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Blackwell Publishing, Ltd.


Heat stability of milk

Riddet Centre, Massey University, Palmerston North, New Zealand

The heat stability of milk has been the subject of a considerable amount of research for about a century. This research has been aimed mainly at understanding the effects of compositional and processing factors on heat stability and elucidating the mechanisms of protein coagulation. This paper provides an overview of the factors that inuence the pH dependence of the heat stability of normal and concentrated milks. The principal heat-induced changes in the milk system that contribute to coagulation are discussed. Current knowledge of the mechanisms of heat coagulation in normal and concentrated milks is also reviewed. Keywords Casein micelle stability, Concentrated milk, Heat coagulation, Heat stability, Milk proteins, Milk.

*Author for correspondence. E-mail:

BACKGROUND The problems of regulating heat stability (that is the relative resistance of milk to coagulation upon sterilization) appeared over a century ago in the manufacture of evaporated (condensed) milk. The idea of commercially preserving milk by sterilizing dates back to 1856 when Gail Borden was granted patents in the United States and England for producing concentrated milk by evaporation in vacuum without addition of sugars and other preservatives. The commercial production of condensed milk increased gradually during the First and Second World Wars, and condensed milk became one of the major dairy products in the 1920s, because of easy transport and a long shelf life. The usual problems faced in those days were that the milk gelled or coagulated during the heat treatment and excessive thickening of the product occurred during storage. These problems were controlled by carrying out various heat-stability tests on the raw milk, and by running pilot sterilization trials on samples from each batch after the addition of various amounts of sodium bicarbonate. Between 1900 and 1960, most of the scientic research focused on solving heat coagulation problems in concentrated (condensed) milk. Studies of factors affecting heat stability were considered to be important because they could be used to predict whether a given milk sample would coagulate after it had been processed into product. Sommer and Hart (1919, 1922) showed that mineral balance was important, and that if a milk sample was too acid (insufcient calcium and magnesium) or too basic (insufcient phosphate and citrate), it would be unstable. Heat stability could, according to the

*Author for correspondence. E-mail: 2004 Society of Dairy Technology

theory, be restored to these milks by appropriately balancing them by adding acids (CaCl2 or HCl) to milks that were too basic and vice versa. The salt balance theory was criticized by Rogers et al. (1921), who showed that the heat stability of condensed milk could not be predicted from the pH and salt balance of the milk or indeed from the heat stability of the raw milk. There was certainly no relationship between the heat stability of raw milk and that of the condensed milk made from it. The effect of preheat treatment (i.e. forewarming) heat treatment given to the milk prior to evaporation was studied in detail in the 1940s (Webb and Bell 1942). Preheating is now a standard commercial practice for the manufacture of condensed or evaporated milk. From the 1960s, studies on heat stability shifted from condensed milk to normal (unconcentrated) milk. This was around the time when the relevance of pH to heat stability was fully revealed by the work of Rose (1961). In the 1970s, research concentrated on the effects of processing and compositional factors on the pH dependence of the heat coagulation time of unconcentrated milk. Some of the more important ndings include understanding the roles of -lactoglobulin and -casein, milk salts and urea in heat coagulation. Advances in analytical methods (e.g. light scattering) and electron microscopy in the 1980s allowed heat-induced interactions in milk proteins to be explored in greater detail and the development of models for heat coagulation in normal and concentrated milk. However, some aspects of the mechanism of heat coagulation have not been completely explained at a molecular level. The extensive literature on the heat stability of milk has been reviewed regularly over the past 111

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40 years (Rose 1963; Fox and Morrissey 1977; Fox 1981a, 1982; Singh 1988, 1995; Van Boekel et al. 1989a,b; Singh and Creamer 1992; OConnell and Fox 2003). ASSESSMENT OF HEAT STABILITY The heat stability of milk refers to the ability of milk to withstand high processing temperatures without visible coagulation or gelation. The most widely used method to assess heat stability, at least for research purposes, involves sealing a milk sample in a glass tube, which is clipped onto a platform and placed in a temperature-controlled oil bath, usually at 140C for normal milk and at 120C for concentrated milk. The platform is rocked at a given rate until a coagulum can be observed visually. The heat coagulation time (HCT) is dened as the time that elapses between placing a sample of milk in an oil bath and the onset of visible coagulation. Other methods for determining heat stability include the ethanol test, a whitening test, a protein sedimentation test and a viscosity determination. However, the correlations between different test methods are generally unsatisfactory and the HCTs determined by these methods often correlate very poorly with the stability of milk on commercial sterilization. From an industry point of view, the use of a pilot-scale or laboratory-scale sterilizer provides more reliable results and prediction of behaviour of milk in commercial plants. H E AT S TA B I L I T Y pH P R O F I L E The HCT of milk is affected by a number of factors, of which pH is the most important. The HCT of most milks shows a sharp maximum at pH values around 6.7 followed by a minimum at pH 6.9; the stability increases again at higher pH value, as shown in Fig. 1. These milks are classied as Type A milks. In Type B milks, the HCT increases as a function of pH, being lower in the region of the maximum and higher in the region of the minimum than for Type A milks. The HCT of concentrated milk (20% non-fat solids) is much lower than that

Figure 1 Heat coagulation (HCT) vs. pH prole for normal skim milk heated at 140C. Type A milk ( ), Type B milk ( ), serum protein-free casein micelle dispersions ( ) or concentrated milk (20% total solids) ( ).

Table 1 Methods for eliminating the minimum from the HCTpH proles of Type A milks Conversion of Type A to Type B Decrease in the assay temperature (150C to 120C) Addition of -casein Removal of whey proteins Reduction in the levels of soluble salts Several additives (e.g. aldehydes, oxidizing agents, polyphenols) Treatment with transglutaminase

of unconcentrated milk, with the maximum occurring in the pH range 6.46.6; the HCT on either side of the maximum remains very low. The minimum in the HCT vs. pH prole of normal milk can be eliminated by altering a number of compositional and processing parameters (Table 1). Articial modication of various milk salts inuences the HCTpH prole; a small reduction in the total calcium and magnesium ion concentration (from 13 to 11 mm) eliminates the minimum in the HCTpH prole whereas addition of these cations decreases the stability throughout the pH range (Morrissey 1969). The addition of phosphate to milk increases the HCT, whereas reducing the soluble phosphate shifts the HCTpH prole to more alkaline values. The addition of citrate shifts the maximum to more acid pH values, and the HCT does not recover on the alkaline side of the maximum. Removal of 40% of the colloidal calcium phosphate (CCP) increases the HCT in the pH range 6.47.4, whereas removal of 60100% of the CCP increases the HCT in the pH range 6.47.0 but has a destabilizing effect at higher pH values (Fox and Hoynes 1975). Increasing the concentration of lactose, to approximately 150% of its normal level, destabilizes a Type A milk throughout the pH range 6.4 7.4 and shifts the minimum to more alkaline pH values (Sweetsur and White 1974). Urea is the only indigenous constituent of milk that has been shown to correlate strongly with natural variations in heat stability. Addition of urea at low concentrations does not affect the HCT in the region of the maximum, but at high concentrations increases the HCT of milk. By contrast, addition of urea to concentrated milk does not affect its HCTpH prole (Muir and Sweetsur 1976). Of the milk proteins, -lactoglobulin and -casein have the greatest impact on the HCTpH prole (Fox and Hoynes 1975; Singh and Fox 1987a).


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-Lactoglobulin is required for the development of a Type A HCTpH prole. The HCT of serum protein-free casein micelles (SPFCMs), dispersed in milk ultraltrate, increases continuously with increasing pH. Addition of -lactoglobulin to an SPFCM dispersion introduces a maximum and a minimum into the HCTpH prole (Fig. 1). Unlike normal milk, the addition of -lactoglobulin to concentrated milk has a destabilizing effect over the entire pH range. Enrichment of the milk with -casein increases the stability in the region of the minimum and converts a Type A milk to a Type B milk (Rose 1961). In addition to the above factors, the HCTpH prole of milk can be modied by numerous additives. Addition of thiol-blocking agents, such as Nethylmaleimide (NEM) or iodoacetamide, markedly reduces the HCT in the region of the maximum (Singh and Fox 1987b). Reducing agents such as -mercaptoethanol destabilize milk over the entire pH range whereas oxidizing agents such as KBrO4 and iodobenzoate eliminate the minimum in the HCTpH prole (Singh and Fox 1985b). Formaldehyde increases the stability throughout the pH range 6.47.4, particularly in the region of the minimum, which is eliminated (Singh and Fox 1985a). Addition of anionic detergents, such as sodium dodecyl sulphate (SDS), to milk increases the stability in the region of the maximum and shifts the HCTpH curve to alkaline pH values whereas cationic detergents, such as cetylmethylammonium bromide, shift the HCTpH prole to more alkaline values while causing a moderate increase in the maximum HCT (Fox and Hearn 1978). Polyphenol-rich extracts of tea, coffee, wine, oak leaves and bark increase the HCT, particularly in the region of the minimum (OConnell et al. 1988; OConnell and Fox 1996). Caffeic acid is the most effective of the polyphenols examined. HEAT-INDUCED CHANGES IN MILK RELATED TO COAGULATION The coagulation of milk on extended heating at high temperatures (120140C) is a consequence

of loss of casein micelle stability, as a result of numerous physical and chemical changes in its components. When we consider the stability/instability of casein micelles, the surface properties rather than the interiors of the micelles are likely to be more important. The surface of the micelle has a number of dissociated carboxyl and some ester phosphate groups, providing a high negative charge (the zeta potential at 20C is 13 mV) and thus electrostatic stabilization. Then there is a diffuse surface layer of exible, hydrophilic polypeptide chains consisting mostly of C-terminal segments of -casein, providing steric stabilization (Holt 1992). This hairy layer of -casein provides a barrier against aggregation unless the hairs are removed by chymosin treatment or the solvent quality is reduced (for example by addition of ethanol). Inside the micelle, the individual casein molecules are associated by hydrophobic and electrostatic bonds in which CCP also plays an important role. Several factors inuence the colloidal stability of milk. Important factors are calcium ions and pH, both of which diminish electrostatic repulsions and possibly alter the conformation of -casein at the micelle surface (indirectly reducing steric repulsions). Heat treatment markedly changes the serum phase environment around the casein micelles (e.g. change in pH and soluble minerals, in particular calcium ions, breakdown of lactose and urea) as well as the casein micelles themselves (association of whey proteins, changes in CCP, dephosphorylation, casein dissociation). Some of these changes are listed in Table 2. It is not known exactly which particular changes are directly responsible for coagulation, predispose the milk to coagulation or are a consequence of the coagulation process. The initial stages of the heat coagulation process must involve a change in colloidal interactions that allows micelles to approach each other and stay together long enough for chemical reactions to take place. CHANGES IN CASEIN MICELLES Heating milk at the heat stability assay temperatures causes denaturation of whey proteins and their interactions with casein micelles. -Casein on

Table 2 Changes in milk during heating and their possible impact on heat stability Changes that promote instability Decrease in pH Deposition of calcium phosphate onto micelles Association of whey proteins with casein micelles Dephosphorylation of casein Dissociation and hydrolysis of caseins, in particular -casein Reduction in zeta potential and hydration Covalent bond formation Changes that enhance stability Reduction in calcium ion activity Association of whey proteins with casein micelles Reduced sensitivity of casein to calcium ions Thermal degradation products of lactose

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Figure 3 Effect of heating time on the zeta potential of particles in skim milk that had been adjusted to pH 6.5 ( ) or 7.1 ( ) and then heated at 140C. (Data from Anema and Klostermeyer 1997). Figure 2 Inuence of pH on the formation of nonsedimentable (100 000 g for 60 min) nitrogen (N) and nonsedimentable 12% TCA-insoluble N-acetylneuraminic acid (NANA) (indicative of -casein) on heating skim milk at 140C for 2 min. Unheated milk: N ( ) or NANA ( ). Heated milk: N ( ) or NANA ( ). (Data from Singh and Fox 1985b, 1986).

the surface of casein micelles is involved in the formation of a specic disulphide-linked complex with -lactoglobulin (Singh and Fox 1987a; Jang and Swaisgood 1990). As the -lactoglobulin aggregates or monomers are considered to form disulphide bonds with -casein, the cystine residues that are located in the para--casein part of the protein must be relatively accessible to incoming proteins. A question still remains as to how whey protein aggregates penetrate the hairy layer to nd the disulphide bonds of -casein. Perhaps there are changes in the -casein hairy layer during heating, or heating causes rearrangement of the micelle surface to allow this interaction to take place. The pH of heating has a large effect on the extent of the association of whey proteins with the casein micelles (Smits and van Brouwershaven 1980; Singh and Fox 1985a,b). At pH values < 6.8, a majority of whey protein complexes remain associated with the casein micelle surface whereas, at higher pH values, these complexes remain in the serum. On heating at pH values > 6.8, not only do the whey protein aggregates remain in the serum but also micellar -casein dissociates in the serum. It is unclear whether the interaction between casein and whey proteins occurs in the micelles and this complex then dissociates into the serum phase or whether the complex is in fact formed in the serum. Nevertheless, the presence of whey protein markedly enhances the dissociation of micellar -casein. Other caseins, s1-, s2- and 114
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-caseins, also dissociate from the micelles upon heating, but to a much lesser extent. Data obtained by Singh and Fox (1985b, 1986) on milk and SPFCMs, heated at 140C for 1 min at different pH values, are shown in Fig. 2. At pH values lower than 6.7, the amounts of soluble -casein (nonsedimentable at 100 000 g for 60 min), represented as 12% TCA-insoluble N-acetylneuraminic acid (NANA) and total soluble protein from heated milk, are lower than in a corresponding unheated milk, whereas at pH values > 6.7, these amounts are greater than in the unheated milk and increase with increasing pH. It appears that -lactoglobulin prevents the dissociation of micellar -casein on heating at pH values < 6.7 but enhances the release of micellar -casein at higher pH values (> 6.9). The effect of heat treatment on the zeta potential is interesting because of similarities between the effects of -lactoglobulin on the HCTpH prole and the zeta potentialpH prole of SPFCM dispersions. Schmidt and Poll (1986) showed that heating SPFCM dispersions at pH 6.7 for 10 min at 120C had only a slight effect on their zeta potentials at room temperature; additions of lactoglobulin before heating led to an increase in the zeta potential in the pH range 6.66.9 but reduced it at higher pH values (7.17.2). Anema and Klostermeyer (1996, 1997) showed that heating milk at 140C at pH 6.5 caused an initial increase in the zeta potential, which then decreased on further heating. However, heating at pH 7.1 markedly reduced the zeta potential initially, which remained relatively constant thereafter (Fig. 3). They proposed that initial changes in the zeta potential are due to association of whey proteins with casein micelles and precipitation of calcium phosphate on to the micelles whereas subsequent changes in the zeta potential are caused by -casein dissociation and dephosphorylation of caseins.

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Clearly, depending on the pH at heating, at least two different kinds of casein particles with different structures and stability are produced. This association of whey proteins with the micelles at pH values below 6.8 would certainly modify the surface of the casein particles, although it is uncertain how this new, complex surface is stabilized. It is possible that whey protein aggregates attached to the casein micelle surface protrude into the serum and thus act as super-steric stabilizers. It has been shown recently that the association of denatured whey protein with the casein micelles increases their size (Anema and Li 2003) and that the zeta potential of the whey protein-coated micelles is greater than that of the native micelles, which would indeed contribute to the stability of these particles. Consequently, the whey protein-coated micelles are more stable to heat, calcium ions, ethanol or rennet than the native casein micelles (Singh and Fox 1986). The -casein-depleted micelles formed by heating milk at pH > 6.8 have a reduced zeta potential and show increased sensitivity to calcium ions, ethanol and heat compared with the native micelles. -Casein in the micelles has been shown to be present as disulphide-linked polymers of approximate molecular weight 300 600 kDa. The determination of the molecular state of the -casein that dissociates from the micelles is complicated by the fact that, on heating, whey proteins interact with it through thioldisulphide interchange reactions. It is unknown whether or not -casein polymers are broken into oligomers and monomers during the high heat treatment of milk. Reduction of disulphide bonds by treatment with mercaptoethanol tends to promote heat-induced dissociation of micellar -casein, indicating that the thermal breakdown of disulphide bonds (if it occurs) is likely to enhance -casein dissociation from the micelles. Singh and Latham (1993) analysed the dissociated protein material by size exclusion chromatography and showed that the soluble protein material formed on heating milk at high pH is composed of smallsized whey protein/-casein aggregates and some monomeric proteins. No monomeric -casein could be detected. It is interesting to note that the dissociated protein material can generally be separated on SDS gel electrophoresis and gel ltration, but that in electrophoresis containing urea buffer systems, these proteins do not separate as discrete bands, indicating modication of charged groups and/or more structural changes to the proteins. The reason why -casein dissociates from the micelles at slightly alkaline pH is not clear. It is likely that, when the surface charge reaches certain critical values, hydrophobic bonds are insufcient to hold -casein on the micelle surface. The dissociation probably occurs as a result of electrostatic repulsions between -casein and other micelle components. Modication of the charge distribu 2004 Society of Dairy Technology

Figure 4 (a) Inuence of pH on the dissociation of micellar -casein on heating milks of 10% ( ), 15% ( ), 20% ( ) or 25% () total solids at 120C for 4 min. (b) Inuence of heating time at 120C on the dissociation of micellar -casein on heating milks of 10% ( ), 15% ( ), 20% ( ) or 25% () total solids at pH 6.55. (From Singh and Creamer 1991a, reproduced with permission from Cambridge University Press).

tion on -casein may also occur as a result of other heat-induced changes, such as Maillard-type reactions. Alternatively, dissociation may involve conversion of CCP to an alternative form that is less capable of binding casein molecules and maintaining the micelle structure (Aoki et al. 1990; Anema and Klostermeyer 1997). As the binding of -casein to the micelles does not involve CCP, it is likely that such a change in the CCP would inuence the dissociation of other caseins but have relatively little effect on -casein. Concentration of milk prior to heating has a marked effect on the dissociation of -casein; the extent of dissociation of -casein at any particular pH increases, with the dissociationpH curve shifting towards lower pH values (Nieuwenhuijse et al. 1991; Singh and Creamer 1991a) (Fig. 4). Thus, in concentrated milk, considerable dissociation of -casein occurs on heating at normal pH, i.e. 6.5 6.6. In a comprehensive study on casein micelle 115

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dissociation, Singh and Creamer (1991b) showed that the casein composition of the dissociated protein was dependent on the heating time at 120C; only -casein dissociated during the initial stages of heating (up to 6 min) but other caseins also dissociated with further heating; after heating for 10 min, the dissociated protein was composed of 70% -casein, 20% -casein and 10% S-caseins. The dissociated caseins existed as aggregates of various sizes, including some monomers. Most of the dissociated -casein was covalently linked to whey proteins. The dissociated -casein appeared to have a charge distribution different from that of native -casein, but - and -caseins were essentially the same as in their native state, as indicated by their separation on urea-containing polyacrylamide gels. On SDS-containing gels, the dissociated caseins showed clear, distinct bands, similar to those of native proteins, indicating that the molecular weights of the proteins were not affected by the heat treatments, at least during the initial stages of heating. Another important change in casein micelles is that the dephosphorylation of caseins would be expected to inuence the casein micelle structure, as the casein phosphate groups are involved in interactions of calcium ions and with CCP and provide negative charges. Loss of phosphate groups may decrease the binding of caseins with CCP, resulting in dissociation of caseins. On the other hand, dephosphorylation of phosphoserine residues may generate reactive intermediate dehydroalanine, which may promote casein cross-linking. Although the real signicance of thermal dephosphorylation is yet to be elucidated, the rate of dephosphorylation does not appear to correlate directly with the rate of heat coagulation. CHANGES IN THE SERUM PHASE It has long been recognized that a crucial change in the serum is the decrease in pH, which plays a key role in creating an environment that favours coagulation of the milk proteins. This is supported by the observation that, if the pH of milk is readjusted occasionally to its original value, coagulation of the milk may be prolonged for at least 3 h (Fox 1981b). The pH of normal milk decreases gradually with increasing heating time at 140C; the pH at coagulation is usually between 5.5 and 6.0 (after cooling the milk to 20C). The decrease in pH is caused by three reactions: 1 thermal oxidation of lactose to organic acids, which accounts for 50% of the pH decrease; 2 hydrolysis of organic phosphate (from phosphoserine in casein), which contributes up to 30% of the decrease in pH; 3 precipitation of tertiary calcium phosphate with a concomitant release of H+. 116
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Although the pH of milk at the point of coagulation (estimated to be about 4.9) approaches that of acid coagulation, heat-induced coagulation is not due merely to an indirect acid-induced coagulation mechanism. This is illustrated by the fact that there is no relationship between the initial pH and the rate of pH decrease during heating; the apparent activation energies for acid production and heat coagulation are very different, and the coagulum formed on heating cannot be redispersed by increasing the pH (Van Boekel et al. 1989a,b; OConnell and Fox 2003). Nevertheless, the decrease in pH is likely to reduce electrostatic repulsions, thus gradually destabilizing the casein micelle system. Heat treatment has been shown to reduce the concentration of soluble phosphate and of both soluble and ionic calcium. The transfer of calcium and soluble phosphate from the serum to the micelles would be expected to shield some of the negative charges on the micelles, reducing the zeta potential and decreasing electrostatic repulsions. The calcium ion activity, which depends on the initial pH of the milk, also decreases upon heating; this decrease is reversible and some or all of the calcium ion activity is recovered after heating. However, this occurs upon cooling, not during heating, and takes at least 24 h. It is interesting that the decrease in pH during heating does not result in an increase in calcium ion activity, but that the calcium ion activity remains more or less constant after heating for a few minutes (Van Boekel et al. 1989a,b). Heating has no effect on monovalent ions, sodium, potassium and chloride, but its effect on citrate is unclear. Another factor of importance in the serum phase is the whey proteins. These proteins are easily denatured by heat treatments above 70C and then react with each other or casein micelles, as discussed earlier. MECHANISM FOR COAGULATION OF MILK PROTEIN Based on many studies, a unied mechanism to explain the HCTpH proles of both normal and concentrated milks has been developed and largely accepted, and ts well with most of the experimental observations (OConnell and Fox 2003). The HCTpH curve is divided into two regions (Fig. 5); region I (pH values below 6.8) is concerned with the stability of whey protein-coated micelles, although the amount of whey proteins that associate with the micelles decreases with pH in this region. At a pH well below the maximum, milk coagulates rapidly because of low pH and high calcium ion activity (decreased electrostatic repulsions). In addition, relatively high amounts of whey proteins associated with the micelles at low pH may promote aggregation of casein particles

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Figure 5 Diagrammatic representation of heat-induced interactions of proteins in normal milk in relation to pHdependence of heat stability. In region I, whey proteins are associated with the casein micelle. The stability of these whey protein-coated micelles increases with pH within this region. In region II, -casein/whey protein complexes are largely in the serum, and the stability of -casein-depleted micelles increases with pH in this region. Coagulation is mainly caused by aggregation of -casein-depleted micelles by calcium.

through cross-linking of whey proteins bound onto different micelles. The occurrence of a maximum (at pH 6.76.8) in the HCTpH prole of normal milk is essentially due to greater stability of whey protein-coated micelles, as the formation of a complex between -casein and -lactoglobulin on the surface of the casein micelles alters the steric and electrostatic interactions and prevents the dissociation of micellar -casein. The details of the coagulation pathway for whey protein-coated micelles are by no means fully understood, but it is likely that further heating causes lowering of the pH, dephosphorylation, covalent bond formation and other reactions. Consequently, the altered micelles can make more frequent contact and probably form covalent cross-links within and between the micelles, resulting in an irreversibly aggregated protein material. At pH values above 6.9 (region II), the stability decreases due to the dissociation of micellar casein, thus reducing the stabilizing effect of this protein. The -casein-depleted micelles are sensitive to calcium ion concentrations. Therefore, the minimum in the HCTpH prole is a result of coagulation of -casein-depleted micelles; coagu 2004 Society of Dairy Technology

lation is essentially salt induced, caused by calcium ions. At higher pH, although dissociation of micellar -casein increases, the HCT increases due to the increase in protein charge and low calcium ion activity. It is also possible that the dissociated -casein may reassociate during extended heating because of a heat-induced decrease in pH. An alternative hypothesis to explain the pH dependence of the heat stability of milk has been proposed recently by OConnell and Fox (2003). From pH about 6.3 to the pH of maximum stability, -lactoglobulin denatures through a calcium-mediated mechanism and enhances the thermal stability of the casein micelles by chelating calcium. At pH values > 6.9, the disulphide bonds of -lactoglobulin and -casein are reduced on heating, which facilitates complex formation. Concurrently, the hydrophobic -barrel of -lactoglobulin is exposed, which results in an increase in the surface hydrophobicity of the -lactoglobulincasein micelle complexes, thereby sensitizing the casein micelles to the destabilizing effect of heat-induced calcium phosphate precipitation. At pH values on the alkaline side of the minimum, stability increases as a function of pH, possibly because of an increase in protein stability with increasing pH (micellar zeta potential and hydration increase as a function of pH) and a decrease in calcium ion activity with increasing pH. However, this hypothesis does not explain the marked dissociation of -casein from the micelles at pH > 6.9, as observed by many researchers. In the region of the minimum, most of the -lactoglobulin is in fact found in the serum and is not associated with the casein micelles. In addition, there is no evidence to suggest that the disulphide bonds of native -lactoglobulin and -casein are reduced at high temperatures, and it is unclear how this reduction would facilitate complex formation. In such a situation, no disulphidesulphydryl interchange reactions between -casein and -lactoglobulin would be expected to occur, which clearly is not the case. Concentrated milks are considerably less heat stable than unconcentrated milks. Because of the lower assay temperatures (120C), many of the heatinduced changes in concentrated milk do not proceed to the same extent as in unconcentrated milk. For example, the degree of dephosphorylation and covalent bond formation, and the decrease in pH, are far smaller than in unconcentrated milk. The coagulum formed on either side of the maximum stability is soluble in dissociating buffers, although some covalent bonds appear to form in the region of the maximum (Nieuwenhuijse et al. 1991). In the pH region 6.56.7, the dissociation of -casein, the extent of which increases with an increase in pH, induces coagulation by altering the surface of the casein micelles, a situation somewhat comparable with that existing for the coagulation of 117

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Table 3 Apparent similarities between the coagulation behaviours of concentrated milk at pH 6.6 (region of maximum stability) and normal milk at pH 6.9 (region of minimum stability) Addition of whey proteins causes destabilization Urea addition up to about 6 mm has no effect The coagulum is dispersible in 6 m urea buffer The coagulation is a two-stage process, as revealed by nitrogen-depletion curves Extensive dissociation of caseins from the micelles, especially -casein, occurs

stability. Practical solutions to the heat stability problems include the following: manipulation of preheat treatments matching the natural pH of milk to that of the heat stability maximum addition of different levels of phosphate addition of buttermilk and phospholipids at appropriate levels combination of the above treatments. REFERENCES

normal milk within the region of minimum stability. Some of the similarities between the coagulation behaviour of concentrated milk in the region of the maximum and that of unconcentrated milk in the region of the minimum are shown in Table 3. At pH values below 6.4, the denatured whey proteins, those in the serum as well as those attached to casein micelles, are susceptible to heat-induced aggregation; high calcium ion activity in this pH range probably promotes the formation of large whey protein aggregates and network structures. At higher pH ( 7.0), the coagulum appears to form a gel-like matrix of protein that is probably derived from whey protein aggregates and the dissociated protein material (Singh et al. 1995). PRACTICAL SIGNIF ICANCE OF HEAT STABILITY The ability of milk to withstand high-temperature treatments without loss of its stability is fairly unique and makes the production possible of many sterilized milk products with a long shelf life. These products include UHT milks and creams, in-can sterilized milks, evaporated milk, sweetened condensed milk and milk powders, especially those intended for reconstitution and recombination into sterilized products (heat-stable powders). Considerable knowledge gained on the heat stability of milk has allowed most of the practical problems to be solved relatively easily by manipulating processing and compositional variables. From an industrial viewpoint, the heat stability of milk of normal concentration has rarely been a problem. However, in recent years, many new liquid milks, fortied with high amounts of calcium, magnesium and zinc, cocoa and tea extracts, have been introduced into the market. As many of these additives have a negative impact on heat stability, these products require very careful manipulation of the formulation to achieve the desired heat stability and it is often difcult to achieve the desired shelf stability. Certain problems regarding the heat stability of concentrated milk, especially full-fat homogenized recombined evaporated milk, remain unsolved. These problems relate largely to seasonal variations and batch-to-batch variations in heat 118
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Anema S G and Klostermeyer H (1996) -Potential of casein micelles from reconstituted skim milk heated at 120C. International Dairy Journal 6 73687. Anema S G and Klostermeyer H (1997) The effect of pH and heat treatment on the -casein content and -potential of particles in reconstituted skim milk. Milchwissenschaft 52 217223. Anema S G and Li Y M (2003) Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size. Journal of Dairy Research 70 7383. Aoki T, Umeda T and Kako Y (1990) Cleavage of the linkage between colloidal calcium phosphate and casein on heating milk at high temperature. Journal of Dairy Research 57 349354. Fox P F (1981a) Heat-induced changes in milk preceding coagulation. Journal of Dairy Science 64 21172137. Fox P F (1981b) Heat stability of milk: signicance of heatinduced acid formation in coagulation. Irish Journal of Food Science Technology 5 111. Fox P F (1982) Heat-induced coagulation of milk. In Developments in Dairy Chemistry. I. Proteins, pp 189 228. Fox P F, ed. London: Applied Science Publishers. Fox P F and Hearn C M (1978) Heat stability of milk: inuence of denaturable proteins and detergents on pH sensitivity. Journal of Dairy Research 45 159172. Fox P F and Hoynes MCT (1975) Heat stability of milk: inuence of colloidal calcium phosphate and -lactoglobulin. Journal of Dairy Research 42 427435. Fox P F and Morrissey P A (1977) Reviews of the progress of dairy science: the heat stability of milk. Journal of Dairy Research 44 627646. Holt C (1992) Structure and stability of bovine casein micelles. Advances in Protein Chemistry 43 64151. Jang H D and Swaisgood H E (1990) Disulphide bond formation between thermally denatured -lactoglobulin and -casein in casein micelles. Journal of Dairy Science 73 900904. Morrissey P A (1969) The heat stability of milk as affected by variations in pH and milk salts. Journal of Dairy Research 36 343351. Muir D D and Sweetsur A W M (1976) The inuence of naturally occurring levels of urea on the heat stability of bulk milk. Journal of Dairy Research 43 495499. Nieuwenhuijse J A, Sjollema A, van Boekel M A J S, van Vliet T and Walstra P (1991) The heat stability of concentrated milk. Netherlands Milk Dairy Journal 45 193224. OConnell J E and Fox P F (1996) Effect of phenolic compounds on the heat stability of milk and concentrated milk. Journal of Dairy Research 66 399407.

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OConnell J E and Fox P F (2003) Heat-induced coagulation of milk. In Advanced Dairy Chemistry. I. Proteins, 3rd edn, pp 879930. Fox P F and McSweeney P L H, eds. London: Kluwer Academic. OConnell J E, Fox P D, Tan-Kintia R and Fox P F (1988) Effects of extracts of tea, coffee and cocoa on the colloidal stability of milk. International Dairy Journal 8 689 693. Rogers L A, Deysher E F and Evans F R (1921) The relation of acidity to the coagulation temperature of evaporated milk. Journal of Dairy Science 4 294309. Rose D (1961) Factors affecting the pH-sensitivity of the heat stability of milk from individual cows. Journal of Dairy Science 44 14051413. Rose D (1963) Heat stability of bovine milk: a review. Dairy Science Abstracts 25 4552. Schmidt D G and Poll J K (1986) Electrokinetic measurements on unheated and heated casein micelle systems. Netherlands Milk Dairy Journal 31 342357. Singh H (1988) Effects of high temperatures on casein micelles. New Zealand Journal of Dairy Science Technology 23 257273. Singh H (1995) Heat-induced changes in caseins including interactions with whey proteins. In Heat-Induced Changes in Milk, pp 8699. Fox P F, ed. Special Issue 9501. Brussels: International Dairy Federation. Singh H and Creamer L K (1991a) Inuence of concentration of milk solids on the dissociation of micellar -casein on heating reconstituted skim milk at 120C. Journal of Dairy Research 58 99105. Singh H and Creamer L K (1991b) Aggregation and dissociation of milk protein complexes in reconstituted skim milk at 120C. Journal of Food Science 56 671677. Singh H and Creamer L K (1992) Heat stability of milk. In Advanced Dairy Chemistry. I. Proteins, pp 621656. Fox P F, ed. London: Elsevier. Singh H and Fox P F (1985a) Heat stability of milk: the mechanism of stabilisation by formaldehyde. Journal of Dairy Research 52 6576. Singh H and Fox P F (1985b) Heat stability of milk: pHdependent dissociation of micellar -casein on heating

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