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Influence of heat treatment of milk on cheesemaking properties
International Dairy Journal 11 (2001) 543–551
Influence of heat treatment of milk on cheesemaking properties Harjinder Singh*, Algane Waungana Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11 222, Palmerston North, New Zealand
Abstract High heat treatment of milk causes denaturation of whey proteins and complex interactions among denatured whey proteins, casein micelles, minerals and fat globules. It is well established that interactions of whey proteins have both positive and negative implications in cheese manufacture. On the one hand, denatured whey proteins could be incorporated into cheese curd, resulting in a higher yield from a given quantity of milk while, on the other, the interactions of whey protein with casein micelles interfere with the rennet coagulation process, resulting in long coagulation times and weak curd structures. The cheeses made from heated milk differ from traditional cheese in body, texture and flavour profiles. This paper reviews current information on the relationships between whey protein interactions in heated milk and rennet coagulation properties, and discusses possible mechanisms involved. Methods that may be used to improve renneting properties of heated milk are described. In addition some recent results on the interactive effects of heat treatment, ultrafiltration concentration and pH on properties of interest to cheesemaking, in particular rennet-induced gel formation, are presented. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Heat treatment; Milk protein interactions; Whey protein denaturation; Rennet coagulation; Ultrafiltration; Cheese
1. Introduction In recent years, the manufacture of various cheese varieties has received considerable interest, primarily because of the potential to improve cheese yield, through incorporation of whey proteins into cheese curd. In addition, high heat treatment of milk results in low bacterial load with an extended shelf life, permitting milk to be transported long distances or held for long periods before cheese manufacture. However, the cheesemaking characteristics of heated milks differ markedly from those typically of pasteurised milk. Milk that has been heat treated at high temperatures shows longer coagulation times and forms weaker, finer curd which retains more water than normal. These effects are considered to arise mainly from the formation of complexes between denatured whey proteins and micellar k-casein, which subsequently modifies the surface characteristics and interactions of casein micelles (Dalgleish, 1992). Heat-induced changes in calcium phosphate equilibrium are also involved. A number of studies have revealed that the adverse effects of heat treatment on rennet coagulation can be overcome, to *Corresponding author. Tel.: +64-6-350-4401; fax: +64-6-3505655. E-mail address: [email protected] (H. Singh).
some extent, by either (i) decreasing the pH to about 6.2, (ii) acidifying milk to below 5.5 followed by neutralization to 6.6, or (iii) adding calcium chloride (Lucey, Gorry, & Fox, 1993b), although the mechanisms by which these effects occur are not fully understood. Ultrafiltration (UF) of milk has been widely used for the manufacture of soft varieties of cheese, but the technology has not proved beneficial in the manufacture of hard and semi-hard cheeses. One of the major problems encountered in the manufacture of semi-hard and hard cheese is that the products tends to very hard and crumbly in texture. Heat treatment in combination with UF may yield opportunities to correct curd firmness problems, improve cheese yields and develop new cheese varieties with different textural and functional characteristics.
2. Effects of heat treatment on milk It is well known that heat treatment of milk during commercial processing operations results in a number of physiochemical changes in the milk constituents (see reviews by Fox, 1981; Singh & Creamer, 1992; Singh, 1995); some of these changes are listed in Table 1. Significant changes occurring upon heating milk above 601C include the denaturation of whey proteins,
0958-6946/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 0 8 5 - 1
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Table 1 Heat-induced changes in milk Whey protein denaturation and aggregation Interactions of whey proteins with casein micelles Reaction between lactose and protein (Maillard reactions) Changes in casein micelle structure Transfer of soluble calcium and phosphate to colloidal phase Changes in fat globule membrane Decrease in pH
interactions between denatured whey proteins and the casein micelles and the conversion of soluble calcium, magnesium and phosphate to the colloidal state. Casein micelles are very stable at high temperatures although changes in zeta potential, size, hydration of micelles, as well as some associationFdissociation reactions do occur at severe heating temperatures (Fox, 1981; Singh, 1988; Singh & Creamer, 1992). The present paper deals mainly with the effects of heat on whey proteins. Thermal denaturation and aggregation of whey proteins have been extensively researched. Upon heating milk above 651C, whey proteins are denatured by the unfolding of their polypeptides, thus exposing the side chain groups originally buried within the native structure. The unfolded proteins then interact with casein micelles or simply aggregate with themselves, involving thiol–disulphide interchange reactions, hydrophobic interactions and ionic linkages. The order of sensitivity of the various whey proteins to heat in milk has been reported to be immunoglobulins >bovine serum albumin >b-lactoglobulin (b-Lg) >a-lactalbumin (a-La) (Larson & Rolleri, 1955; Dannenburg & Kessler, 1988; Oldfield, Singh, Taylor, & Pearce, 1998b). Ionic strength, pH and the concentrations of calcium and protein markedly influence the extent of denaturation of the whey proteins (Hillier, Lyster, & Cheeseman, 1979; Park & Lund, 1984; Dannenburg & Kessler, 1988; Oldfield et al., 1998b). Increasing the calcium concentration up to 4 mg/mL tends to retard the denaturation of both b-Lg and a-La, but further increase has little effect. Increasing protein concentration decreases the rate of denaturation of b-Lg, but increases the rate of denaturation of a-La (Hillier et al., 1979). Heat denaturation of whey proteins is also influenced by lactose and other sugars, polyhydric alcohols, and protein modifying agents (Hillier et al., 1979; Bernal & Jelen, 1985; Donovan & Mulvihill, 1987). Denatured whey proteins have been shown to associate with k-casein on the surface of the casein micelles, giving the appearance under an electron microscope of threadlike appendages, protruding from the micelles (Creamer & Matheson, 1980; Mohammad & Fox, 1987). The principal interaction is considered to be between b-Lg and k-casein and involves both disulphide and hydrophobic interactions (Smits & van Brouwershaven, 1980; Singh & Fox, 1987). Not all the
denatured whey proteins complex with the casein micelles. Some remain in the serum where they may form aggregates with other whey proteins or with serum k-casein. The extent of association of denatured whey protein with casein micelles is markedly dependent on the pH of the milk prior to heating, levels of soluble calcium and phosphate, milk solids concentration and type of heating system (water bath, indirect or direct heating system). Heating at pH values less than 6.7 results in a greater quantity of denatured whey proteins associating with casein micelles, whereas, at higher pH values, whey protein\k-casein complexes dissociate from the micelle surface, apparently due to dissociation of kcasein (Singh & Fox, 1985, 1986). Recent studies (Oldfield, Singh, & Taylor, 1998a; Corredig & Dalgleish, 1996) suggest that indirect heating tends to result in greater proportions of b-Lg and a-La associating with micelles than when using a direct heating system (e.g. direct steam injection). The mechanism of b-Lg denaturation and their association with casein micelles in milk systems have been proposed recently by Oldfield et al. (1998b) who suggested that at least three possible species of denatured b-Lg that could associate with micelles; (i) unfolded monomeric b-Lg, (ii) self-aggregated b-Lg and (iii) b-Lg/a-La aggregates. The relative rates of association of these species with the casein micelles depend on temperature and heating rate, which in turn affect the relative rates of unfolding and the formation of the various aggregated species. The b-Lg aggregates, which have been shown to be stiff and rod-like (Griffin, Griffin, Martin, & Price, 1993), would protrude from the micelles surface, providing steric effects for further bLg association. In addition, these aggregates may have their reactive sulphydryl group buried within the interior of the aggregate, and therefore unavailable for sulphydryl–disulphide interchange reactions with micellar kcasein. In contrast, unfolded monomeric b-Lg molecules would be expected to penetrate the k-casein hairy layer with greater ease and have a readily accessible sulphydryl group. The formation of unfolded b-Lg may be promoted by prolonged heating times at low temperatures, or by heating at a slower rate to the required temperature. However, at high temperatures and fast heating rates, all whey proteins begin to unfold in a relatively short period of time, thus presenting more opportunity for unfolded monomeric b-Lg to selfaggregate, which consequently is likely to associate with the casein micelles less efficiently.
3. Rennet coagulation properties of heated milks The effects of whey protein denaturation and association with casein micelles on rennet coagulation properties have been studied (Dalgleish, 1990; Waungana,
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Singh, & Bennett, 1996). It is believed that the heat treatment of milk results in longer rennet coagulation times (RCTs) and reduced strength of rennet gels (Morrissey, 1969; Banks, 1990; Dalgleish, 1992). Dynamic testing, which involves an oscillatory applied strain or stress, can provide very useful information on the gel formation process. The rheological parameters, such as elastic storage modulus (G0 ), viscous loss modulus (G00 ) and loss tangent characterising the rennet gels may be derived from such measurements. Typical changes in G0 with time during the course of renneting skim milks (at 321C) preheated at a range of temperatures (80–1401C for 4 s) are shown in Fig. 1. Gelation time (defined as the time when storage modulus reached >1 Pa) increases and G0 decreases as the severity of heat treatment increases (Fig. 1; Waungana et al., 1996). Milks heated at 1401C for 4 s fail to form a gel within 120 min. The extent of denaturation of b-Lg was plotted as a function of gelation time or G0 (Waungana et al., 1996; Fig. 2a). It is clear that increasing the level of denaturation of b-lactoglobulin to about 60% has little influence on gelation time, but gelation time increases very markedly with further increase in b-Lg denaturation. In contrast, the G0 of rennet gels is much more sensitive to the presence of denatured b-lactoglobulin than the gelation time. For example, the G0 values of rennet gels decrease almost linearly with increase in the degree of denaturation of b-lactoglobulin from 10% to 65%. Similar trends are observed for the relationship between the extent of b-lactoglobulin association with the micelles and gelation time, although increases in gelation time are observed at lower levels of blactoglobulin association than those observed for the
denatured protein (Fig. 2b). As the amount of blactoglobulin associated with micelles increases from 0% to 50%, there is an almost linear decrease in G0 : Milks containing >50% b-Lg associated with micelles have very low values of G0 : The decrease in the rate of gel development and final G0 values could be attributed to the association of whey protein aggregates with casein micelles surfaces through formation of a b-Lg/k-casein complex which may protrude from the micelle surface, as discussed earlier. This association would affect the close approach of the reactive sites formed on the micelles by the action of rennet. After k-casein conversion into para-k-casein, aggregation would occur mostly between micelles not fully covered with b-Lg, resulting in formation of fewer bridges with fewer and weaker bonds. A considerable proportion of b-Lg aggregates remains in the serum after heating. The role of these aggregates in rennetinduced gel formation is not clear, but they are likely to interfere with the aggregation and gel formation processes, possibly reducing the number and strength
Fig. 1. The effects of temperature for a holding time of 4 s on the storage modulus (G0 ) as a function of time after addition of rennet in skim milk. Heating temperatures: unheated milk (J), 80, (K), 90 (&), 100 (’), 110 (m), 120 (n), 130 (+) or 1401C ( ). All samples were renneted at pH 6.5 at 321C. Taken with permission from Elsevier Science, Waungana et al. (1996) Food Research International, 29, 715– 721.
Fig. 2. (a) Relationship between heat denaturation of b-lactoglobulin and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was >1 Pa) (’) in normal skim milk. (b) Relationship between heat-induced association of b-lactoglobulin with the casein micelles and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was >1 Pa) (’) in normal skim milk. Data from Waungana et al. (1996).
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of the contacts between adjacent aggregate chains within the gel network. A very large decrease in gel stiffness at high levels of b-Lg denaturation (>65%) could be attributed to the marked decrease in the extent and rate of k-casein hydrolysis, possibly due to chemical damage to k-casein molecules caused by severe heat treatments (Leaver, Law, Horne, & Banks, 1995).
4. Methods for improving the rennet coagulation properties of heated milks The rennet coagulation properties of heated milk may be at least partly restored either by (i) acidification of heated milk to pH values below 6.2, (ii) acidification of heated milk to low pH values (B5.5) followed by reneutralization to the neutral pH range, often termed as pH cycling, or (iii) heating at elevated pH combined with pH cycling and CaCl2 addition. Acidification of heated milks (70–1401C) to below 6.2 results in shorter RCTs and increased gel firmness, with a maximum firmness at pH 6.2, probably by reducing charge repulsion and increasing ionic calcium concentrations (Singh, Shalabi, Fox, Flynn, & Berry, 1988; Lucey et al., 1993b). Singh et al. (1988) heated milk at 901C for 10 min before adjusting the pH from 6.6 down to values in the range 5.1–6.3, holding at the low pH for 2 h at 201C and then readjusting to pH 6.6 before measurement of RCT at 301C. They reported marked reductions in RCT of such pH-cycled milk. van Hooydonk, Koster, and Boerrigter (1987), who used more severe heat treatment (5 s at 1401C, direct UHT), observed reductions in RCT of about 14% upon acidification to pH 5.5 followed by re-neutralization after 24 h. Lucey, Gorry, and Fox (1993a) reported reductions in RCT for milks heated at temperatures up to 1001C for 10 min. More severely heated milk (1201C for 10 min) did not coagulate even after acidification to low pH values and re-neutralization to pH. In a more recent work (Waungana, 1995), it was shown that in skim milk heated at temperatures in the range 80–1401C for 4 s, gelation time decreases in all samples upon pH cycling (skim milk acidified to pH 5.5, held for 2 h, and subsequently neutralised to pH 6.5) compared with the control (skim milk adjusted to pH 6.5); the decrease in gelation time is greatest when the milks are heated either at 1001C for 4 s prior to renneting (Fig. 3a). At 1401C, there is only B10% reduction in gelation time. pH cycling also affects the G0 values of rennet gels formed from heated milks (Fig. 3b). In the unheated (control) milk, pH cycling has an adverse effect on G0 and this persists to a smaller extent in the sample heated at 801C. The most significant increase in G0 is observed in the sample heated at 1001C; samples that had received more severe heat treatments
prior to renneting also form slightly firmer gels upon renneting after pH cycling, although the effect is not as marked as that observed at 1001C. Singh et al. (1988) reported that rennet gel formed from milk heated at 901C for 10 min, acidified to pH 5.5 and re-adjusted to pH 6.6, is slightly firmer than that from the original, unheated milk. Lucey et al. (1993a), however, found that acidification of heated milk (1001C for 10 min) to pH values of B5.5 before re-neutralization to pH 6.7 results in greatly improved RCT but gel firmness is not completely restored to that of raw milk. The concept of pH cycling as a means of improving the rennet coagulation properties of heated milks was initiated by experiments performed by Banks and Muir (1985). They showed that sterilised starter milk, which was acidified by lactic acid and subsequently neutralised by addition to the cheese milk, is fully incorporated into a rennet gel. They suggested that this was due to the disruption of micelles at low pH, which would make the hidden k-casein liable to conversion and cause the casein
Fig. 3. (a) Gelation times of skim milks heated at temperatures in the range 80–1401C for 4 s (&). Heated skim milks adjusted to pH 5.5, left for 2 h at 201C, and re-adjusted to 6.5 before renneting (’). All samples were renneted at pH 6.5 at 321C. (b) Storage modulus of skim milks heated at temperatures in the range 80–1401C for 4 s (&). Heated skim milks adjusted to pH 5.5, left for 2 h at 201C, and readjusted to 6.5 before renneting (’). All samples were renneted at pH 6.5 at 321C.
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micelles to participate in the gel formation process. It has since been shown, however, that the pH cycling procedure does not result in the release of any additional casein macropeptide (CMP) (van Hooydonk et al., 1987) or 12% TCA soluble N-acetylneuramic acid (NANA) (Singh et al., 1988) during the primary enzymic phase of the coagulation of heated milks. van Hooydonk et al. (1987) suggested that acidification to pH 5.5 solubilises a substantial amount of both the original colloidal calcium phosphate (CCP) and the heat-induced Ca/P complexes. Subsequent neutralization probably leads to the reformation of Ca/P complexes with composition and properties more like the original CCP, and that this may be the most important factor responsible for the improved rennet coagulation properties of heated and pH cycled milks. Singh et al. (1988) reported that pH cycling affected the rennet coagulability of heated milk by increasing the [Ca2+], (an effect similar to that obtained by small additions of CaCl2), and thus promoting aggregation of renneted micelles. Overall, it may be concluded that pH cycling only partly restores the rennet coagulation properties of heated milks. The extent to which these are restored is dependent on the severity of the heat treatment employed. Gelation time appears to be more sensitive to pH cycling than gel firmness. The coagulation properties of milks heated at temperatures of about 1001C for 4 s appear to be mostly restored by pH cycling but the properties of more severely heated milks (X1201C) are less affected. Imafidon and Farkye (1993a) reported that milk adjusted to pH 7.3 before heating (911C for 16 s) followed by acidification to pH 6.4 before renneting has a mean RCT of B9 min. After acidification to pH 5.5 and overnight storage at 41C before re-neutralization to pH 6.4 (pH cycling) RCT decreases by B50%. They also reported faster curd-firming rates and slightly firmer gels in samples that are pH cycled to pH 6.4 than in those directly acidified to the same pH. However, when milk is pH cycled to its original pH of 6.6 before setting, the curds formed are very weak. Waungana (1995) found that milk adjusted to pH 7.3, heated at 1401C for 4 s and then pH cycled has the shortest gelation time and highest G0 values of all the heat treated milks. These values, however, are still far short of those of an unheated control, and that the rennet coagulation properties of severely heated milks are not satisfactorily restored as a result of increasing the pH before heating followed by an acidification and re-neutralisation step. The accelerating effects of calcium chloride on the rennet coagulation of unheated milk are well documented (Mehaia & Cheryan, 1983; van Hooydonk, Hagedoorn, & Boerrigter, 1986; Singh et al., 1988) and CaCl2 is frequently added to cheese milk to enhance rennet coagulation during commercial cheese manufacture.
Waungana (1995) showed that addition of CaCl2 results in a decrease in gelation time and formation of stronger gels in both the heated (1401C for 4 s) and unheated milk systems (Table 2). G0 values increase by 17% and 28%, respectively, upon addition of 1 and 2 mm CaCl2 to the control milk. Added CaCl2 has a major effect on G0 of heated milks; G0 values increase from 2 Pa (no CaCl2) to 86 Pa at 2 mm CaCl2 addition. pH cycling of the heated milks before addition of CaCl2 causes a further reduction in gelation time, but has a negligible effect on G0 : The decrease in gelation time and increase in G0 observed in both the heated and unheated milk samples upon addition of CaCl2 is probably due to increased aggregation of renneted micelles caused by increased [Ca2+], arising largely from the charge reduction on casein micelles by binding of Ca2+ ions, thus reducing electrostatic resistance to aggregation of casein micelles. Moreover, van Hooydonk et al. (1986) showed that the degree of k-casein conversion at the coagulation point decreases with an increasing amount of CaCl2 concentration and this may also account for the reduction in gelation time.
5. Effects of heat treatment on rennet coagulation properties of UF concentrated milks It is generally accepted that there is a very steep increase in the rate of curd firming and strength of rennet gels made from UF concentrated milks (Schmutz & Puhan, 1978; Green, 1990) and that heat treatment of UF milk lowers the strength of rennet gels (Sharma, Hill, & Goff, 1990; McMahon, Yousif, & Kalab, 1993; Guinee, O’Callaghan, Pudja, & O’Brien, 1996; Waungana et al., 1996). Recent work by Waungana, Singh, and Bennet (1998) showed that significant decreases in both the rate of gel firming and final gel firmness occur upon heating UF
Table 2 Effect of CaCl2 addition, heat treatment and pH cycling on rennet coagulation propertiesa Sample
pH cycle
CaCl2 (mm)
Gelation time (min)
Storage modulus G0 (Pa)
Control Control Control 1401C (4 s) 1401C (4 s) 1401C (4 s) 1401C (4 s) 1401C (4 s) 1401C (4 s)
F F F F F F 5.5–6.5 5.5–6.5 5.5–6.5
0.00 1.00 2.00 0.00 1.00 2.00 0.00 1.00 2.00
7.0 3.5 2.0 62.5 8.0 4.5 54.5 6.5 3.5
122 147 170 2 58 86 10 57 79
a pH cycling: pH adjusted to 5.5, held for 2 h and pH readjusted to 6.5. Samples were renneted at 321C at pH 6.5.
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concentrates (3 concentration) at temperatures between 801C and 1401C for 4 s. Denaturation of b-Lg up to B75% has little effect on the onset of gelation in UF skim milk, but higher levels of denaturation decrease gelation times (Fig. 4a). However, G0 decreases almost linearly as the degree of denaturation of b-Lg increases up to B75% with a further increase in denaturation causing a sharper decrease in G0 (Fig. 4b). Similar trends are observed between the levels of b-Lg association with the micelles and G0 or gelation time (Fig. 4b). It is clear that the concentration of milk by UF counteracts the effects of heat treatment on the rennet coagulation properties of milk since rennet gels may still be formed from severely heated UF concentrates. The gels formed from the UF concentrates are much firmer than the corresponding unconcentrated milks at comparable levels of b-Lg denaturation and association. This is probably because UF of milk increases the protein concentration and lowers the volume of the
aqueous phase, which lowers the mean free distance between casein micelles, and so increases the aggregation velocity of renneted micelles. This coupled with the fact that in UF concentrates there is a higher level of colloidal calcium which results in coagulation being initiated at a lower extent of k-casein hydrolysis (Garnot & Corre, 1980; Dalgleish, 1981; McMahon et al., 1993). 5.1. High heat treatment of milk before or after UF concentration Waungana et al. (1998) studied the effects of UHT treatment before or after UF concentration and reported that UHT treatment of milk prior to concentration by UF results in concentrates that have slower curd firming rates and longer times to reach a given G0 value as compared to the concentrates where UHT treatment is given after UF (Table 3). It is not clear, however, why the G0 values of the rennet gels formed from milk that was heated before UF are considerably lower than those from milk that is concentrated before heat treatment. It is possible that the nature of the b-lactoglobulin/k-casein complexes and the way in which they associate with casein micelles upon heating of UF concentrates is different to that when normal milk is heat treated. In addition, differences in the mineral equilibria between the concentrated and unconcentrated milks may influence heat-induced changes in minerals and proteins during heating. Waungana et al. (1998) also showed that pH cycling of heated 3 UF concentrate delays the onset of gelation and greatly reduces the curd firming rate and final G0 values compared to that of heated but not pHcycled concentrates. When pH cycling of heated milk is carried out before UF, the onset of gelation is further delayed and the curd firming rate is much lower compared to that of 3 UF concentrate prepared from heated milk that is not pH cycled (Table 3).
Table 3 Effect of high heat treatment (1401C for 4 s) before or after ultrafiltration and pH cycling on rennet coagulation properties of skim milka
Fig. 4. (a) Relationship between heat denaturation of b-lactoglobulin and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was >1 Pa) (’) in 3 UF concentrated skim milk. (b) Relationship between heat-induced association of blactoglobulin with the casein micelles and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was > 1 Pa) (’) in 3 UF concentrated skim milk. Data from Waungana et al. (1996).
Sample
Gelation time (min)
Storage modulus G0 (Pa)
Skim milk UF concentrate Heated skim milk Heated UF concentrate Heated milk concentrated by UF pH-cycled heated milk pH-cycled heated UF concentrate Heated milk, pH-cycled and then UF concentrated
10.5 19.0 No gel 21.0 25.5 58.0 28.0 31.0
140 2354 No gel 312 186 9 139 85
a pH cycling: pH adjusted to 5.5, held for 2 h and pH readjusted to 6.5. Samples were renneted at 321C at pH 6.5.
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When heated milk is pH cycled before UF, there is presumably an increase in [Ca2+] which is considered to be responsible for its improved rennet coagulation properties (Singh et al., 1988; Lucey et al., 1993a). During subsequent UF treatment of this milk, this [Ca2+] is presumably lost in the milk permeate, resulting in poorer rennet coagulation properties (possibly due to reduced rate of aggregation in the secondary phase) of such treated milk compared to that which is not pH cycled. Since pH cycling of the concentrates after UF and heat treatment do not improve their rennet coagulation properties, it appears that the explanations are more complex than proposed above, and probably involves changes in the equilibrium of mineral salts (especially calcium), state of micellar calcium phosphate and the nature of the associations between b-lactoglobulin and k-casein. Further research is needed on the influence of technological treatments such as heating, UF and pH cycling before renneting on the equilibrium of milk salts, as well as on the nature of associations between b-lactoglobulin and k-casein.
6. Cheese manufacture from heated milk In addition to the effects of heat treatment on rennet coagulation and gel stiffness, the detrimental effects of casein–whey protein interactions during heating on syneresis of rennet curd are well documented (Walstra, van Dijk, & Geurts, 1985). The association of whey proteins at the micelle surface sterically impedes the fusion of rennet-altered micelles resulting in less shrinkage of the paracasein network. There is also an increase in the water binding capacity of the paracasein– whey protein complex. There have been a few studies on the manufacture of cheese from milks heated under fairly mild conditions to give whey protein denaturation levels of up to B35% (Marshall, 1986; Cheshire cheese; Guinee, Fenelon, Mulholland, O’Kennedy, O’Brien, & Reville, 1998; reduced fat (B16–18%) Cheddar cheese). Because of the inferior coagulation and syneresis properties of high heat-treated milk, higher set-to-cut times during cheesemaking are generally observed. Curds from heat treated milks tend to be soggy, ragged in appearance with poor matting ability. There is a poor curd fusion of the network, with more porous matrices. Increasing heat treatment of milk results in an increase in concentration of whey proteins, moisture, moisture in non-fat substances (MNFS) in the cheese. There is a lower level of calcium and phosphorus in cheese made from heated milk, which is essentially due to the reduction in cheese dry matter and increase in the level of whey proteins. Acidification or pH-cycling approach has been used in the manufacture of cheese from severely heated milks by a number of workers (Banks, Stewart, Muir, & West,
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1987; Banks, 1988, 1990; Imafidon & Farkye, 1993b). Banks et al. (1987) made Cheddar cheeses from milks heated at 1101C for 60 s (66% whey protein denaturation) by changing the pH of milk to 5.8 prior to renneting. With this procedure, the yield increase of 4.7% (on a dry solids basis) was possible. These cheeses showed minor defects with respect to moisture retention and texture, but a more serious defect was the development of an intense bitter off flavour during ripening. This bitterness may be eliminated, to a large extent, by a considerable reduction in the level of rennet used in cheese manufacture (Banks, 1988). Further studies by Banks (1990), Banks, Law, Leaver, and Horne (1993) involved manipulations of pH and processing conditions to achieve whey protein incorporation with a process more similar to traditional cheddar methodology. Milks were heated at 851C or 901C for 1 min and pH adjusted 6.2 prior to renneting. Rennet levels were reduced to 90% of that in the control. On a dry solids basis, the yield increase at 851C and 901C was 3.61% and 3.93%, respectively. Cheddar cheese flavour intensity was generally reduced in cheeses prepared from high heat treatment milks. The level of off-flavour development, predominantly described as bitter, is higher in cheeses made from heated milk, particularly after nine months maturation. The textures were found to be comparable with samples produced from pasteurised milk. The difference in the overall acceptability of the cheese are therefore related to the flavour defect, and other varieties of cheese which are not extensively ripened, e.g. Cheshire and acid set Mozzarella have been made successfully from heat treated milk. Imafidon et al. (1993b) made Cheddar cheeses from milk heated at 911C for 16 s, followed by pH cycling (i.e. acidified to pH 5.5, held overnight at 41C and then neutralised to pH 6.2) before rennet addition. Cheese made from high heat treated milk had higher moisture content (40.5%) than that made from normal pasteurised milk (38.1%). On a dry matter basis, the protein content of cheese made from heated milk was higher (41%) compared with 38% for cheese made from normal milk. This represented B2.6% more protein recovery in cheese made from the high-heat treated milk. No information was provided on the texture and sensory properties of the cheeses. Limited information is available on the effects of heat treatment of cheese milk on the proteolysis during cheese ripening. Experiments by Calvo, Leaver, Law, and Banks (1992) on the ripening of Cheddar cheese made from severely heated milks (1101C for 60 s with pH adjustment to 5.8), showed that the breakdown of bcasein is more extensive than in cheese made from milk heated at 631C for 30 min. No evidence of whey protein breakdown during maturation was found. In contrast, Benfeldt, S rensen, Elleg(aard, and Petersen (1997)
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reported that cheeses (Danbo 45+) made from heated milks show decreased proteolysis of b- and as2 -caseins, which is attributed to reduced plasmin activity in the cheese as a consequence of the thermal inactivation of plasminogen activation system and heat-induced interactions between the plasminogen activation system and b-Lg. Heat treatment does not affect the proteolysis of as1 -casein and only alters slightly the breakdown of para-k-casein. The effects of heat treatment of milk combined with UF on the manufacture of semi-hard cheeses, were studied by Guinee et al. (1995). Standardised milks, heated at 72–1001C to denature 5–63% of the whey protein, were ultrafiltered to yield retentates with protein and fat levels of B18.5% and 14%, respectively. Retentates were converted into semi-hard cheeses using specialised coagulation and gel-cutting equipment, with scalding and further syneresis carried out in conventional cheese vats. High heat treatment of milk necessitated a reduction in set pH, an increase in set temperature and an increase in curd scalding temperature. Increasing levels of whey protein denaturation result in cheeses having higher moisture, S/M and whey protein levels, lower ex-brine pH values and lower rates of pH increase during a 182 day ripening period. Cheeses with high levels of denatured whey proteins also show poorer curd fusion and lower yield (fracture) values during ripening. Higher levels of denatured whey proteins in cheese were associated with a higher degree of primary proteolysis, as revealed by PAGE, levels of WSN and 12% TCA N. However, heat treatment resulted in slower formation of small peptides (B1000 Da) and free amino acids. Examination of various cheeses by SEM showed that as the intensity of heat treatment to which the milk was subjected increases, the protein matrices of cheese become coarser and less homogeneous/continuous in appearance and contain numerous small holes and/or cracks. However, there were no significant differences between the extent of heat treatment and appearance, flavour body and overall impression of the cheeses, as judged by trained cheese sensory panel.
7. Conclusions Considerable progress has been made towards understanding how heat treatment of milk affects rennet coagulation times, rheological and physical properties of rennet gels. However, much less is known about the effects of heating on texture, flavour, functionality and ripening characteristics of cheeses. Heat treatment of milk combined with UF and pH manipulations may offer new possibilities for the production of soft cheeses, semi-hard cheeses and novel hybrid cheeses with varying textures, flavours and functionality. However, there still
remains a great deal to be understood on the complex interactions of proteins, fat and minerals during these combined processes. It must be emphasised that further research in this area should be approached more with the aim of developing novel products as opposed to trying to emulate the flavours and textures of existing products as is the current trend.
Acknowledgements We are grateful to the ministry of Foreign affairs for a scholarship to A.W. and, to David Oldfield for useful discussions.
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H. Singh, A. Waungana / International Dairy Journal 11 (2001) 543–551 Fox, P. F. (1981). Heat-induced changes in milk preceding coagulation. Journal of Dairy Science, 64, 2127–2137. Garnot, P., & Corre, C. (1980). Influence of milk protein concentration on the gelling activity of chymosin and bovine pepsin. Journal of Dairy Research, 47, 103–111. Green, M. L. (1990). The cheesemaking potential of milk concentrated up to four-fold by ultrafiltration and heated in the range 90–971C. Journal of Dairy Research, 57, 549–557. Griffin, W. G., Griffin, M. C. A., Martin, S. R., & Price, J. (1993). Molecular basis for thermal aggregation of bovine b-lactoglobuin A. Journal of Chemical Society of Faraday Transactions, 89, 3395– 3406. Guinee, T. P., Fenelon, M. A., Mulholland, E. O., O’Kennedy, B. T., O’Brien, N., & Reville, W. J. (1998). The influence of milk pasteurization temperature and pH at curd milling on the composition, texture and maturation of reduced fat Cheddar cheese. International Journal of Dairy Technology, 51, 1–10. Guinee, T. P., Pudja, P. D., Reville, W. J., Harrington, D., Mulholland, E. O., Cotter, M., & Cogan, T. M. (1995). Composition, microstructure and maturation of semi-hard cheese from high protein ultrafiltered milk retentates with different levels of denatured whey protein. International Dairy Journal, 5, 543–568. Guinee, T. P., O’Callaghan, D. J., Pudja, P. D., & O’Brien, N. (1996). Rennet coagulation properties of retentates obtained by ultrafiltrration of skim milks heated to different temperatures. International Dairy Journal, 6, 581–596. Hillier, R. M., Lyster, R. L. J., & Cheeseman, G. C. (1979). Thermal denaturation of a-lactalbumin and b-lactoglobuin in cheese whey: Effect of total solid concentration and pH. Journal of Dairy Research, 46, 95–102. Imafidon, G. I., & Farkye, N. Y. (1993a). Rennet coagulability of high heat-treated milk influenced by time of pH adjustment. Journal of Food Science, 58, 1300–1302. Imafidon, G. I., Farkye, N. Y. (1993b). Composition of Cheddar cheese made from high-heat treated milk. In Cheese yield and factors affecting its control, Special issue 9402 (pp. 433–438). Brussels: International Dairy Federation. Larson, B. L., & Rolleri, C. D. (1955). Heat denaturation of the specific serum proteins in milk. Journal of Dairy Science, 38, 351–360. Leaver, J., Law, A. J. R., Horne, D. S., & Banks, J. M. (1995). Influence of heating regime and pH on the primary phase of renneting of whole milk. International Dairy Journal, 5, 129–140. Lucey, J. A., Gorry, C., & Fox, P. F. (1993a). Rennet coagulation properties of heated milk. Agricultural Science in Finland, 2, 361–368. Lucey, J. A., Gorry, C., Fox, P. F. (1993b). Methods for improving the rennet coagulation properties of heated milk. In Cheese yield and factors affecting its control, Special issue 9402 (pp 448–456). Brussels: International Dairy Federation. Marshall, R. J. (1986). Increasing cheese yields by high heat treatment of milk. Journal of Dairy Research, 53, 313–322. McMahon, D. J., Yousif, B. H., & Kalab, M. (1993). Effect of whey protein denaturation on structure of casein micelles and their rennetability after ultra-high temperature processing with or without ultrafiltration. International Dairy Journal, 3, 239–256. Mehaia, M. A., & Cheryan, M. (1983). The secondary phase of milk coagulation. Effect of calcium, pH and temperature on clotting activity. Milchwissenschaft, 38, 137–140. Mohammad, K. S., & Fox, P. F. (1987). Heat-induced microstructural changes in casein micelles before and after heat coagulation. New Zealand Journal of Dairy Science and Technology, 22, 191–203.
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Morrissey, P. A. (1969). The rennet hysteresis of heated milk. Journal of Dairy Research, 36, 333–341. Oldfield, D. J., Singh, H., & Taylor, M. W. (1998a). Association of blactoglobulin and a-lactalbumin with the casein micelles in skim milk heated in a ultra-high temperature plant. International Dairy Journal, 8, 765–770. Oldfield, D. J., Singh, H., Taylor, M. W., & Pearce, K. N. (1998b). Kinetics of denaturation and aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) plant. International Dairy Journal, 8, 311–318. Park, K. H., & Lund, D. B. (1984). Calorimetric study of thermal denaturation of b-lactoglobuin. Journal of Dairy Science, 67, 1699– 1706. Schmutz, M., & Puhan, Z. (1978). Rennet coagulation of ultrafiltered milk. Schwei. Milchw. Forschung, 7, 69–72. Sharma, S. K., Hill, A. R., & Goff, H. D. (1990). The effect of heat treatment of ultrafiltered milk on its coagulation properties. Milchwissenschaft, 45, 432–435. Singh, H. (1988). Review: Effects of high temperatures on casein micelles. New Zealand Journal of Dairy Science and Technology, 23, 257–273. Singh, H. (1995). Heat-induced changes in casein, including interactions with whey proteins. International Dairy Federation Bulletin, 238, 86–104. Singh, H., & Creamer, L. K. (1992). Heat stability of milk. In P. F. Fox (Ed.), Advanced dairy chemistry Volume 1. Proteins (pp. 621– 656). London: Elsevier Science Publishers Ltd.. Singh, H., & Fox, P. F. (1985). Heat stability of milk: pH dependant dissociation of micellar k-casein on heating milk at ultra-high temperatures. Journal of Dairy Research, 52, 529–538. Singh, H., & Fox, P. F. (1986). Heat stability of milk: Further studies on the pH dependent dissociation of micellar k-casein on heating milk at ultra-high temperatures. Journal of Dairy Research, 53, 237–248. Singh, H., & Fox, P. F. (1987). Heat stability of milk: Role of blactoglobulin in the pH-dependant dissociation of micellar kcasein. Journal of Dairy Research, 54, 509–521. Singh, H., Shalabi, S. I., Fox, P. F., Flynn, A., & Berry, A. (1988). Rennet coagulation of heated milk: Influence of pH adjustment before or after heating. Journal of Dairy Research, 55, 205–215. Smits, P., & van Brouwershaven, J. H. (1980). Heat induced association of b-lactoglobulin and casein micelles. Journal of Dairy Research, 47, 313–325. van Hooydonk, A. C. M., Hagedoorn, H. G., & Boerrigter, I. J. (1986). The effect of various cations on the renneting of milk. Netherlands Milk and Dairy Journal, 40, 369–390. van Hooydonk, A. C. M., Koster, P. G., & Boerrigter, I. J. (1987). The renneting properties of heated milks. Netherlands Milk and Dairy Journal, 41, 369–390. Walstra, P., van Dijk, H. J. M., & Geurts, T. J. (1985). The syneresis of curd. 1. General considerations and literature review. Netherlands Milk and Dairy Journal, 39, 209–246. Waungana, A. (1995). Rennet coagulation properties of heated milks. Master of Technology thesis, Massey University, Palmerston North, New Zealand. Waungana, A., Singh, H., & Bennet, R. J. (1996). Influence of denaturation and aggregation of b-lactoglobulin on rennet coagulation properties of skim milk and ultrafiltered milk. Food Research International, 29, 715–721. Waungana, A., Singh, H., & Bennet, R. J. (1998). Rennet coagulation properties of skim milk concentrated by ultrafiltration: Effects of heat treatment and pH adjustment. Food Research International, 31, 645–651.
Influence of heat treatment of milk on cheesemaking properties Harjinder Singh*, Algane Waungana Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11 222, Palmerston North, New Zealand
Abstract High heat treatment of milk causes denaturation of whey proteins and complex interactions among denatured whey proteins, casein micelles, minerals and fat globules. It is well established that interactions of whey proteins have both positive and negative implications in cheese manufacture. On the one hand, denatured whey proteins could be incorporated into cheese curd, resulting in a higher yield from a given quantity of milk while, on the other, the interactions of whey protein with casein micelles interfere with the rennet coagulation process, resulting in long coagulation times and weak curd structures. The cheeses made from heated milk differ from traditional cheese in body, texture and flavour profiles. This paper reviews current information on the relationships between whey protein interactions in heated milk and rennet coagulation properties, and discusses possible mechanisms involved. Methods that may be used to improve renneting properties of heated milk are described. In addition some recent results on the interactive effects of heat treatment, ultrafiltration concentration and pH on properties of interest to cheesemaking, in particular rennet-induced gel formation, are presented. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Heat treatment; Milk protein interactions; Whey protein denaturation; Rennet coagulation; Ultrafiltration; Cheese
1. Introduction In recent years, the manufacture of various cheese varieties has received considerable interest, primarily because of the potential to improve cheese yield, through incorporation of whey proteins into cheese curd. In addition, high heat treatment of milk results in low bacterial load with an extended shelf life, permitting milk to be transported long distances or held for long periods before cheese manufacture. However, the cheesemaking characteristics of heated milks differ markedly from those typically of pasteurised milk. Milk that has been heat treated at high temperatures shows longer coagulation times and forms weaker, finer curd which retains more water than normal. These effects are considered to arise mainly from the formation of complexes between denatured whey proteins and micellar k-casein, which subsequently modifies the surface characteristics and interactions of casein micelles (Dalgleish, 1992). Heat-induced changes in calcium phosphate equilibrium are also involved. A number of studies have revealed that the adverse effects of heat treatment on rennet coagulation can be overcome, to *Corresponding author. Tel.: +64-6-350-4401; fax: +64-6-3505655. E-mail address: [email protected] (H. Singh).
some extent, by either (i) decreasing the pH to about 6.2, (ii) acidifying milk to below 5.5 followed by neutralization to 6.6, or (iii) adding calcium chloride (Lucey, Gorry, & Fox, 1993b), although the mechanisms by which these effects occur are not fully understood. Ultrafiltration (UF) of milk has been widely used for the manufacture of soft varieties of cheese, but the technology has not proved beneficial in the manufacture of hard and semi-hard cheeses. One of the major problems encountered in the manufacture of semi-hard and hard cheese is that the products tends to very hard and crumbly in texture. Heat treatment in combination with UF may yield opportunities to correct curd firmness problems, improve cheese yields and develop new cheese varieties with different textural and functional characteristics.
2. Effects of heat treatment on milk It is well known that heat treatment of milk during commercial processing operations results in a number of physiochemical changes in the milk constituents (see reviews by Fox, 1981; Singh & Creamer, 1992; Singh, 1995); some of these changes are listed in Table 1. Significant changes occurring upon heating milk above 601C include the denaturation of whey proteins,
0958-6946/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 0 8 5 - 1
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Table 1 Heat-induced changes in milk Whey protein denaturation and aggregation Interactions of whey proteins with casein micelles Reaction between lactose and protein (Maillard reactions) Changes in casein micelle structure Transfer of soluble calcium and phosphate to colloidal phase Changes in fat globule membrane Decrease in pH
interactions between denatured whey proteins and the casein micelles and the conversion of soluble calcium, magnesium and phosphate to the colloidal state. Casein micelles are very stable at high temperatures although changes in zeta potential, size, hydration of micelles, as well as some associationFdissociation reactions do occur at severe heating temperatures (Fox, 1981; Singh, 1988; Singh & Creamer, 1992). The present paper deals mainly with the effects of heat on whey proteins. Thermal denaturation and aggregation of whey proteins have been extensively researched. Upon heating milk above 651C, whey proteins are denatured by the unfolding of their polypeptides, thus exposing the side chain groups originally buried within the native structure. The unfolded proteins then interact with casein micelles or simply aggregate with themselves, involving thiol–disulphide interchange reactions, hydrophobic interactions and ionic linkages. The order of sensitivity of the various whey proteins to heat in milk has been reported to be immunoglobulins >bovine serum albumin >b-lactoglobulin (b-Lg) >a-lactalbumin (a-La) (Larson & Rolleri, 1955; Dannenburg & Kessler, 1988; Oldfield, Singh, Taylor, & Pearce, 1998b). Ionic strength, pH and the concentrations of calcium and protein markedly influence the extent of denaturation of the whey proteins (Hillier, Lyster, & Cheeseman, 1979; Park & Lund, 1984; Dannenburg & Kessler, 1988; Oldfield et al., 1998b). Increasing the calcium concentration up to 4 mg/mL tends to retard the denaturation of both b-Lg and a-La, but further increase has little effect. Increasing protein concentration decreases the rate of denaturation of b-Lg, but increases the rate of denaturation of a-La (Hillier et al., 1979). Heat denaturation of whey proteins is also influenced by lactose and other sugars, polyhydric alcohols, and protein modifying agents (Hillier et al., 1979; Bernal & Jelen, 1985; Donovan & Mulvihill, 1987). Denatured whey proteins have been shown to associate with k-casein on the surface of the casein micelles, giving the appearance under an electron microscope of threadlike appendages, protruding from the micelles (Creamer & Matheson, 1980; Mohammad & Fox, 1987). The principal interaction is considered to be between b-Lg and k-casein and involves both disulphide and hydrophobic interactions (Smits & van Brouwershaven, 1980; Singh & Fox, 1987). Not all the
denatured whey proteins complex with the casein micelles. Some remain in the serum where they may form aggregates with other whey proteins or with serum k-casein. The extent of association of denatured whey protein with casein micelles is markedly dependent on the pH of the milk prior to heating, levels of soluble calcium and phosphate, milk solids concentration and type of heating system (water bath, indirect or direct heating system). Heating at pH values less than 6.7 results in a greater quantity of denatured whey proteins associating with casein micelles, whereas, at higher pH values, whey protein\k-casein complexes dissociate from the micelle surface, apparently due to dissociation of kcasein (Singh & Fox, 1985, 1986). Recent studies (Oldfield, Singh, & Taylor, 1998a; Corredig & Dalgleish, 1996) suggest that indirect heating tends to result in greater proportions of b-Lg and a-La associating with micelles than when using a direct heating system (e.g. direct steam injection). The mechanism of b-Lg denaturation and their association with casein micelles in milk systems have been proposed recently by Oldfield et al. (1998b) who suggested that at least three possible species of denatured b-Lg that could associate with micelles; (i) unfolded monomeric b-Lg, (ii) self-aggregated b-Lg and (iii) b-Lg/a-La aggregates. The relative rates of association of these species with the casein micelles depend on temperature and heating rate, which in turn affect the relative rates of unfolding and the formation of the various aggregated species. The b-Lg aggregates, which have been shown to be stiff and rod-like (Griffin, Griffin, Martin, & Price, 1993), would protrude from the micelles surface, providing steric effects for further bLg association. In addition, these aggregates may have their reactive sulphydryl group buried within the interior of the aggregate, and therefore unavailable for sulphydryl–disulphide interchange reactions with micellar kcasein. In contrast, unfolded monomeric b-Lg molecules would be expected to penetrate the k-casein hairy layer with greater ease and have a readily accessible sulphydryl group. The formation of unfolded b-Lg may be promoted by prolonged heating times at low temperatures, or by heating at a slower rate to the required temperature. However, at high temperatures and fast heating rates, all whey proteins begin to unfold in a relatively short period of time, thus presenting more opportunity for unfolded monomeric b-Lg to selfaggregate, which consequently is likely to associate with the casein micelles less efficiently.
3. Rennet coagulation properties of heated milks The effects of whey protein denaturation and association with casein micelles on rennet coagulation properties have been studied (Dalgleish, 1990; Waungana,
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Singh, & Bennett, 1996). It is believed that the heat treatment of milk results in longer rennet coagulation times (RCTs) and reduced strength of rennet gels (Morrissey, 1969; Banks, 1990; Dalgleish, 1992). Dynamic testing, which involves an oscillatory applied strain or stress, can provide very useful information on the gel formation process. The rheological parameters, such as elastic storage modulus (G0 ), viscous loss modulus (G00 ) and loss tangent characterising the rennet gels may be derived from such measurements. Typical changes in G0 with time during the course of renneting skim milks (at 321C) preheated at a range of temperatures (80–1401C for 4 s) are shown in Fig. 1. Gelation time (defined as the time when storage modulus reached >1 Pa) increases and G0 decreases as the severity of heat treatment increases (Fig. 1; Waungana et al., 1996). Milks heated at 1401C for 4 s fail to form a gel within 120 min. The extent of denaturation of b-Lg was plotted as a function of gelation time or G0 (Waungana et al., 1996; Fig. 2a). It is clear that increasing the level of denaturation of b-lactoglobulin to about 60% has little influence on gelation time, but gelation time increases very markedly with further increase in b-Lg denaturation. In contrast, the G0 of rennet gels is much more sensitive to the presence of denatured b-lactoglobulin than the gelation time. For example, the G0 values of rennet gels decrease almost linearly with increase in the degree of denaturation of b-lactoglobulin from 10% to 65%. Similar trends are observed for the relationship between the extent of b-lactoglobulin association with the micelles and gelation time, although increases in gelation time are observed at lower levels of blactoglobulin association than those observed for the
denatured protein (Fig. 2b). As the amount of blactoglobulin associated with micelles increases from 0% to 50%, there is an almost linear decrease in G0 : Milks containing >50% b-Lg associated with micelles have very low values of G0 : The decrease in the rate of gel development and final G0 values could be attributed to the association of whey protein aggregates with casein micelles surfaces through formation of a b-Lg/k-casein complex which may protrude from the micelle surface, as discussed earlier. This association would affect the close approach of the reactive sites formed on the micelles by the action of rennet. After k-casein conversion into para-k-casein, aggregation would occur mostly between micelles not fully covered with b-Lg, resulting in formation of fewer bridges with fewer and weaker bonds. A considerable proportion of b-Lg aggregates remains in the serum after heating. The role of these aggregates in rennetinduced gel formation is not clear, but they are likely to interfere with the aggregation and gel formation processes, possibly reducing the number and strength
Fig. 1. The effects of temperature for a holding time of 4 s on the storage modulus (G0 ) as a function of time after addition of rennet in skim milk. Heating temperatures: unheated milk (J), 80, (K), 90 (&), 100 (’), 110 (m), 120 (n), 130 (+) or 1401C ( ). All samples were renneted at pH 6.5 at 321C. Taken with permission from Elsevier Science, Waungana et al. (1996) Food Research International, 29, 715– 721.
Fig. 2. (a) Relationship between heat denaturation of b-lactoglobulin and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was >1 Pa) (’) in normal skim milk. (b) Relationship between heat-induced association of b-lactoglobulin with the casein micelles and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was >1 Pa) (’) in normal skim milk. Data from Waungana et al. (1996).
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of the contacts between adjacent aggregate chains within the gel network. A very large decrease in gel stiffness at high levels of b-Lg denaturation (>65%) could be attributed to the marked decrease in the extent and rate of k-casein hydrolysis, possibly due to chemical damage to k-casein molecules caused by severe heat treatments (Leaver, Law, Horne, & Banks, 1995).
4. Methods for improving the rennet coagulation properties of heated milks The rennet coagulation properties of heated milk may be at least partly restored either by (i) acidification of heated milk to pH values below 6.2, (ii) acidification of heated milk to low pH values (B5.5) followed by reneutralization to the neutral pH range, often termed as pH cycling, or (iii) heating at elevated pH combined with pH cycling and CaCl2 addition. Acidification of heated milks (70–1401C) to below 6.2 results in shorter RCTs and increased gel firmness, with a maximum firmness at pH 6.2, probably by reducing charge repulsion and increasing ionic calcium concentrations (Singh, Shalabi, Fox, Flynn, & Berry, 1988; Lucey et al., 1993b). Singh et al. (1988) heated milk at 901C for 10 min before adjusting the pH from 6.6 down to values in the range 5.1–6.3, holding at the low pH for 2 h at 201C and then readjusting to pH 6.6 before measurement of RCT at 301C. They reported marked reductions in RCT of such pH-cycled milk. van Hooydonk, Koster, and Boerrigter (1987), who used more severe heat treatment (5 s at 1401C, direct UHT), observed reductions in RCT of about 14% upon acidification to pH 5.5 followed by re-neutralization after 24 h. Lucey, Gorry, and Fox (1993a) reported reductions in RCT for milks heated at temperatures up to 1001C for 10 min. More severely heated milk (1201C for 10 min) did not coagulate even after acidification to low pH values and re-neutralization to pH. In a more recent work (Waungana, 1995), it was shown that in skim milk heated at temperatures in the range 80–1401C for 4 s, gelation time decreases in all samples upon pH cycling (skim milk acidified to pH 5.5, held for 2 h, and subsequently neutralised to pH 6.5) compared with the control (skim milk adjusted to pH 6.5); the decrease in gelation time is greatest when the milks are heated either at 1001C for 4 s prior to renneting (Fig. 3a). At 1401C, there is only B10% reduction in gelation time. pH cycling also affects the G0 values of rennet gels formed from heated milks (Fig. 3b). In the unheated (control) milk, pH cycling has an adverse effect on G0 and this persists to a smaller extent in the sample heated at 801C. The most significant increase in G0 is observed in the sample heated at 1001C; samples that had received more severe heat treatments
prior to renneting also form slightly firmer gels upon renneting after pH cycling, although the effect is not as marked as that observed at 1001C. Singh et al. (1988) reported that rennet gel formed from milk heated at 901C for 10 min, acidified to pH 5.5 and re-adjusted to pH 6.6, is slightly firmer than that from the original, unheated milk. Lucey et al. (1993a), however, found that acidification of heated milk (1001C for 10 min) to pH values of B5.5 before re-neutralization to pH 6.7 results in greatly improved RCT but gel firmness is not completely restored to that of raw milk. The concept of pH cycling as a means of improving the rennet coagulation properties of heated milks was initiated by experiments performed by Banks and Muir (1985). They showed that sterilised starter milk, which was acidified by lactic acid and subsequently neutralised by addition to the cheese milk, is fully incorporated into a rennet gel. They suggested that this was due to the disruption of micelles at low pH, which would make the hidden k-casein liable to conversion and cause the casein
Fig. 3. (a) Gelation times of skim milks heated at temperatures in the range 80–1401C for 4 s (&). Heated skim milks adjusted to pH 5.5, left for 2 h at 201C, and re-adjusted to 6.5 before renneting (’). All samples were renneted at pH 6.5 at 321C. (b) Storage modulus of skim milks heated at temperatures in the range 80–1401C for 4 s (&). Heated skim milks adjusted to pH 5.5, left for 2 h at 201C, and readjusted to 6.5 before renneting (’). All samples were renneted at pH 6.5 at 321C.
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micelles to participate in the gel formation process. It has since been shown, however, that the pH cycling procedure does not result in the release of any additional casein macropeptide (CMP) (van Hooydonk et al., 1987) or 12% TCA soluble N-acetylneuramic acid (NANA) (Singh et al., 1988) during the primary enzymic phase of the coagulation of heated milks. van Hooydonk et al. (1987) suggested that acidification to pH 5.5 solubilises a substantial amount of both the original colloidal calcium phosphate (CCP) and the heat-induced Ca/P complexes. Subsequent neutralization probably leads to the reformation of Ca/P complexes with composition and properties more like the original CCP, and that this may be the most important factor responsible for the improved rennet coagulation properties of heated and pH cycled milks. Singh et al. (1988) reported that pH cycling affected the rennet coagulability of heated milk by increasing the [Ca2+], (an effect similar to that obtained by small additions of CaCl2), and thus promoting aggregation of renneted micelles. Overall, it may be concluded that pH cycling only partly restores the rennet coagulation properties of heated milks. The extent to which these are restored is dependent on the severity of the heat treatment employed. Gelation time appears to be more sensitive to pH cycling than gel firmness. The coagulation properties of milks heated at temperatures of about 1001C for 4 s appear to be mostly restored by pH cycling but the properties of more severely heated milks (X1201C) are less affected. Imafidon and Farkye (1993a) reported that milk adjusted to pH 7.3 before heating (911C for 16 s) followed by acidification to pH 6.4 before renneting has a mean RCT of B9 min. After acidification to pH 5.5 and overnight storage at 41C before re-neutralization to pH 6.4 (pH cycling) RCT decreases by B50%. They also reported faster curd-firming rates and slightly firmer gels in samples that are pH cycled to pH 6.4 than in those directly acidified to the same pH. However, when milk is pH cycled to its original pH of 6.6 before setting, the curds formed are very weak. Waungana (1995) found that milk adjusted to pH 7.3, heated at 1401C for 4 s and then pH cycled has the shortest gelation time and highest G0 values of all the heat treated milks. These values, however, are still far short of those of an unheated control, and that the rennet coagulation properties of severely heated milks are not satisfactorily restored as a result of increasing the pH before heating followed by an acidification and re-neutralisation step. The accelerating effects of calcium chloride on the rennet coagulation of unheated milk are well documented (Mehaia & Cheryan, 1983; van Hooydonk, Hagedoorn, & Boerrigter, 1986; Singh et al., 1988) and CaCl2 is frequently added to cheese milk to enhance rennet coagulation during commercial cheese manufacture.
Waungana (1995) showed that addition of CaCl2 results in a decrease in gelation time and formation of stronger gels in both the heated (1401C for 4 s) and unheated milk systems (Table 2). G0 values increase by 17% and 28%, respectively, upon addition of 1 and 2 mm CaCl2 to the control milk. Added CaCl2 has a major effect on G0 of heated milks; G0 values increase from 2 Pa (no CaCl2) to 86 Pa at 2 mm CaCl2 addition. pH cycling of the heated milks before addition of CaCl2 causes a further reduction in gelation time, but has a negligible effect on G0 : The decrease in gelation time and increase in G0 observed in both the heated and unheated milk samples upon addition of CaCl2 is probably due to increased aggregation of renneted micelles caused by increased [Ca2+], arising largely from the charge reduction on casein micelles by binding of Ca2+ ions, thus reducing electrostatic resistance to aggregation of casein micelles. Moreover, van Hooydonk et al. (1986) showed that the degree of k-casein conversion at the coagulation point decreases with an increasing amount of CaCl2 concentration and this may also account for the reduction in gelation time.
5. Effects of heat treatment on rennet coagulation properties of UF concentrated milks It is generally accepted that there is a very steep increase in the rate of curd firming and strength of rennet gels made from UF concentrated milks (Schmutz & Puhan, 1978; Green, 1990) and that heat treatment of UF milk lowers the strength of rennet gels (Sharma, Hill, & Goff, 1990; McMahon, Yousif, & Kalab, 1993; Guinee, O’Callaghan, Pudja, & O’Brien, 1996; Waungana et al., 1996). Recent work by Waungana, Singh, and Bennet (1998) showed that significant decreases in both the rate of gel firming and final gel firmness occur upon heating UF
Table 2 Effect of CaCl2 addition, heat treatment and pH cycling on rennet coagulation propertiesa Sample
pH cycle
CaCl2 (mm)
Gelation time (min)
Storage modulus G0 (Pa)
Control Control Control 1401C (4 s) 1401C (4 s) 1401C (4 s) 1401C (4 s) 1401C (4 s) 1401C (4 s)
F F F F F F 5.5–6.5 5.5–6.5 5.5–6.5
0.00 1.00 2.00 0.00 1.00 2.00 0.00 1.00 2.00
7.0 3.5 2.0 62.5 8.0 4.5 54.5 6.5 3.5
122 147 170 2 58 86 10 57 79
a pH cycling: pH adjusted to 5.5, held for 2 h and pH readjusted to 6.5. Samples were renneted at 321C at pH 6.5.
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concentrates (3 concentration) at temperatures between 801C and 1401C for 4 s. Denaturation of b-Lg up to B75% has little effect on the onset of gelation in UF skim milk, but higher levels of denaturation decrease gelation times (Fig. 4a). However, G0 decreases almost linearly as the degree of denaturation of b-Lg increases up to B75% with a further increase in denaturation causing a sharper decrease in G0 (Fig. 4b). Similar trends are observed between the levels of b-Lg association with the micelles and G0 or gelation time (Fig. 4b). It is clear that the concentration of milk by UF counteracts the effects of heat treatment on the rennet coagulation properties of milk since rennet gels may still be formed from severely heated UF concentrates. The gels formed from the UF concentrates are much firmer than the corresponding unconcentrated milks at comparable levels of b-Lg denaturation and association. This is probably because UF of milk increases the protein concentration and lowers the volume of the
aqueous phase, which lowers the mean free distance between casein micelles, and so increases the aggregation velocity of renneted micelles. This coupled with the fact that in UF concentrates there is a higher level of colloidal calcium which results in coagulation being initiated at a lower extent of k-casein hydrolysis (Garnot & Corre, 1980; Dalgleish, 1981; McMahon et al., 1993). 5.1. High heat treatment of milk before or after UF concentration Waungana et al. (1998) studied the effects of UHT treatment before or after UF concentration and reported that UHT treatment of milk prior to concentration by UF results in concentrates that have slower curd firming rates and longer times to reach a given G0 value as compared to the concentrates where UHT treatment is given after UF (Table 3). It is not clear, however, why the G0 values of the rennet gels formed from milk that was heated before UF are considerably lower than those from milk that is concentrated before heat treatment. It is possible that the nature of the b-lactoglobulin/k-casein complexes and the way in which they associate with casein micelles upon heating of UF concentrates is different to that when normal milk is heat treated. In addition, differences in the mineral equilibria between the concentrated and unconcentrated milks may influence heat-induced changes in minerals and proteins during heating. Waungana et al. (1998) also showed that pH cycling of heated 3 UF concentrate delays the onset of gelation and greatly reduces the curd firming rate and final G0 values compared to that of heated but not pHcycled concentrates. When pH cycling of heated milk is carried out before UF, the onset of gelation is further delayed and the curd firming rate is much lower compared to that of 3 UF concentrate prepared from heated milk that is not pH cycled (Table 3).
Table 3 Effect of high heat treatment (1401C for 4 s) before or after ultrafiltration and pH cycling on rennet coagulation properties of skim milka
Fig. 4. (a) Relationship between heat denaturation of b-lactoglobulin and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was >1 Pa) (’) in 3 UF concentrated skim milk. (b) Relationship between heat-induced association of blactoglobulin with the casein micelles and storage modulus (G0 ) (&) or gelation time (defined as the time when the value of G0 was > 1 Pa) (’) in 3 UF concentrated skim milk. Data from Waungana et al. (1996).
Sample
Gelation time (min)
Storage modulus G0 (Pa)
Skim milk UF concentrate Heated skim milk Heated UF concentrate Heated milk concentrated by UF pH-cycled heated milk pH-cycled heated UF concentrate Heated milk, pH-cycled and then UF concentrated
10.5 19.0 No gel 21.0 25.5 58.0 28.0 31.0
140 2354 No gel 312 186 9 139 85
a pH cycling: pH adjusted to 5.5, held for 2 h and pH readjusted to 6.5. Samples were renneted at 321C at pH 6.5.
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When heated milk is pH cycled before UF, there is presumably an increase in [Ca2+] which is considered to be responsible for its improved rennet coagulation properties (Singh et al., 1988; Lucey et al., 1993a). During subsequent UF treatment of this milk, this [Ca2+] is presumably lost in the milk permeate, resulting in poorer rennet coagulation properties (possibly due to reduced rate of aggregation in the secondary phase) of such treated milk compared to that which is not pH cycled. Since pH cycling of the concentrates after UF and heat treatment do not improve their rennet coagulation properties, it appears that the explanations are more complex than proposed above, and probably involves changes in the equilibrium of mineral salts (especially calcium), state of micellar calcium phosphate and the nature of the associations between b-lactoglobulin and k-casein. Further research is needed on the influence of technological treatments such as heating, UF and pH cycling before renneting on the equilibrium of milk salts, as well as on the nature of associations between b-lactoglobulin and k-casein.
6. Cheese manufacture from heated milk In addition to the effects of heat treatment on rennet coagulation and gel stiffness, the detrimental effects of casein–whey protein interactions during heating on syneresis of rennet curd are well documented (Walstra, van Dijk, & Geurts, 1985). The association of whey proteins at the micelle surface sterically impedes the fusion of rennet-altered micelles resulting in less shrinkage of the paracasein network. There is also an increase in the water binding capacity of the paracasein– whey protein complex. There have been a few studies on the manufacture of cheese from milks heated under fairly mild conditions to give whey protein denaturation levels of up to B35% (Marshall, 1986; Cheshire cheese; Guinee, Fenelon, Mulholland, O’Kennedy, O’Brien, & Reville, 1998; reduced fat (B16–18%) Cheddar cheese). Because of the inferior coagulation and syneresis properties of high heat-treated milk, higher set-to-cut times during cheesemaking are generally observed. Curds from heat treated milks tend to be soggy, ragged in appearance with poor matting ability. There is a poor curd fusion of the network, with more porous matrices. Increasing heat treatment of milk results in an increase in concentration of whey proteins, moisture, moisture in non-fat substances (MNFS) in the cheese. There is a lower level of calcium and phosphorus in cheese made from heated milk, which is essentially due to the reduction in cheese dry matter and increase in the level of whey proteins. Acidification or pH-cycling approach has been used in the manufacture of cheese from severely heated milks by a number of workers (Banks, Stewart, Muir, & West,
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1987; Banks, 1988, 1990; Imafidon & Farkye, 1993b). Banks et al. (1987) made Cheddar cheeses from milks heated at 1101C for 60 s (66% whey protein denaturation) by changing the pH of milk to 5.8 prior to renneting. With this procedure, the yield increase of 4.7% (on a dry solids basis) was possible. These cheeses showed minor defects with respect to moisture retention and texture, but a more serious defect was the development of an intense bitter off flavour during ripening. This bitterness may be eliminated, to a large extent, by a considerable reduction in the level of rennet used in cheese manufacture (Banks, 1988). Further studies by Banks (1990), Banks, Law, Leaver, and Horne (1993) involved manipulations of pH and processing conditions to achieve whey protein incorporation with a process more similar to traditional cheddar methodology. Milks were heated at 851C or 901C for 1 min and pH adjusted 6.2 prior to renneting. Rennet levels were reduced to 90% of that in the control. On a dry solids basis, the yield increase at 851C and 901C was 3.61% and 3.93%, respectively. Cheddar cheese flavour intensity was generally reduced in cheeses prepared from high heat treatment milks. The level of off-flavour development, predominantly described as bitter, is higher in cheeses made from heated milk, particularly after nine months maturation. The textures were found to be comparable with samples produced from pasteurised milk. The difference in the overall acceptability of the cheese are therefore related to the flavour defect, and other varieties of cheese which are not extensively ripened, e.g. Cheshire and acid set Mozzarella have been made successfully from heat treated milk. Imafidon et al. (1993b) made Cheddar cheeses from milk heated at 911C for 16 s, followed by pH cycling (i.e. acidified to pH 5.5, held overnight at 41C and then neutralised to pH 6.2) before rennet addition. Cheese made from high heat treated milk had higher moisture content (40.5%) than that made from normal pasteurised milk (38.1%). On a dry matter basis, the protein content of cheese made from heated milk was higher (41%) compared with 38% for cheese made from normal milk. This represented B2.6% more protein recovery in cheese made from the high-heat treated milk. No information was provided on the texture and sensory properties of the cheeses. Limited information is available on the effects of heat treatment of cheese milk on the proteolysis during cheese ripening. Experiments by Calvo, Leaver, Law, and Banks (1992) on the ripening of Cheddar cheese made from severely heated milks (1101C for 60 s with pH adjustment to 5.8), showed that the breakdown of bcasein is more extensive than in cheese made from milk heated at 631C for 30 min. No evidence of whey protein breakdown during maturation was found. In contrast, Benfeldt, S rensen, Elleg(aard, and Petersen (1997)
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reported that cheeses (Danbo 45+) made from heated milks show decreased proteolysis of b- and as2 -caseins, which is attributed to reduced plasmin activity in the cheese as a consequence of the thermal inactivation of plasminogen activation system and heat-induced interactions between the plasminogen activation system and b-Lg. Heat treatment does not affect the proteolysis of as1 -casein and only alters slightly the breakdown of para-k-casein. The effects of heat treatment of milk combined with UF on the manufacture of semi-hard cheeses, were studied by Guinee et al. (1995). Standardised milks, heated at 72–1001C to denature 5–63% of the whey protein, were ultrafiltered to yield retentates with protein and fat levels of B18.5% and 14%, respectively. Retentates were converted into semi-hard cheeses using specialised coagulation and gel-cutting equipment, with scalding and further syneresis carried out in conventional cheese vats. High heat treatment of milk necessitated a reduction in set pH, an increase in set temperature and an increase in curd scalding temperature. Increasing levels of whey protein denaturation result in cheeses having higher moisture, S/M and whey protein levels, lower ex-brine pH values and lower rates of pH increase during a 182 day ripening period. Cheeses with high levels of denatured whey proteins also show poorer curd fusion and lower yield (fracture) values during ripening. Higher levels of denatured whey proteins in cheese were associated with a higher degree of primary proteolysis, as revealed by PAGE, levels of WSN and 12% TCA N. However, heat treatment resulted in slower formation of small peptides (B1000 Da) and free amino acids. Examination of various cheeses by SEM showed that as the intensity of heat treatment to which the milk was subjected increases, the protein matrices of cheese become coarser and less homogeneous/continuous in appearance and contain numerous small holes and/or cracks. However, there were no significant differences between the extent of heat treatment and appearance, flavour body and overall impression of the cheeses, as judged by trained cheese sensory panel.
7. Conclusions Considerable progress has been made towards understanding how heat treatment of milk affects rennet coagulation times, rheological and physical properties of rennet gels. However, much less is known about the effects of heating on texture, flavour, functionality and ripening characteristics of cheeses. Heat treatment of milk combined with UF and pH manipulations may offer new possibilities for the production of soft cheeses, semi-hard cheeses and novel hybrid cheeses with varying textures, flavours and functionality. However, there still
remains a great deal to be understood on the complex interactions of proteins, fat and minerals during these combined processes. It must be emphasised that further research in this area should be approached more with the aim of developing novel products as opposed to trying to emulate the flavours and textures of existing products as is the current trend.
Acknowledgements We are grateful to the ministry of Foreign affairs for a scholarship to A.W. and, to David Oldfield for useful discussions.
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