the dissociation of the β-lactoglobulin dimer to monomer species and followed by the unfolding of the native structure, exposing the buried side groups including the free thiol at Cys121. The exposure of this free Cys121 then initiated a series of intermolecular thiol-disulfide exchange reactions with other denatured whey proteins and with κ-casein on the casein micelle surface (Snoeren and van der Spek, 1977; Iametti et al., 1996; Hoffmann and van Mil, 1997; Verheul et al., 1998; Vasbinder and de Kruif, 2003; Creamer et al., 2004). However, studies on pure β-lactoglobulin indicated that, in the early stages of the denaturation process, nonnative monomeric β-lactoglobulin species, which may be intermediates in the intermolecular aggregation processes, are formed. These nonnative monomeric species are stable on subsequent cooling and can be separated by alkaline PAGE techniques (Manderson et al., 1998; Hong and Creamer, 2002) or by gel permeation chromatography (Iametti et al., 1996; Croguennec et al., 2003, 2004). It was hypothesized that the nonnative monomers were formed from intramolecular thiol-disulfide exchange reactions between the free Cys121 of β-lactoglobulin and the Cys106-Cys119 and/or Cys66-Cys160 disulfide bonds within the same β-lactoglobulin monomer (Iametti et al., 1996; Manderson et al., 1998; Hong and Creamer, 2002; Croguennec et al., 2003, 2004). With the advent of sensitive mass spectrometric techniques, the identification of the Cys residues involved in disulfide-bonded protein species was possible. The strategy for the identification of specific disulfide bonds involves a number of steps (Gillece Castro and Stults, 1996; Gorman et al., 2002; Lowe et al., 2004; Kehoe et al., 2007). For the sample under analysis,

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9. The whey proteins in milk

the protein species are first hydrolyzed under conditions where no further thiol-disulfide bond exchange reactions are likely to occur. The peptides formed from this hydrolysis are separated, usually by reverse phase HPLC, and the mass of the individual peptides is determined by mass spectrometry (MS). The identification of individual peptides can be achieved by comparing the measured masses with those of expected peptides for the hydrolysis of the protein under study. For the disulfide-bonded peptides, usually, other criteria also need to be satisfied, such as the peptides being present in nonreduced hydrolyzates but being absent in the reduced system. Further confirmation can be gained by the use of tandem MS, where single molecular ions are isolated and analyzed in the first mass analyzer and are then passed into a collision cell where fragmentation of the peptide is induced by collision with an inert gas (collisioninduced dissociation [CID]) and the fragments are characterized in the second mass analyzer. From the mass of the fragments, the sequence of the amino acids in the peptides can be determined, providing conclusive characterization of the peptides (Gorman et al., 2002; Lowe et al., 2004; Kehoe et al., 2007). Using these types of mass spectrometric techniques, a stable nonnative β-lactoglobulin monomer with a free thiol group at position Cys119 rather than the natural position of Cys121 was found in heated β-lactoglobulin solutions (Croguennec et al., 2003), confirming that intramolecular thiol-disulfide exchange within monomeric β-lactoglobulin could occur. It was suggested that this β-lactoglobulin with the free thiol at Cys119 may be the activated monomer that was proposed as the starting point for intermolecular aggregation reactions leading to large polymers, although it was equally possible that unfolded protein with a free thiol at the natural position of Cys121 was that activated monomer (Croguennec et al., 2003, 2004; Creamer et al., 2004). A more recent investigation on the disulfide-bonding patterns in heated β-lactoglobulin found that a significant proportion of Cys160 was in the reduced form after heating β-lactoglobulin in solution (Creamer et al., 2004; Kehoe et al., 2007), indicating that the Cys66-Cys160 disulfide bond was broken during the early stages of heating. This may occur concurrently with the interchange of the free thiol from Cys121 to Cys119. It was suggested that a monomeric β-lactoglobulin species with a free thiol at Cys160 may be (one of ) the reactive species involved in the intermolecular thiol-disulfide bonding that is responsible for crosslinking in heat-induced whey protein aggregates because of its position near the C-terminal end of the protein. Attempts have been made to identify the specific Cys residues involved in disulfide bonds formed between κ-casein and β-lactoglobulin when these proteins are heated together. Livney and Dalgleish (2004) compared masses of peptides from tryptic digests of heated κ-casein/ β-lactoglobulin mixtures with theoretical values and concluded that Cys106/119/121 of β-lactoglobulin were involved in disulfide bonds with both Cys11 and Cys88 of κ-casein (note that the hydrolysis pattern does not allow the separation of the three Cys106/119/121 residues of β-lactoglobulin unless CID is used for sequencing). Although some peptides involving Cys66 and Cys160 of β-lactoglobulin and the two Cys residues of κ-casein were also identified based on mass comparisons, the high abundance of disulfide-bonded peptides containing Cys106/119/121 led these authors to conclude that β-lactoglobulin with a free thiol at Cys119/121 was the predominant species that was involved in intermolecular disulfide bonding. The potential disulfide-bonded species were characterized based on mass analysis alone;

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no confirmatory experiments were performed, such as comparing reduced with nonreduced systems to ensure that the proposed intermolecular peptides were disulfide bonded, or confirming sequences by CID-MS to preclude misidentification of similarly massed peptides. In a novel study, Lowe et al. (2004) used an activated monomeric κ-casein with reduced thiol groups that were blocked with thionitrobenzoate (TNB). β-Lactoglobulin was added to the mixture, and the system was heated under very mild conditions (60°C). The TNB groups on the thiols of κ-casein are good leaving groups, and when a reactive thiol from β-lactoglobulin is exposed, it is capable of interacting with the activated TNB groups on κ-casein in a specific 1:1 oxidative reaction, forming a disulfide-bonded complex and releasing the TNB as a brightly colored compound. This approach allowed the formation of specific disulfide bonds between κ-casein and β-lactoglobulin under mild heating conditions. Because of the chemical nature of the reaction, it limited further thiol-disulfide exchange reactions, which allowed specific interactions between β-lactoglobulin and κ-casein to be monitored during the early stages of the denaturation of β-lactoglobulin. The interacted β-lactoglobulin-κ-casein complexes were hydrolyzed with trypsin and were separated by reverse phase HPLC followed by MS. In addition, disulfide bonding was confirmed by comparing the HPLC traces of nonreduced systems with those of reduced systems, and the identities of some peptides were confirmed by sequencing using CID-MS. Although it was possible to identify disulfide bonds between Cys106/119/121 of β-lactoglobulin and Cys88 of κ-casein, Cys160 of β-lactoglobulin was found to have formed disulfide bonds with both Cys11 and Cys88 of κ-casein as major products (Fig. 9.9). This supported the earlier findings on pure β-lactoglobulin that intramolecular thiol-disulfide exchange may precede the intermolecular reactions and that the nonnative monomeric form of β-lactoglobulin species with a free thiol at Cys160 is likely to be (one of ) the reactive monomer species that initiates intermolecular thiol-disulfide exchange reactions (Creamer et al., 2004; Kehoe et al., 2007). Lowe et al. (2004), using the techniques developed for the model system, expanded the study to examine the specific disulfide bonds involved in aggregation between β-lactoglobulin and κ-casein in heated milk systems. Interestingly, no disulfide bonds between Cys106/119/121 of β-lactoglobulin and either of the Cys residues of κ-casein could be found, even though the disulfide bond between Cys88 of κ-casein and Cys106/119/121 of β-lactoglobulin was readily identified in the model system. In the heated milk system, it was found that Cys160 of β-lactoglobulin formed disulfide bonds with both Cys88 and Cys11 of κ-casein, as was found in the model system (Fig. 9.9). In independent studies, a similar disulfide bond between Cys160 of β-lactoglobulin and Cys88 of κ-casein was identified in a heated model goat milk system consisting of isolated casein micelles and β-lactoglobulin suspended in milk ultrafiltrate, although it appears that no attempts were made to isolate and characterize other intermolecular disulfide bonds between these protein species (Henry et al., 2002). From these observations, it was concluded that, in the model system of β-lactoglobulin and activated κ-casein, nonnative monomeric β-lactoglobulin species with a free thiol at either Cys119 or Cys121 (but probably not Cys106) could be the reactive monomer that is involved in intermolecular thiol-disulfide exchange reactions, as Cys119/121 was involved in disulfide bonds with κ-casein. However, further intramolecular thiol-disulfide exchange reactions in β-lactoglobulin must precede or occur concurrently with the intermolecular reactions, as disulfide bonds between Cys160 of β-lactoglobulin and the two Cys residues of κ-casein were also observed as major products in the model system (Fig. 9.9).

352

9. The whey proteins in milk

S-S

p

p

p

k-Casein Confirmed tryptic peptides CID-MS confirmed peptides Cys11

Cys88

Found in Fou n

d in

Fo un d

heated m ilk and m

odel syst

hea

ted

Confirmed by CID MS Not confirmed

Foun d in

milk

Cys66

ated

m

od el

em

in he

sy

milk

and

st

mod

el sy

stem

em

Cys106

Found in model system Cys119/121

Cys160

Peptides only in reduced hydrolyzate Hydrolyzate peptides from model system and heated milk

b-Lactoglobulin SS SS

1

20

40

60

80

100

120

140

160

170

Residue number

FIG. 9.9 Diagram indicating the identified peptides on the linear sequences of κ-casein and β-lactoglobulin and the

intermolecular disulfide bonds formed between κ-casein and β-lactoglobulin on heating model systems and milk. The horizontal box lines represent the protein sequence, the lines over the boxes represent the peptides, and SdS indicates the presence of a disulfide bond. Arrows indicate the major proteolytic sites, the chymosin site for κ-casein, and the rapid tryptic sites for β-lactoglobulin. Potential glycosylation and phosphorylation sites are indicated for κ-casein. Reproduced with permission from Lowe, E.K., Anema, S.G., Bienvenue, A., Boland, M.J., Creamer, L.K., Jimenez-Flores, R., 2004. Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine β-lactoglobulin and κ-casein. J. Agric. Food Chem. 52, 7669–7680. Copyright (2004) American Chemical Society.

In the heated milk system, no peptides involving Cys106/119/121 and the two Cys residues of κ-casein were isolated. Only peptides involving Cys160 and Cys66 with both Cys residues of κ-casein were found (Fig. 9.9). As Cys160 and Cys66 are involved in a disulfide bond in native β-lactoglobulin, this indicates that intramolecular thiol-disulfide exchange reactions in β-lactoglobulin precede the intermolecular thiol-disulfide exchange reactions and that a β-lactoglobulin (monomeric) species with a free thiol group at Cys160 may play a significant role in the interprotein disulfide bonding that occurs in heated milk or whey protein systems. The differences in reaction products between the model systems and milk may be a consequence of factors such as the heating conditions, the nature of the reactions (oxidative interaction compared with thiol-disulfide interchange reactions), and the fact that the κ-casein in milk is found on the casein micelles whereas in the model system, it is not (Lowe et al., 2004). Because of the C-terminal location of Cys160 in β-lactoglobulin, when this Cys is in the free thiol form and not linked to Cys66, it may be able to productively react with the disulfide bonds of κ-casein to give stable whey protein-κ-casein aggregates (Lowe et al., 2004).

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353

Relationships between denaturation/interactions of the whey proteins in heated milk and the functional properties of milk products When milk is heated, there are numerous changes, including changes to the proteins, the milk salts (such as changes in the mineral equilibria between the colloidal and serum phases), and lactose. Many of the changes can involve more than one of the milk constituents (International Dairy Federation, 1995). The changes can be irreversible or reversible to various extents depending on the changes being monitored and the conditions of the heat treatment. Although the changes to the protein system are an important determinant of the functional properties of milk products, all other changes to the milk system should also be considered to obtain a full understanding of the relationships between heat treatments, interactions, and functional performance. However, as there are limited examples of changes to components other than the proteins and the functional behavior of milk products, this review is restricted to some examples of the relationships between the changes in the milk protein system and the functional performance of the milk.

Examples of the relationships between whey protein denaturation and the functional properties of milk In the early days of milk powder manufacture, it was recognized that the level of whey protein denaturation could be used as an index for the extent of heat treatment the milk had received during the manufacture of the milk powders. The functional properties of the milk products were related to some extent to the heat treatment that the milk had received during processing and therefore the level of whey protein denaturation (Harland and Ashworth, 1947; Larson et al., 1951; Harland et al., 1952). Even as early as 1952, the concept of “tailor-made” milk powders was discussed, where powders were processed to provide specific requirements, such as low heat powders for beverage applications and cottage cheese manufacture, and high heat powders for bakery applications (Harland et al., 1952). Although there were no standards of quality or processing at this time, it was recognized that the proper control of processing conditions, particularly preheating of the milk, was necessary to produce satisfactory products. Measurement of the level of whey protein denaturation could be used as an objective method for determining the suitability of milk (powder) products for particular commercial and functional applications. The heat treatment of milk, whether in liquid milk applications or prior to drying for milk powder manufacture, remains one of the major processes for manipulating the functional properties of milk products. Products such as milk powders are still generally classified according to the heat treatments received during manufacture using one of the derivatives of the WPNI test (Singh and Newstead, 1992; Kelly et al., 2003). With the extensive research on the denaturation of the whey proteins and the ability to predict the denaturation levels after defined heat treatments, it could be envisaged that the level of denaturation of the whey proteins could be used as an indicator of the functional properties of milk products. In a broad sense, this is true. For example, certain heat classifications of milk powders will give improved functionality over other classes of milk powders for particular applications.

354

9. The whey proteins in milk

Some of the general applications of different heat-classified milks and their functional uses, in particular for milk powder products, have been summarized in numerous publications (Singh and Newstead, 1992; International-Dairy-Federation, 1995, 1996; Kelly et al., 2003). However, an infinite number of temperature and heating time combinations to denature the whey proteins when milk is heated are available. As a consequence, specific correlations between the level of whey protein denaturation and the functional properties of milk across all possible heating conditions and milk sources do not exist. For example, the WPNI method was developed for assessing the suitability of milk powders for use in bakery applications; however, it was noted that a powder with a low WPNI did not always correspond to good baking qualities (Harland and Ashworth, 1947). Some of these variations are due to factors such as natural variations in the initial whey protein levels in the milk (Harland et al., 1955; Sanderson, 1970b); however, others are due to the methods of heat treatment during milk processing. As such, the WPNI or level of whey protein denaturation is, at best, a guide for the suitability of powders for specific applications or an in-factory guide on processing conditions. Many manufacturers impose additional specifications to the milk powders to ensure suitability in their specific applications (Sanderson, 1970c; Singh and Creamer, 1991a). Some of the most detailed studies on the relationship between the functional performance of milk and the heat treatment conditions or whey protein denaturation levels have been reported for acid gel or yogurt systems. Parnell-Clunies et al. (1986) showed correlations between the level of whey protein denaturation and the firmness and apparent viscosity of yogurt, regardless of the method used to heat the milk (batch [85°C], high temperature, short time [98°C], and UHT [140°C] heating systems for different holding times). However, other properties, such as water-holding capacity/syneresis, were more dependent on the heating system used, and it was concluded that high levels of whey protein denaturation in milk were not necessarily associated with an improved water-holding capacity in yogurt. In extensive studies, Dannenberg and Kessler (1988a,c) examined the relationship between the denaturation level of whey proteins in the milk and the functional performance (firmness, flow properties, and syneresis) of the milk in set yogurt applications. There was a clear relationship between whey protein denaturation and yogurt firmness, with a higher firmness at higher levels of whey protein denaturation. Similar results were obtained for the flow properties of the yogurt. However, very high levels of whey protein denaturation appeared to be detrimental, with a decrease in the firmness and flow properties at denaturation levels above about 95% (Dannenberg and Kessler, 1988c). For syneresis of the yogurt, a negative relationship between whey protein denaturation and the level of serum expelled from the yogurt was observed (Dannenberg and Kessler, 1988a). Despite the apparent correlations between denaturation and firmness, flow properties, or syneresis, there were significant variations at each denaturation level, indicating that the temperature of heating used to denature the whey proteins, rather than just the whey protein denaturation level, may be an important factor in determining the functional performance in acid gels. In a study on reconstituted whole milk, McKenna and Anema (1993) also observed a positive correlation between the denaturation of the whey proteins in the milk and the firmness of the yogurt made from the milk regardless of whether the heat treatment was performed before or after powder manufacture (Fig. 9.10A). However, when individual heating conditions were examined, it was also noted that excessive heat treatment of the milk could be

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355

FIG. 9.10 Relationship between the level of whey protein denaturation in reconstituted whole milk and (A) the firmness and (B) the syneresis of acid gels prepared from the heated milks. The milks were (●) heated only before powder manufacture, (.) heated only after reconstitution, or () heated both before powder manufacture and after reconstitution. Plotted from the data of McKenna, A.B., Anema, S.G., 1993. The effect of thermal processing during whole milk powder manufacture and after its reconstitution on set-yoghurt properties. In: International Dairy Federation Special Issue 9303. Protein & Fat Globule Modifications by Heat Treatment, Homogenization & Other Technological Means for High Quality Dairy Products. International Dairy Federation, Brussels, Belgium, pp. 307–316.

detrimental to the firmness of the set yogurt (McKenna and Anema, 1993). A less clear relationship between syneresis and the level of whey protein denaturation was observed, with the level of syneresis appearing to have a greater dependence on the heating conditions (temperature, time, and before/after reconstitution) than on the level of denaturation itself (Fig. 9.10B; McKenna and Anema, 1993), which supports the findings of Parnell-Clunies et al. (1986).

Examples of the relationships between the level of interactions of whey proteins with κ-casein/casein micelles and the functional properties of milk A major limitation in using whey protein denaturation as an index of the functional properties of milk is that it does not consider the subsequent interaction reactions of the denatured whey proteins. These interactions will be dependent on the conditions of denaturation such as temperature and time as well as on the properties of the milk such as pH, concentration, and composition. It is more complex to investigate these aggregation reactions, as there are potentially numerous pathways and there is great difficulty in isolating and characterizing the specific reaction products. However, in recent years, some effort has been made in identifying the interaction reactions of the denatured whey proteins with other components in milk and, in some cases, their effects on the functional properties of the milk.

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9. The whey proteins in milk

Acid-induced aggregation/gelation of heated milk Anema et al. (2004a) showed that small changes in the pH of milk from the natural value at the time of heating markedly affected the properties of acid gels prepared from these heated milks. During acidification of the heated pH-adjusted milks, the acid gelation profiles were progressively shifted to higher firmness as the pH at heating was increased (Fig. 9.11A) so that the final firmness of the acid gels (measured as the storage modulus, G0 , at pH 4.2) was almost doubled as the pH at heating was increased from pH 6.5 to pH 7.1 (Fig. 9.11). The effect was particularly pronounced in the milks that were heated for times sufficient to fully denature the whey proteins (Fig. 9.11B). This effect of small changes in the pH of the milk at the time of heating on the firmness of acid gels prepared from heated milk has been independently confirmed (Lakemond and van Vliet, 2005; Rodriguez del Angel and Dalgleish, 2006; Lakemond and van Vliet, 2008a,b). Large-strain deformation experiments were also conducted on the set gels. In these experiments, the strain was increased at a constant rate, and the stress was monitored until the gel structure yielded and the stress decreased. The maximum in the strain versus stress curves FIG. 9.11 (A) Changes in firmness (G0 ) with time after the addition of glucono-δ-lactone (GDL) for heated (90°C/30 min) skim milk samples. (B) Changes in the final firmness (final G0 ) for acid gels prepared from milk samples heated for various times at 90°C. The pH values of the milk samples prior to heating were ●, pH 6.5; , pH 6.55; ., pH 6.6; 4, pH 6.65; , pH 6.7; □, pH 6.9; and ⧫, pH 7.1. In all samples, the pH was readjusted back to pH 6.7 before addition of GDL, and this pH was reduced to about pH 4.2 after 5.5 h. Reproduced with permission from Anema, S.G., Lee, S.K., Lowe, E.K., Klostermeyer, H., 2004a. Rheological properties of acid gels prepared from heated pH-adjusted skim milk. J. Agric. Food Chem. 52, 337–343. Copyright (2004) American Chemical Society.

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357

FIG. 9.12 Stress versus strain curves for acid gels prepared from heated (90°C/15 min) skim milk samples. The pH values of the milk samples prior to heating were ●, pH 6.5; , pH 6.6; ., pH 6.7; 4, pH 6.9; and , pH 7.1. The maximum in the stress represents the breaking point of the gel, and the stress and the strain at this point are considered to be the breaking stress and the breaking strain, respectively.

▪

was considered to be the point at which the gel structure broke (Fig. 9.12). The yield properties of the final set acid gels were affected by the pH at which the milk was heated. The yield stresses of the gels increased markedly, and the yield strains of the gels decreased slightly as the pH at which the milk was heated was increased (Fig. 9.12; Anema, 2008a; Lakemond and van Vliet, 2008a). In addition to influencing the final firmness of the acid gels, the pH at the heat treatment of the milk also influenced the pH at which the milk started to gel/aggregate during acidification (Vasbinder and de Kruif, 2003; Anema et al., 2004a; Anema et al., 2004b; Lakemond and van Vliet, 2005; Rodriguez del Angel and Dalgleish, 2006; Lakemond and van Vliet, 2008a,b). On subsequent acidification of milk samples that were heated over a pH range from 6.5 to 7.1, samples heated at higher pH (pH 7.1) started to gel at significantly higher pH than samples heated at lower pH. These effects were very dependent on the temperature at which the milks were acidified (Anema et al., 2004b). In further detailed studies on the properties of the acid gels prepared from milks heated at different pHs (Lakemond and van Vliet, 2008a), it was shown that the permeability coefficients of the gels increased as the pH of the milks at heating increased. In addition, adding thiol-blocking agents to the milks during acid gelation did not affect the firmness of the gels prepared from milks heated at low pH (about pH 6.20) but markedly reduced the firmness of the gels prepared from milks heated at higher pH (about pH 6.90). Based on the permeability, the effects of thiol-blocking agents, and the small and large-strain rheological results of the acid gels prepared from pH-adjusted heated milks, it was concluded that acid gels from milks heated at low pH had a finer stranded structure with a higher strand curvature and that the gels contained fewer (intermolecular) disulfide bonds. These differences in gel properties accounted for the lower firmness of the gels prepared from milks heated at lower pH (Lakemond and van Vliet, 2008a). In concentrated milk, a similar effect of the pH of the milk at heating on acid gel firmness and yield stress was observed, with a marked increase in final gel firmness (Fig. 9.13A and B) and yield stress (Fig. 9.14A and B) as the pH of the milk at heating was increased from the natural pH and a marked decrease in firmness when the pH of the milk at heating was

358

9. The whey proteins in milk

FIG. 9.13 (A) Changes in storage modulus, G0 , with time after GDL addition for heated (80°C/30 min) 10% TS skim milk samples. ●, pH 6.48; , pH 6.55; ., pH 6.59; 4, pH 6.67; , pH 6.90; and □, pH 7.10. (B) Changes in storage modulus, G0 , with time after GDL addition for heated (80°C/30 min) 20% TS skim milk samples. ●, pH 6.28; , pH 6.39; ., pH 6.48; 4, pH 6.75; and , pH 6.95. (C) Percentage change in final G0 versus change in pH at heating from the natural pH of the milk. ●,  10% TS milk samples; ., 4 15% TS milk samples; , □ 20% TS milk samples; and ⧫, ◇ 25% TS milk samples. Open symbols, samples at the natural pH; filled symbols, samples that were pH adjusted before heating. Reproduced with permission from Anema, S.G., 2008a. Effect of milk solids concentration on the gels formed by the acidification of heated pH-adjusted skim milk. Food Chem. 108, 110–118. Copyright (2008) Elsevier.

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359

FIG. 9.14 (A) Stress versus strain curves for acid gels prepared from heated (80°C/30 min) 10% TS skim milk samples. ●, pH 6.48; , pH 6.55; ., pH 6.59; 4, pH 6.67; , pH 6.90; and □, pH 7.10. (B) Stress versus strain curves for acid gels prepared from heated (80°C/30 min) 20% TS skim milk samples. ●, pH 6.28; , pH 6.39; ., pH 6.48; 4, pH 6.75; and , pH 6.95. (C) Percentage change in breaking stress versus change in pH at heating from the natural pH of the milk. ●, 10% TS milk samples; ., 15% TS milk samples; , 20% TS milk samples; and ⧫, 25% TS milk samples. Reproduced with permission from Anema, S.G., 2008a. Effect of milk solids concentration on the gels formed by the acidification of heated pH-adjusted skim milk. Food Chem. 108, 110–118. Copyright (2008) Elsevier.

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decreased (Anema, 2008a). As the natural pH of milk decreases when milk is concentrated, the percentage changes in firmness or yield stress of the gels from that obtained at the natural pH were plotted against the relative change in pH; it was found that the firmnesses (Fig. 9.13C) or yield stresses (Fig. 9.14C) of the gels at all milk concentrations fell on a single curve. This indicates that changing the pH of milk at heating had the same effect on the final firmnesses or yield stresses of the acid gels at all milk concentrations (Anema, 2008a). When low levels of starch (up to 1% w/w) were added to milks prior to heating and acidification, the effect of pH on gel firmness was similar to that of milk without starch addition; however, the firmness of the gels increased as the starch level increased (Oh et al., 2007). The gelatinized starch absorbed the aqueous phase in the milk and consequently increased the density of the protein network in the acid gels, increasing the firmness in a similar fashion to increasing the concentration of the milk. However, when higher levels of starch were added to the milk (>1% w/w), the pH effect diminished, although the gels were still firmer as the starch level increased (Oh et al., 2007). At these high levels of starch, the viscosity of the milk increased markedly after heating because of the gelatinization of the starch and the leaching of amylose into the continuous phase. It was proposed that the high viscosity of the continuous phase affected the diffusion of protein components during heating and subsequent acidification and that this in turn changed the network structure formed in the acid gel, diminishing the importance of serum-phase components (Oh et al., 2007). The changes in acid gel firmness or yield stress on changing the pH at heating of the milk could not be related solely to the level of whey protein denaturation (Fig. 9.11A). Small changes in the pH of the milk before heating markedly affect the distribution of the denatured whey proteins and κ-casein between the colloidal and serum phases in milk at its natural concentration (Figs. 9.4 and 9.5; Anema and Klostermeyer, 1997; Anema and Li, 2003a,b; Vasbinder and de Kruif, 2003; Rodriguez del Angel and Dalgleish, 2006; Lakemond and van Vliet, 2008b), and similar effects were observed in concentrated milks (Anema, 2008a; Chandrapala et al., 2010). Although heating milk prior to acidification markedly increased the firmness of the acid gels (i.e., acid gels prepared from heated milks always had a considerably higher firmness than acid gels prepared from unheated milks (Dannenberg and Kessler, 1988c; Lucey et al., 1997; Lucey and Singh, 1998)), the distribution of the denatured whey proteins and κ-casein between the colloidal and serum phases also appeared to influence the firmness of the acid gels. When the final firmness of the acid gels was plotted against the level of nonsedimentable denatured whey proteins in the milk, the results for all pH values fell on a single curve for milk at its natural concentration (Fig. 9.15). Similarly for concentrated milks, when the percentage change in final gel firmness (Fig. 9.16A) or yield stress (Fig. 9.16B) was plotted against the level of nonsedimentable denatured whey proteins in the milk, the results for all pHs and milk concentrations fell on a single curve (Anema et al., 2004a; Anema, 2008a). From these results, it was concluded that, although the denatured whey proteins that associate with the micelles have a significant effect on the final firmness of acid gels, the denatured whey proteins that remain in the serum appear to have a more dominant influence over the final firmness than those associated with the casein micelles (Anema et al., 2004a; Anema, 2008a). There was no relationship between the final gel strength and level of whey protein denaturation in the milk used to prepare the acid gel. For example, in samples where virtually all the whey proteins were denatured, there was a very large range of final gel strengths

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FIG. 9.15 Comparison between the final firmness (final G0 ) and the level of denatured whey protein (open symbols) and the level of nonsedimentable denatured whey protein (gray-filled symbols) for acid gels prepared from heated (90°C/30 min) skim milk samples. The pH values at heating of the milks were (●, ), pH 6.5; ( , □), pH 6.55; (▲, 4), pH 6.6; (., 5), pH 6.65; (⧫, ◇), pH 6.7; ( , ⊙), pH 6.9; and ( , &  ), pH 7.1. Reproduced with permission from Anema, S.G., Lee, S.K., Lowe, E.K., Klostermeyer, H., 2004a. Rheological properties of acid gels prepared from heated pH-adjusted skim milk. J. Agric. Food Chem. 52, 337–343. Copyright (2004) American Chemical Society.

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FIG. 9.16 (A) Relationship between the change

in final G0 for acid skim milk gels and the level of nonsedimentable denatured whey protein in heated (80°C/30 min), pH-adjusted skim milk. (B) Relationship between the change in breaking stress for acid skim milk gels and the level of nonsedimentable denatured whey protein in heated (80°C/30 min), pH-adjusted skim milk. ●, 10% TS milk samples; ., 15% TS milk samples; , 20% TS milk samples; and ⧫, 25% TS milk samples. Reproduced with permission from Anema, S.G., 2008a. Effect of milk solids concentration on the gels formed by the acidification of heated pH-adjusted skim milk. Food Chem. 108, 110–118. Copyright (2008) Elsevier.

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(Fig. 9.15). However, the final gel strength for acid gels prepared from milks in which all the denatured whey proteins were in the serum phase (i.e., 100% nonsedimentable denatured whey proteins) was found to be essentially a factor of two higher than that for acid gels prepared from milks in which all the whey proteins were associated with the casein micelles (i.e., 0% nonsedimentable denatured whey proteins; Fig. 9.15; Anema et al., 2004a; Anema, 2008a). This effect was observed over a wide range of milk concentrations (Anema, 2008a). Rodriguez del Angel and Dalgleish (2006) separated the nonsedimentable whey protein-κcasein aggregates from milks heated at different pHs using size exclusion chromatography and related the peak area of these aggregates to the firmness of the acid gels. They also concluded that the gel firmness appeared to be strongly dependent on the formation of soluble complexes in the milks and that there appeared to be a linear relationship between the level of soluble aggregates in the heated milk and the final strength of the acid gels. Based on these results, a hypothesis on the roles of the nonsedimentable and micelle-bound denatured whey protein-κ-casein aggregates has been developed (Anema et al., 2004a,b; Rodriguez del Angel and Dalgleish, 2006; Donato et al., 2007a; Anema, 2008a; Lakemond and van Vliet, 2008a,b). The increased pH of gelation and the increased acid gel strength of heated milk when compared with unheated milk have been attributed to the incorporation of the whey proteins and casein (micelles) in the acid gel structure during the acidification of milk (Lucey et al., 1997; Lucey, 2002; Graveland-Bikker and Anema, 2003). In milk, the casein is insoluble at its isoelectric point (about pH 4.6), whereas the native whey proteins remain soluble at all pHs. Therefore, unheated milk starts to aggregate when the milk pH approaches the isoelectric point of casein, and visible gelation is observed at about pH 4.9. However, for heated milk, the denatured whey proteins are insoluble at their isoelectric points (about pH 5.3 for β-lactoglobulin, the major whey protein). Therefore, on the acidification of heated milk, the proteins will start to aggregate at a much higher pH, closer to the isoelectric points of the whey proteins. As a consequence, the contribution of the denatured whey proteins to the acid gel structure and the firmness of the acid gels is markedly higher than that observed for unheated milk (Lucey et al., 1997; Graveland-Bikker and Anema, 2003). The pH at heating the milk will produce casein micelle particles with markedly different compositions (Figs. 9.5 and 9.6; Anema and Li, 2003b; Vasbinder and de Kruif, 2003; Anema, 2008a). Therefore, on the acidification of milks heated at different pHs, different aggregation and gelation behaviors are observed. Originally, it was hypothesized that, for milks heated at high pH, the serum-phase denatured whey protein-κ-casein complexes may aggregate separately and at a higher pH than the casein micelles because of the higher isoelectric points of the serum-phase protein components when compared with the κ-casein-depleted casein micelles. Based on this, it was proposed that the pH at which aggregation occurs will be progressively shifted to higher pH as the heating pH and the concentration of the serum-phase denatured whey protein-κ-casein complexes is increased (Anema et al., 2004a; Rodriguez del Angel and Dalgleish, 2006; Guyomarc’h et al., 2009). However, subsequent experimental evidence does not support this hypothesis. In a study in which the serum-phase and colloidal-phase protein aggregates were labeled with different fluorescent dyes before remixing and acidification, it was not possible to identify separate aggregation stages of the different fractions at the early stages of gelation. In addition, the final gel had colocalized serum-phase and colloidal-phase aggregates (Guyomarc’h et al., 2009).

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A further detailed study examined the reassociation behavior of the caseins and whey proteins during the slow acidification of skim milk that was adjusted to pHs from 6.5 to 7.1 before heating at 20–120°C for 10 min (Anema and Li, 2015). Prior to acidification, these skim milk samples had various levels of denatured whey protein and different levels of κ-casein and denatured whey proteins distributed between the serum and colloidal phases. On slow acidification, the dissociated caseins along with the denatured whey proteins aggregated with the κ-casein-depleted casein micelles once the pH was below about 5.4. There was no evidence for the separate aggregation of the denatured whey protein-κ-casein complexes or the κ-caseindepleted casein micelles (Fig. 9.17). As both serum-phase and colloidal-phase components are involved in the aggregation/gelation process at pHs below 5.4, it was unlikely that the earlier gelation of milks with high levels of serum-phase denatured whey protein-κ-casein complexes was due to a two-step gelation process. These two studies indicate that the earlier gelation of milk samples heated at high pH was a consequence of the destabilization of the entire milk protein system (Guyomarc’h et al., 2009; Anema and Li, 2015). Thus, the serum-phase denatured whey protein-κ-casein complexes aggregate at a higher pH because of the higher isoelectric points of these serum-phase complexes. In addition, the dissociation of κ-casein from the casein micelles will reduce the density of the surface hairy layer. This may cause the surface hairy layer to collapse at a higher pH, or this layer may have a reduced efficiency in stabilizing the casein micelles as the pH is decreased. As a consequence, the serum-phase denatured whey protein-κ-casein complexes and the κ-casein-depleted casein micelles aggregate at a pH that is markedly higher than that observed for the native casein micelles or for casein micelles in milk heated at a lower pH and therefore with low levels of serum-phase denatured whey protein-κ-casein complexes (Fig. 9.17). The firmness of acid gels can be related to the number and the properties of the contact points between the protein components in the acid gel (van Vliet and Keetals, 1995; Lucey et al., 1997; Mellema et al., 2002; Lakemond and van Vliet, 2008a). As the pH at heating of the milk is increased, the level of serum-phase denatured whey protein-κ-casein complexes increases, and therefore, there are a greater number of particles to aggregate during the subsequent acidification to form the acid gels. There is also the potential for the formation of a more complex acid gel structure when the milk is heated at high pH, at which there are high levels of serum-phase denatured whey protein-κ-casein complexes, than when the milk is heated at low pH, when most of the denatured whey protein and κ-casein are associated with the casein micelles. In the latter case, the acid gel process will probably involve only entire whey protein-casein micelle complexes. Therefore, there may be fewer contact points in the acid gels formed from milk with the denatured whey proteins associated with the micelles than in those formed from milk with soluble denatured whey proteins, and hence, a gel with a lower firmness is observed (Anema et al., 2004a,b; Lakemond and van Vliet, 2008a). The large-strain deformation properties also give some indication of the types of bonds involved in the acid gel network. As the pH at heating was increased, the breaking stress of the acid gels prepared from the heated milks was found to increase markedly; however, the breaking strain was virtually unchanged (Figs. 9.12 and 9.14; Anema, 2008a; Lakemond and van Vliet, 2008a). For a gel to break on increasing the strain, the strands within the gel network are first straightened and then stretched until rupture of the strands or the bonds within the strands (van Vliet and Walstra, 1995; Mellema et al., 2002; Lakemond and van Vliet, 2008a). Therefore, the breaking strain is dependent on factors such as the degree of curvature of the

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FIG. 9.17 Levels of casein and whey proteins in sera of treated milks. The milk samples had an initial pH of (A) 6.5 or (B) pH7.1. The proteins measured were (i) αS-casein, (ii) β-casein, (iii) κ-casein, (iv) β-lactoglobulin, and (v) α-lactalbumin. The sera were obtained from milks that were treated at (●) 20°C, () 60°C, (.) 80°C, (4) 100°C, and ( ) 120°C for 10min before acidification. Reproduced with permission from Anema, S.G., Li, Y., 2015. Reassociation of dissociated caseins upon acidification of heated pH-adjusted skim milk. Food Chem. 174, 339–347. Copyright (2015) Elsevier.

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strands, with a higher breaking strain when the strands have greater curvature. As the breaking strain of the acid gels changed only slightly with the pH at heating of the milk, despite the marked change in final firmness (Figs. 9.12 and 9.14), this indicates that the relative curvature of the individual strands within the gel network was similar for all acid gel samples. The types of bonds involved in the acid gel network will have an influence on the breaking stress (van Vliet and Walstra, 1995; van Vliet, 1996; Mellema et al., 2002; Lakemond and van Vliet, 2008a). The breaking of strands containing covalent bonds requires a greater force than the breaking of strands held together by noncovalent bonds, as covalent bonds have higher bond energies. Therefore, a change in the number or distribution of covalent bonds within the gel network may explain the differences in breaking stress as the pH of the milk at heating was changed (Figs. 9.12 and 9.14). It seems unlikely that the difference in breaking stress can be due to a greater degree of disulfide bonding within the gelled sample; although thiol-disulfide exchange reactions may continue during acidification (Vasbinder et al., 2003b), the physical number of disulfide bonds is unlikely to be markedly different between the samples. The denatured whey proteins, along with some of the κ-casein, are progressively transferred to the serum phase when the pH of the milk is increased before heating (Figs. 9.5 and 9.6). As these interactions involve disulfide bonding, this indicates that the interaction between the denatured whey proteins and κ-casein is transferred from the colloidal phase (casein micelle) to the serum phase as the pH of the milk at heating is increased. On subsequent acidification, both nonsedimentable and colloidal-phase denatured whey proteins are incorporated in the acid gel structure. The nonsedimentable denatured whey protein-κ-casein complexes can form strands that may be involved in interconnecting the colloidal particles. As the nonsedimentable aggregates are disulfide bonded, those samples heated at high pH and with high levels of nonsedimentable whey protein-κ-casein aggregates will have a greater number of these strands interconnecting the residual casein micelles. In contrast, samples heated at lower pH will have the denatured whey proteins predominantly associated with the casein micelles and therefore will have fewer of the whey proteinκ-casein aggregates interconnecting the colloidal particles. Therefore, samples heated at higher pH may have a greater number of disulfide bonds interconnecting the colloidal particles and therefore a higher breaking stress, whereas for samples heated at lower pH, most of the disulfide bonds are on the colloidal particles, and fewer disulfide bonds interconnect the colloidal particles, and this may explain the lower breaking stress (Figs. 9.12 and 9.14). Chymosin-induced aggregation/gelation of heated milk It should be noted that the pH of milk has a marked effect on the action of chymosin in destabilizing the system (Walstra and Jenness, 1984; Walstra et al., 1999). In all studies in which the pH of the milk at heating is discussed in relation to chymosin treatment, after the heat treatment, the pHs of the milks were readjusted back to the natural pH so that effects of pH on the enzyme activity were eliminated. Changing the pH of milk at heating changed the interactions between denatured whey proteins and the casein micelles; however, this did not appear to influence the chymosininduced gelation behavior of heated milk to any great extent (Anema et al., 2007, 2011; Kethireddipalli et al., 2010, 2011). The rate of release of glycomacropeptide (GMP) was similar in all milks regardless of whether they were unheated or heated, and the pH at heating had almost no effect on GMP release (Vasbinder et al., 2003a; Anema et al., 2007; Kethireddipalli

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et al., 2011). When profiles of chymosin-induced gelation were considered, all heated milks had very long gelation times and formed very weak gels compared with unheated milks (Fig. 9.18; Anema et al., 2007, 2011; Kethireddipalli et al., 2010). These results indicated that there was a retardation in the chymosin-induced gelation regardless of whether the denatured whey proteins were associated with the caseins micelles (as observed on heating milk at low pH) or in the serum phase and associated with κ-casein that had dissociated from the casein micelles (as observed on heating milk at high pH). The inhibition of the gelation process was therefore not due to steric or charge effects of the denatured whey proteins that are associated with κ-casein at the casein micelle surface. The denatured whey proteins, whether as serum-phase complexes with κ-casein or associated with κ-casein at the casein micelle surface, interfere with the aggregation process and therefore increase the gelation time (Anema et al., 2007; Kethireddipalli et al., 2010). This was confirmed by experiments in which serum and colloidal phases from unheated and heated milks were exchanged, as it was shown that denatured whey proteins, whether in the serum phase or associated with casein micelles, inhibited the chymosin-induced gelation of milk. However, this inhibition may be more complex as it was also shown that heated casein micelles in the absence of whey proteins and nonprotein serum components from heated milks also inhibited gelation (Kethireddipalli et al., 2010). From these observations, it was concluded that the inhibition of chymosin-induced gelation was complex for heated milks and may have been due to combined effects of heat on casein micelles, denatured whey proteins (both serum phase and colloidal phase), and nonprotein serum components (Kethireddipalli et al., 2010). The chymosin-induced gelation of pH-adjusted, heated milks did not reveal differences; however, this is not an objective measure of the destabilization of the system as it requires the formation of an interconnected network structure, and it is possible for the casein micelles to be destabilized without forming a gel. By monitoring the particle size changes of the milk during chymosin treatment, it was possible to examine the early stages of the aggregation process (Anema et al., 2011). For all milk samples, after adding chymosin, the size initially decreased slightly because of the cleavage of GMP from κ-casein (lag phase) and then increased as the destabilized particles aggregated (aggregation phase; Fig. 9.19). FIG. 9.18 Changes in storage modulus (G0 ) with time after addition of rennet to unheated milks (solid symbols) and skim milk samples heated at 90°C for 30 min (open symbols). The pH values at heating of the milks were (●, ) pH 6.5; (.,4) pH 6.7; ( , □) pH 6.9; and (⧫, ◇) pH 7.1. All samples were readjusted back to the natural pH (pH 6.67) before the addition of rennet (40 μL of 1:3 diluted rennet per 1.3 mL of milk). Reproduced with permission from Anema, S.G., Lee, S.K., Klostermeyer, H., 2011. Rennet-induced aggregation of heated pH-adjusted skim milk. J. Agric. Food Chem. 59, 8413–8422. Copyright (2011) American Chemical Society.

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FIG. 9.19 Changes in particle size on the rennet treatment of (A) unheated skim milk, (B) skim milk heated at 90°C for 2.5 min, and (C) skim milk heated at 90°C for 30 min. Milk samples (20 μL) were diluted in Ca-imidazole buffer (1 mL) before the addition of rennet (10 μL of 1:120 diluted rennet). The pHs of the milk samples were (●) 6.5, () 6.6, (.) 6.7, (4) 6.9, and ( ) 7.1. (D) Effect of pH on the time taken for the particle size to increase by 50 nm from the initial size (T50) after the addition of rennet to the milk samples. The milk samples were unheated (●) or heated at 90°C for () 1 min, (.) 2.5 min, (4) 5 min, ( ) 10 min, (□) 15 min, and (⧫) 30 min. Reproduced with permission from Anema, S.G., Lee, S.K., Klostermeyer, H., 2011. Rennet-induced aggregation of heated pH-adjusted skim milk. J. Agric. Food Chem. 59, 8413–8422. Copyright (2011) American Chemical Society.

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Milks heated at temperatures below those at which whey proteins denature (60°C) had short lag phases and rapid aggregation phases; these were similar to those from unheated milks and were only slightly affected by the pH of the milk at heating. However, milks heated at higher temperatures (>60°C) and at low pH (e.g., pH 6.5) had extremely long lag phases, and once destabilized, the particles aggregated at a slow rate. Increasing the pH at heating shortened the lag phase and increased the rate at which the particles aggregated. In fact, samples heated at pH 7.1 had lag phases and aggregation rates not dissimilar to those of unheated milks (Anema et al., 2011). Similarly, for milks heated at 90°C for different times, the lag phase decreased, and the rate of aggregation increased markedly as the pH of the milk at heating was increased from pH 6.5 to pH 7.1. In fact, the lag phases and aggregation rates for the milks heated at pH 7.1 were not substantially different from those for unheated milks (Fig. 9.19). The effect of pH was greater as the heating time was increased, predominantly as a result of an increased lag phase and a decreased aggregation rate of the samples heated at lower pH when compared with those heated at higher pH (Anema et al., 2011; Fig. 9.19). For milk samples heated at high temperatures, there were positive correlations between the aggregation time and the level of whey protein or κ-casein that was associated with the casein micelles (Fig. 9.20). It was proposed that casein micelles that were substantially coated in denatured whey proteins and κ-casein (obtained on heating milks at low pH) were more resistant to chymosin-induced aggregation than casein micelles with low levels of denatured whey proteins and κ-casein on the casein micelle surface (as observed on heating milks at high pH; Anema et al., 2011). It was also proposed that, although the casein micelles in heated milks aggregated on chymosin treatment, the denatured whey proteins, whether associated with the micelles or in the serum phase, inhibited the gelation of the milk by stabilizing the surface of aggregated particles and preventing or slowing the formation of an interconnected network structure (Anema et al., 2011). Sedimentation and age gelation of UHT milk samples UHT milk is microbiologically sterile and can usually be stored for long periods; however, many chemical and physical changes can occur over time, and these can cause instability such as excessive sedimentation or age gelation. Excessive sedimentation is the formation of a thick layer of proteinaceous material at the bottom of the pack, and this can occur relatively soon after the manufacture of the milk. For age gelation, the milk is usually stable for a period of several months, after which the contents can gel spontaneously to form a three-dimensional proteinaceous network within the pack. Both excessive sedimentation and age gelation are unacceptable and effectively end the shelf life of the UHT milk. A large number of factors are known to affect the sedimentation propensity of UHT milk, such as milk composition, milk concentration, the type (indirect or direct) and severity of the UHT treatment, the pH and ionic calcium levels of the milk, and whether additives such as calcium chelators have been added to the milk (Deeth and Lewis, 2016, 2017; Anema, 2018b). Recent studies have shown that both indirect and direct UHT processing of milk cause significant levels of κ-casein to dissociate from the casein micelles, and this dissociated κ-casein associates with the denatured whey proteins in the serum phase. The sediments from these UHT milk samples had a similar composition regardless of the pH or ionic calcium level of the milk or whether the sediment level was high or low. The sediment was formed from the

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FIG. 9.20 Relationship between the time taken for the particle size to increase by 50 nm (T50) and (A) the percentage of micelle-bound β-lactoglobulin, (B) the percentage of micelle-bound α-lactalbumin, (C) the percentage of micelle-bound κ-casein (all data), and (D) the percentage of micelle-bound κ-casein (selected data with high levels of denatured whey proteins). The milk samples were heated either at 90°C for various times (open symbols) or at different temperatures for 30 min (filled symbols). The pHs of the samples were (, ●) 6.5, (◇) 6.55, () 6.6, (⊠) 6.65, (□, ) 6.7, (4, ▲) 6.9, and (5, .) 7.1. Reproduced with permission from Anema, S.G., Lee, S.K., Klostermeyer, H., 2011. Rennet-induced aggregation of heated pH-adjusted skim milk. J. Agric. Food Chem. 59, 8413–8422. Copyright (2011) American Chemical Society.

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κ-casein-depleted casein micelles and had only low levels of denatured whey proteins, although the level was higher for sediment from indirect UHT milk than for that from direct UHT milk (Fig. 9.21; Gaur, 2017; Gaur et al., 2018).

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FIG. 9.21 Electrophoretic traces of (A) commercial indirect UHT milk samples, their supernatants, and their sediments and (B) commercial direct UHT milk samples, their supernatants, and their sediments. The red lines represent the milk samples, and the blue lines (dark gray in print version) represent their respective supernatants (i) or sediments (ii). The peaks correspond to (a) α lactalbumin, (b) β-lactoglobulin, (c) β-casein, (d) αS1-casein, and (e) κ-casein. (C) Percentages of αs-casein + β-casein combined (gray bar), κ-casein (red bar; dark gray in print version), and β-lactoglobulin + α lactalbumin combined (green bar; light gray in print version) in commercial indirect UHT (CI-1, CI-2, and CI-3) and direct UHT (CD-1, CD-2, and CD-3) milk (M), supernatant (SUP), and sediment (SED) samples. Reproduced with permission from Gaur, V., Schalk, J., Anema, S.G., 2018. Sedimentation in UHT milk. Int. Dairy J. 78, 92–102. Copyright (2018) Elsevier.

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The ionic calcium levels and the pH of the milk have a major impact on sedimentation, with increased sedimentation when the ionic calcium exceeds a certain critical level and/or when the pH is below a certain critical level (Fig. 9.22; Lewis et al., 2011; Anema, 2018b; Gaur et al., 2018). These results suggest that the κ-casein-depleted micelles are metastable and that, at sufficiently high ionic calcium levels or low pH values, the metastable micelles associate in the serum via calcium-mediated aggregation reactions, increasing their size and therefore their propensity to sediment. A number of mechanisms are known to cause age gelation (Datta and Deeth, 2001; Deeth and Lewis, 2016, 2017; Anema, 2018b). Two mechanisms involve proteolytic degradation of the proteins by either plasmin or heat-stable exogenous enzymes prior to the onset of gelation. These mechanisms have been studied in some detail and are reasonably well understood (Datta and Deeth, 2001; Deeth and Lewis, 2016, 2017; Anema, 2018b). In another “physicochemical” mechanism, age gelation can occur in the complete absence of proteolysis. The factors leading to this type of gelation are less well understood. A mechanism for age FIG. 9.22

Relationship between (A) pH and sediment level and (B) ionic calcium and sediment level for UHT milk samples: (●) direct UHT milk samples, () indirect UHT milk samples, (.) direct UHT milk sample with added sodium hexametaphosphate. The inserts show the indirect UHT milk samples on an expanded scale. Reproduced with permission from Gaur, V., Schalk, J., Anema, S.G., 2018. Sedimentation in UHT milk. Int. Dairy J. 78, 92–102. Copyright (2018) Elsevier.

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gelation proposed by McMahon (1996), McMahon and Alleyne (1994) suggested that the β-lactoglobulin-κ-casein complex, initially on the micelle surface after UHT treatment, progressively dissociated from the casein micelles into the serum phase during storage. These β-lactoglobulin-κ-casein complexes formed the gel network on prolonged storage, entrapping the remaining κ-casein-depleted casein micelles when extensive gelation had occurred. However, a recent study has indicated that this mechanism may not be correct (Anema, 2017a). High levels of the β-lactoglobulin-κ-casein complex were in the serum phase immediately after the UHT treatment of milk, and these levels hardly changed during storage, even at the point of gelation or beyond. In addition, the gelled material was composed of κ-caseindepleted casein micelles, with little κ-casein or denatured whey proteins associated with this gelled phase. A new hypothesis was proposed, in which the UHT treatment forms metastable κ-casein-depleted casein micelles immediately after processing. If the ionic calcium levels are high and/or the pH levels are low at the time of processing, then sediments form rapidly, as detailed earlier (Anema, 2017a, 2018b). However, if the calcium activity and/or the pH are outside certain limits at the time of processing, the milk will appear to be stable for long periods. On storage, changes to the milk occur, such as a pH decline, changes in the ionic calcium levels, and a slow settling of the κ-casein-depleted casein micelles to the bottom of the pack. The high concentration of κ-casein-depleted casein micelles at the bottom along with the changes in pH and ionic calcium levels lead to the aggregation and eventual gelation of the milk, starting at the bottom of the pack and progressing upward as more κ-caseindepleted casein micelles settle. All milk samples will eventually gel by this mechanism; however, in most cases, the gelation will occur beyond the shelf life of the milk. It is the age gelation that occurs within the shelf life of the milk that causes complaint. From the studies on sedimentation (Gaur, 2017; Gaur et al., 2018) and the study on the physicochemical age gelation (Anema, 2017a) of UHT milk, it is evident that a self-consistent mechanism can be proposed to explain these two phenomena, as outlined in Fig. 9.23. The propensity for a UHT milk to sediment or gel is dependent on the pH and/or ionic calcium level at the time of manufacture. If the pH is below a critical level and/or the ionic calcium is above a critical level at the time of manufacture, the κ-casein-depleted casein micelles will aggregate in the milk serum through calcium-mediated bridging reactions, and these aggregates will sediment fairly rapidly during storage. However, if the pH and/or the ionic calcium levels are outside these critical levels after manufacture, the milk will not sediment significantly on storage. However, the slow settling of the κ-casein-depleted casein micelles along with changes to the pH and/or ionic calcium levels during storage will, at some stage, cause aggregation and gelation, starting at the bottom of the pack and progressing upward. Concentrate viscosity Skim milk samples at low pH (approximately pH 6.5) and heated at 90°C for up to 30 min had high levels of denatured whey proteins associated with the casein micelles and had a markedly higher viscosity than similarly heated milk samples at higher pH (Fig. 9.8B, Anema et al., 2004c). If similar viscosity differences could be achieved at higher milk solid concentrations, this may allow for more efficient production of milk powders as milk samples that maintain a lower viscosity could be evaporated to higher total solids before spray drying. A detailed study examined the rheological properties of concentrates (up to about 45% total solids) that were prepared by evaporating skim milk samples that were heated (90°C/30 min)

Relationships between denaturation/interactions and functional properties

FIG. 9.23

373

A self-consistent mechanism for the age gelation and sedimentation of UHT milk during storage. (1) Casein micelles and native whey proteins in unheated milk, with κ-casein on the casein micelle surface. (2) On UHT processing, κ-casein dissociates from the casein micelles, forming whey protein-κ-casein complexes in the serum and κ-caseindepleted casein micelles. (3) If the calcium activity is low and/or the pH is high during processing, the milk is initially stable. During storage, the environment changes so that the pH and the calcium activity move into an unstable range. The κ-casein-depleted casein micelles aggregate to form a gel. The whey protein-κ-casein complexes remain in the serum on initial gelation but may be trapped in the gel during extensive gelation. (4) If the calcium activity is high and/or the pH is low during processing, the milk is unstable. Aggregation of the κ-casein-depleted casein micelles occurs over days/weeks to form large particles that rapidly sediment. The whey protein-κ-casein complexes remain in the serum. These mechanisms were developed, with permission, from the results of Anema, S.G., 2017a. Storage stability and age gelation of reconstituted ultra-high temperature skim milk. Int. Dairy J. 75, 56–67. Gaur, V., 2017. Sedimentation Reduction in UHT Milk. PhD Thesis, University of Canterbury, Christchurch, New Zealand, and Gaur, V., Schalk, J., Anema, S.G., 2018. Sedimentation in UHT milk. Int. Dairy J. 78, 92–102. Copyright (2018) Elsevier.

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at different pHs (pH 6.5–7.1). The viscosities of the concentrates followed power law behavior and therefore could be described by consistency coefficients and flow behavior indices (Anema et al., 2014). Milk concentrates of up to 40% total solids did have higher consistency coefficients than concentrates from unheated skim milk, whereas those from skim milk heated at pH 7.1 had lower consistency coefficients than those from the unheated milk. The concentrates from milk heated at intermediate pH had consistency coefficients between those from the milks heated at pH 6.5 and pH 7.1. These effects were observed for concentrates produced from the skim milk at the pH of heating and for those from the milk samples that were readjusted back to the natural pH after heating and before concentration. However, if the milk was concentrated to above 40% total solids, the consistency coefficients of the concentrates from heated milk samples converged so that they were similar regardless of the pH at heating, and they were significantly higher than those of the concentrates prepared from unheated milk (Fig. 9.24). It is proposed that, when the denatured whey proteins associate with the casein micelles, they increase the voluminosity of the micelles, leading to an increased viscosity and therefore consistency coefficient at all milk concentrations. In contrast, when the denatured whey proteins and κ-casein are in the serum, they are part of the continuous phase and do not contribute to the viscosity and therefore consistency coefficient until the milk is at very high concentrations (>40% total solids).

Examples of the effect of denaturing whey proteins separately from casein micelles on the functional properties of milk Compared with heated milks, different effects on functional properties can be observed when the whey proteins are denatured and aggregated separately from the casein micelles and then remixed. However, the effects of adding predenatured whey proteins to casein micelle suspensions or milk on the functional properties are dependent on the conditions under which the whey proteins are denatured. Lucey et al. (1998) showed that acid gels prepared FIG. 9.24 Consistency coefficients of skim milk concentrates measured at 20°C (filled symbols) and 40°C (open symbols). Concentrates were from (⧫) unheated skim milk or skim milk heated at (●, ) pH 6.5, (▲, 4) pH 6.7, and ( , □) pH 7.1 and 90°C for 30 min before being concentrated to different TS levels. Reproduced with permission from Anema, S.G., Lowe, E.K., Lee, S.K., Klostermeyer, H., 2014. Effect of the pH of skim milk at heating on milk concentrate viscosity. Int. Dairy J. 39, 336–343. Copyright (2014) Elsevier.

▪

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375

from milk samples in which the whey proteins were denatured in the presence of casein micelles had a markedly higher firmness than acid gels prepared from milk samples in which the whey proteins in the milk serum were predenatured and then added back to the casein micelle suspensions. In fact, in many cases, the samples with denatured whey proteins added back to the casein micelles produced acid gels with firmness similar to or only slightly higher than those of acid gels prepared from unheated milks. In another study, Schorsch et al. (2001) prepared model milk systems in which the whey proteins in a simulated milk serum either were heated in the presence of casein micelles or were heated separately and added back to the casein micelles. Acid gels were prepared from these model milk systems. It was shown that the acid-induced gelation occurred at a higher pH and in a shorter time when the whey proteins were denatured separately from the casein micelles than when the whey proteins were heated in the presence of the casein micelles. However, the gels formed were weaker and more heterogeneous because of the particulate nature of the denatured whey proteins. It was suggested that the large denatured whey protein aggregates, as formed when the whey proteins in the milk serum were heated separately from the casein micelles, hinder the formation of a casein gel network when the milk is subsequently acidified and that a weak acid gel with a heterogeneous structure results. When the whey proteins are heated in the presence of the casein micelles, the denatured whey proteins interact with the κ-casein at the casein micelle surface, and on subsequent acidification, the denatured whey proteincasein micelle complexes aggregate to form a firmer acid gel with a more homogeneous structure (Schorsch et al., 2001). This proposal is supported by early studies, which showed that the aggregation of the denatured whey proteins, and in particular β-lactoglobulin, formed large aggregate species when heated in the absence of κ-casein, whereas aggregation was limited when the whey proteins were heated in the presence of κ-casein (McKenzie et al., 1971). In contrast, if the whey proteins are denatured at relatively low protein concentrations, at low ionic strengths, and at a pH far from the isoelectric point (pH > 6.5), then soluble denatured whey protein polymers can be formed. The polymers are linear and can be induced to gel when salt is added or the pH is reduced (Britten and Giroux, 2001; Gustaw et al., 2006, 2009). When these whey protein polymers were added to heated skim milk and the preparations were acidified, the acid gels formed had markedly higher firmness and water-holding capacities than those from the skim milk or from milks heated with equivalent levels of native whey proteins (Britten and Giroux, 2001; Gustaw et al., 2006, 2009). The firmnesses and waterholding capacities were markedly higher than when the whey proteins were denatured in the milk serum and then added back to casein micelle suspensions or milk before acidification (Lucey et al., 1998; Schorsch et al., 2001). The isoelectric points of denatured whey protein complexes were chemically modified through succinylation or methylation of carboxyl groups (Morand et al., 2012a). These modified denatured whey protein complexes were added to whey protein-free milk suspensions to produce model heated milk systems, and the milks were subsequently acidified to form gels. The pH of gelation of these milks increased markedly as the isoelectric point of the whey protein complexes increased, supporting the proposition that it is the higher isoelectric point of the whey proteins that causes heated milks to gel at markedly higher pH than unheated milks (Lucey et al., 1997; Anema et al., 2004a). Interestingly, the final firmness of the gels was not markedly affected by the isoelectric point of the complexes (Morand et al., 2012a).

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In similar studies, the hydrophobicities of denatured whey protein complexes were altered by acylation of lysine amino acids using anhydrides of various carbon chain lengths (Morand et al., 2012b). These complexes were also added to whey protein-free milk suspensions, and the milks were acidified to form gels. Increasing the hydrophobicity of the whey protein complexes also increased the pH at which the milks gelled and also increased the maximum firmness of the gels, although this maximum was not always at the same pH. Taken together, these results indicate that both the isoelectric properties and the hydrophobicity of the serumphase complexes influence the acid gelation properties of heated milk systems (Morand et al., 2011, 2012a,b).

Conclusions A considerable amount of work has gone into understanding the irreversible denaturation reactions of the whey proteins in heated milk systems. These detailed studies have produced models that allow reasonably accurate prediction of the level of whey protein denaturation in milks under a wide range of heating conditions, even in milk samples with markedly modified concentrations and compositions. However, with a few exceptions, monitoring of the whey protein denaturation levels provides only a crude indication of the functionality of the milk system. As a consequence, more recent research efforts have focused on trying to understand the specific interaction reactions of the denatured whey proteins with other proteins in the milk system. Early indications suggest that these types of studies on the interactions of denatured whey proteins may provide greater insights into the functional properties of heated milk products than can be obtained by monitoring just whey protein denaturation levels. These initial studies on protein interactions have been conducted under relatively narrowly defined conditions (temperatures, heating times, pH, milk concentrations, and milk compositions). It is likely that changes to these variables will markedly influence the interaction behavior and will explain the changes in functional behavior when the heating conditions are changed (even though the whey protein denaturation levels may be similar). Although studies on understanding the specific interactions between milk proteins, particularly in complex systems such as milk, are extremely difficult, these types of studies should continue to give useful insights into the behavior of milk proteins during heating and the functional behavior of the heated milk products.

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