The effect of UHT processing and storage on milk proteins

C H A P T E R

10 The effect of UHT processing and storage on milk proteins Hilton C. Deeth School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, Australia

Introduction The high processing temperatures and long storage times at ambient temperature of ultrahigh-temperature (UHT)-processed milk put huge demands on the stability of a reasonably fragile biological product. The temperature-time conditions of both processing and storage vary considerably. Ambient temperatures of storage (and transport) can vary from well below 0°C in cold countries to
50°C in tropical zones and some storage facilities. When these variable conditions are considered in conjunction with the variability in the raw material involved, it is not surprising that the proteins in the product can undergo several changes before the product is consumed. It is essential that the nature of these changes is understood so that those detrimental to the stability and nutritive value of the product can be minimized. The changes in proteins during processing and storage are caused by chemical reactions, physicochemical interactions, and enzymatic activity. However, changes can also be caused by microbiological contamination; this source of change is not addressed in this chapter. UHT milk has a shelf life of 6–9 months although some companies are placing a 12-month use-by date on their product. Given the numerous ways in which the product can deteriorate during storage, even under ideal conditions, as outlined in this chapter, it is apparent that the product is outside its comfort zone when stored for more than perhaps 6, and certainly 9, months. However, it is noted that the legal expiry date for UHT milk in some countries is less than 6 months [e.g., 90 days (Pizzano et al., 2012)]. Several different UHT-processed milk products are now produced. This chapter primarily concerns UHT milk, but other products are mentioned as some of the changes in the milk proteins are more relevant to some of these products. Good examples of these are UHT concentrated milks, flavored milks, and lactose-hydrolyzed milks.

Milk Proteins https://doi.org/10.1016/B978-0-12-815251-5.00010-4

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10. The effect of UHT processing and storage on milk proteins

The UHT process The UHT process, that is, the heating of milk at high temperature for a short period of time followed by aseptic packaging, was introduced to produce a shelf-stable milk that had superior properties to in-container sterilized milk. The thermal treatment is in the range 135–150°C for holding times of 1–10 s (Deeth and Smithers, 2018). These conditions have been developed to achieve what is known as “commercial sterility,” meaning that no microorganisms will grow in the product under the normal conditions of storage (Burton, 1988; Lewis and Heppell, 2000; Deeth and Lewis, 2017). The minimum heating conditions for producing a commercially sterile product are generally recognized to be 135°C for 10.1 s or equivalent, which is sufficient to cause a 9-log reduction of thermophilic bacterial spores. These conditions are equal to a B* of 1, where B* is a bacteriological index (Kessler, 1981). UHT processes should be equivalent to a B* of
1.0. A related bacteriological index is the F0, which is sometimes used in UHT processing but is more commonly used in canning technology. It refers to a 12-log reduction of spores of Clostridium botulinum and is equivalent to heating at 121.1°C for 1 min. A UHT process should be equivalent to an F0 of
 3.0. The bacteriological indices differ from the chemical index, C*, whereby a C* of 1.0 relates to the heat input that would cause a 3% reduction in the vitamin thiamine. It is more of a theoretical construct than a practical guide as virtually no one measures the loss of thiamine. The heating conditions for a C* of 1.0 are 135°C for 30.5 s or equivalent. A general recommendation is that the C* of a UHT process should be  1.0. Thus, it is recommended that any UHT heat treatment should be equivalent to a B* of
1.0 and a C* of  1.0; however, most commercial UHT plants operate at B* and C* values that are considerably greater than 1.0 (Tran et al., 2008), with the conditions of many considered to be excessive (Cattaneo et al., 2008). The chemical index, C*, is useful for juxtaposing bacterial and chemical changes, but it provides only a guide to the specific chemical changes caused by a heat treatment. As each chemical change follows its own kinetics, the changes in individual components, such as the denaturation of the whey proteins, can be estimated only by taking into account the individual kinetics. There are two main broad types of UHT heating: direct and indirect. Direct heating involves the direct mixing of the milk with saturated steam (free of entrained water droplets) (Lewis and Heppell, 2000) and the subsequent removal of the added water in a vacuum flash vessel. In indirect processes, the heating medium, steam or hot water, heats the milk indirectly by conduction and convection through a stainless steel barrier in the form of a heat exchanger. Indirect processes, using either plate or tubular heat exchangers, are most commonly used. The main difference between the two processes that is relevant to their effects on milk proteins can be readily seen in their temperature-time profiles (Fig. 10.1A–C); direct processes heat and cool the milk to and from the sterilization temperature very quickly (<0.5 s), whereas the rate of heating in indirect processes is much slower. Because of the different kinetics involved in bacterial inactivation and chemical changes, the temperature-time profiles can provide considerable information on these changes. Of primary importance is the fact that high-temperature, short-time thermal treatments produce greater bacterial inactivation compared with chemical change than do lower temperature, longer time treatments. Thus, if a direct process and an indirect process had the same B*

387

The UHT process

160 140

Temperature (°C)

120 100 80 60 40 20 0

0

50

100

150

200

160

160

140

140

120

120

100 80 60

100 80 60

40

40

20

20

0

0 0

50

100

150

Time (s)

(B)

250

Time (s)

Temperature (°C)

Temperature (°C)

(A)

0

20

40

60

80

Time (s)

(C)

FIG. 10.1

Temperature-time profiles for (A) an indirect UHT plant with a 60 s preheat hold time, (B) a direct UHT plant with a 60 s preheat hold time, and (C) a direct UHT plant with no preheat hold time.

(i.e., they would cause the same bacterial inactivation), the direct process would produce considerably less chemical change (i.e., would have a lower C*) than the indirect process. As discussed later, this translates to, among other changes, lower release of volatile sulfur compounds and hence less cooked flavor production, in the directly treated milk. As an illustration of the different effects of indirect and direct UHT processes, the profiles shown in Fig. 10.1A and B have the same B* value (2.95) but different C* values; the indirect process, Fig. 10.1A, has a C* of 1.1, whereas the corresponding direct process, Fig. 10.1B, has a C* of 0.37.

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10. The effect of UHT processing and storage on milk proteins

Aseptic packaging of the milk sterilized by the UHT thermal process is an integral component of all UHT systems. It is this operation that enables UHT milk to be packaged without contamination and to achieve a long shelf life at ambient temperature (Robertson, 2002). The importance of aseptic packaging can be appreciated from the fact that the spoilage rate of UHT products is, in general, determined by the performance of the aseptic filling system. The system involves an aseptic tank (A-tank), into which the product is placed after sterilization and before being packaged, and a filling machine. The whole system requires absolute sterility, which includes the sterility of all contact surfaces after the holding tube, complete exclusion from the product of the air surrounding the filling machine, and a positive air pressure in the filling machine using sterile air. A further requirement of aseptic packaging is that the package into which the product is filled not only must be sterile and exclude microbiological contamination but also should exclude light and air (oxygen), which can damage the milk’s components including the proteins (Smet et al., 2009). Multilayered paperboard cartons meet these requirements fully, whereas plastic bottles and plastic pouches fully or partially meet these requirements, depending on the composition of the plastic used (Robertson, 2011, 2013). A third component, which is integral to most UHT systems, is the homogenizer. This ensures that any fat is thoroughly emulsified and does not separate from the rest of the product during storage. Homogenization causes the production of a new membrane, consisting predominantly of casein, around the disrupted fat globules (Garcia-Risco et al., 2002). This has implications for the stability of the product because these casein-enveloped milk fat globules can behave like large casein micelles (Stoeckel et al., 2016). Garcia-Risco et al. (2002) reported that the homogenization of milk before UHT treatment promoted whey protein denaturation and reduced protease levels. They considered that increased complex formation between κ-casein and β-lactoglobulin (β-Lg) and reduced proteinase activity could be responsible for reduced proteolytic degradation during storage.

Protein changes during processing and storage There are several indications that the proteins in UHT milk and milk products change during processing and storage. These indications include both physical, often visible, changes and chemical changes, which can be determined by chemical analysis of the product. A good example of a physical change during processing is the phenomenon of fouling, which largely involves deposition of proteins on heat exchanger surfaces, and a good example of a physical change during storage is the formation of a proteinaceous sediment. Chemical analysis of UHT milk during storage can be very telling. This was shown graphically by Al-Saadi and Deeth (2008) and Gaucher et al. (2008) in the form of reversed-phase (RP) highperformance liquid chromatography (HPLC) chromatograms. In each case, storage, particularly at elevated temperature (
40°C), changed the peaks for the individual proteins in the chromatograms from sharp and well separated at manufacture to very broad and converged and with lower overall intensity after storage. Similarly, changes were observed in the Fourier transform infrared spectra of UHT milk during storage (Grewal et al., 2017b,c). The chemical reactions of the proteins responsible for these changes and the physical changes resulting

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from these chemical reactions are explored in this section. As many of the changes are initiated during processing and proceed during storage of the product, they are not separated here according to the processing or storage stages. The changes in the proteins in UHT milk have been reviewed extensively. In addition to the coverage in books by Burton (1988), Lewis and Heppell (2000), and Deeth and Lewis (2017), reviews specifically covering aspects of this topic include those by Hill (1988), Harwalkar (1992), McMahon (1996), Nieuwenhuijse and van Boekel (2003), and Deeth and Lewis (2016).

Denaturation of whey proteins and subsequent protein-protein interactions The denaturation of proteins refers to the disruption of hydrogen bonds and hydrophobic interactions and the subsequent destruction of secondary and tertiary structures. In the context of the whey proteins during UHT processing, heat-induced denaturation commonly refers also to subsequent protein-protein interactions. Reported levels of denaturation usually refer to the reduction in the levels of acid-insoluble whey proteins. The major whey proteins in milk vary in their susceptibility to heat denaturation. The most heat sensitive are the immunoglobulins followed by, in order of decreasing sensitivity, bovine serum albumin (BSA), β-Lg, α-lactalbumin (α-La), and proteose peptones, the last being not affected by heat (Donovan and Mulvihill, 1987). Most UHT treatments completely denature immunoglobulins and denature most of the BSA; however, as these proteins are present at much lower concentrations than β-Lg and α-La, the effects of their denaturation and interaction with other proteins are small compared with those of β-Lg and α-La. In fact, the denaturation of the whey proteins in milk is dominated by the denaturation of β-Lg, which represents about 50% of the whey proteins, although α-La is also significant (Oldfield et al., 1998; Wijayanti et al., 2014, 2018). The whey protein denaturation process β-Lg is a globular protein that exists naturally as a dimer with a molecular weight of  36,000 Da at the normal pH of milk. When subjected to heat, β-Lg unfolds and dissociates into two monomers, each of which contains a disulfide bond and, importantly, an exposed (reactive) free sulfhydryl group, which is buried in the native structure. At temperatures above  70°C, this denaturation is irreversible, because the reactive monomer forms disulfide bonds with other β-Lg molecules, α-La molecules, and, significantly, also with κ-casein through thiol-disulfide interactions. Some interaction with the other cystine-containing casein, αs2-casein, may also occur (Corredig and Dalgleish, 1996a). In addition, the interactions involve hydrophobic bonding through the hydrophobic parts of β-Lg that are exposed by the denaturation. As α-La has no free dSH group, it reacts with β-Lg via thiol-disulfide interactions. At high temperatures, one of the disulfide bonds can be disrupted to release reactive free dSH groups, which can initiate and propagate irreversible aggregation in a similar manner to the naturally occurring free dSH in β-Lg (Chaplin and Lyster, 1986). BSA has a free sulfhydryl group and could take part in intermolecular interactions. However, Havea et al. (2001) reported that, when a mixture of β-Lg and BSA was heated at 75°C for 15 min, BSA formed homopolymers. Most of the BSA aggregated during heating to 75°C and remained inert

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during heating at higher temperatures, at which β-Lg formed intermolecular aggregates. On this basis, BSA may be involved to only a limited degree in intermolecular complex formation in UHT milk. An important factor that is related to the β-Lg denaturation process is the dissociation of κ-casein from the casein micelle. Anema (2017) reported that the percentage of the total κ-casein that dissociated from the micelle during the UHT processing of reconstituted skim milk (total solids 12%–15%) at pH  6.6 was 30%–60%. Therefore, the κ-casein with which denatured β-Lg interacts can be either free in the serum phase or part of the casein micelle. Whether the β-Lg reacts with the serum κ-casein before or after it dissociates from the micelle has been debated (Oldfield et al., 1998; Corredig and Dalgleish, 1999; Anema, 2009), although Anema (2008) showed that the release of κ-casein from the micelle preceded the denaturation of β-Lg. pH is a major factor in determining how much of the denatured whey protein remains bound to the κ-casein in the casein micelle and how much becomes associated with κ-casein in the serum phase (Corredig and Dalgleish, 1996a,b; Anema and Li, 2003). When milk is heated at pH  6.5, the denatured whey proteins preferentially attach to the casein micelle, whereas, at higher pH, > 6.8, most of the whey proteins are found in the serum attached to κ-casein (Kudo, 1980). The amount of attached whey proteins is very sensitive to small changes in pH. Oldfield et al. (1998) reported a maximum association of whey proteins with casein micelles of  55% during heating in a direct steam injection UHT pilot plant at temperatures from 70°C to 130°C. However, this appears to conflict with an earlier study by Corredig and Dalgleish (1996b), which reported that almost all of the whey proteins bound to casein micelles during heating in a water bath to 70–90°C at the natural pH of milk, 6.75–6.8. Oldfield et al. (1998) considered that the different results from those in their work could have been due to the much slower rate of heating. This explanation is consistent with the report that more whey proteins associate with the casein micelles during indirect UHT heating than during direct UHT heating (Corredig and Dalgleish, 1996b). Almost all of the β-Lg attached to the casein micelles in milk processed on an indirect plant but much less associated with the micelles in milk directly processed at the same nominal holding temperature and time. The heating method also influences the relative amounts of β-Lg and α-La that become attached to the micelle. Rapid heating, such as in direct UHT processing, results in a high β-Lg:α-La ratio, whereas slower heating, such as in indirect UHT treatments, results in a lower ratio, that is, more α-La and less β-Lg become attached (Mottar et al., 1989). The ratio is also influenced by the temperature of heating. Oldfield et al. (1998) found that, in the range 80–130°C, β-Lg was the first to associate with the casein micelles and was followed by α-La on prolonged heating. Curiously, at temperatures < 80°C, β-Lg and α-La attached to the micelles simultaneously. β-Lg is denatured to a lesser extent but α-La is denatured to a similar extent in concentrated milk compared with single-strength milk (McKenna and O’Sullivan, 1971). The denaturation rates of both β-Lg and α-La decrease with an increase in the level of nonprotein solids but increase with an increase in the protein content. It has been suggested that these effects cancel each other out for α-La but that the effect of the nonprotein components is greater than that of the proteins for β-Lg (Anema, 2009).

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Whey protein denaturation levels in UHT milk Table 10.1 shows some reported levels of undenatured whey proteins in commercial UHT milk produced in direct and indirect UHT plants. It is clear that, whereas a wide range of values has been reported, the values for direct milks are consistently lower than those for indirect milks (Morales et al., 2000; Elliott et al., 2003, 2005). The variation in the results largely reflects the different UHT processing conditions, but different methodologies may also contribute to the variability. The other inference from these data is that the levels of residual α-La are comparable with and sometimes higher than those of β-Lg. This reflects the greater stability of α-La, given that its concentration in milk is less than half that of β-Lg. The kinetics of the denaturation of β-Lg and α-La have been reported by several authors (Lyster, 1970; Dannenberg and Kessler, 1988; Anema et al., 1996; Reddy et al., 1999). Using the kinetic data of Lyster (1970) and Dannenberg and Kessler (1988) and the temperature-time profiles of the plants, Tran et al. (2008) calculated the denaturation percentages of β-Lg and α-La in milk processed by 22 commercial UHT plants. The percentages calculated using the data of Dannenberg and Kessler (1988) were higher than those based on the data of Lyster (1970). The denaturation of β-Lg was calculated to be close to 100% in the commercial indirect plants but to be 74%–92% in the direct plants. For α-La, the calculated denaturation percentages were 25%–90% for indirect plants and 27%–58% for direct plants. The denaturation percentages for β-Lg and α-La, calculated using the Lyster (1970) kinetics data, for the indirect and direct profiles shown in Fig. 10.1A and B, were 94% and 58% (indirect) and 82% and 25% (direct), respectively. Denaturation in the preheat and high-temperature sections of UHT plants During UHT processing, the extent of irreversible denaturation and the associated proteinprotein interactions of the whey proteins depend on the severity of processing in terms of temperature, the duration of heating, and the temperature-time profile of the UHT plant. As the denaturation of β-Lg commences at  70°C, much of the denaturation occurs in the preheat section of indirect UHT plants and in direct plants if a preheat holding step is included. This is illustrated in Table 10.2, which shows the calculated denaturation percentages of β-Lg and α-La in milk at three points (after the preheat section, after the sterilization holding tube, and after cooling) during processing on an indirect UHT plant with preheating at 90°C for 60 s, a direct plant with the same preheating, and a direct plant with no preheat hold. The temperature-time profiles of the plants are shown in Fig. 10.1A–C. The calculated B* of the three plants is the same, that is, 2.95. This shows that, where such preheating is used, TABLE 10.1 Acid-soluble whey proteins (mg/L) in commercial UHT milks UHT process type Analyte

Direct

Indirect

References

α-Lactalbumin

356  176 63–1210

1131  340 220–1036

Elliott et al. (2005) Andreini et al. (1990)

β-Lactoglobulin

190  52 142–154

1404  516 736–792

Elliott et al. (2005) Recio et al. (1996)

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10. The effect of UHT processing and storage on milk proteins

TABLE 10.2 Calculated denaturation levels (%)a of β-lactoglobulin and α-lactalbumin during UHT processing on indirect and direct UHT plantsb β-Lactoglobulin

α-Lactalbumin

Indirect (with preheat hold)

Direct (with preheat hold)

Direct (no preheat hold)

Indirect (with preheat hold)

Direct (with preheat hold)

Direct (no preheat hold)

At end of preheating including preheat hold (immediately before sterilization holding tube)

91

72

13

44

11

2

At end of sterilization holding tube

93

81

66

50

23

14

At end of cooling

93

82

67

58

25

16

Position in UHT plant

a

Based on kinetics of Lyster (1970). The temperature-time profiles are shown in Fig. 10.1A–C; B* for all plants, 2.95. Sterilization holding tube conditions: indirect, 140°C for 4 s; direct, 144°C for 4.05 s. Preheat hold conditions: 90°C for 60 s. b

most of the denaturation occurs prior to the sterilization holding tube; however, in the direct plant in which no preheat holding is used, most of the denaturation occurs in the sterilization holding tube. On the basis of the data in Table 10.2, the extent of the denaturation of β-Lg is not necessarily a good indicator of the severity of the overall heat treatment. This is significant as the Aschaffenberg turbidity test (Aschaffenburg, 1950), which gives an estimate of undenatured whey protein, is sometimes used an indicator of the total heat treatment that a UHT milk has received. Furthermore, the extent of the denaturation of β-Lg in some UHT plants, especially indirect plants, is often
95%, and hence, discrimination between plants on the basis of their heat input using this criterion could be difficult. Table 10.2 shows that the calculated extent of the denaturation of α-La is much lower than that of β-Lg and that, even under quite severe heating conditions, as in an indirect UHT plant with preheating at 90°C for 60 s, the overall level is not excessive. Because of this, the denaturation percentage of α-La has been suggested to be a better index of heat treatment than that of β-Lg (Reddy et al., 1999; Tran et al., 2008; Pizzano et al., 2012). The preheat conditions used in Table 10.2 reflect those that have been recommended for the inactivation of plasmin (Newstead et al., 2006; Anema, 2017). They may also be suitable for minimizing heat exchanger fouling. It should be noted that a preheat holding section is not normally included for the direct UHT processing of milk but is included in indirect plants (Tran et al., 2008; SPX, 2013). However, as discussed in the section on The plasmin system, a preheat holding section in all UHT plants could minimize or even prevent the development of plasmin-related defects. Measurement of whey protein denaturation The extent of whey protein denaturation is typically mentioned in terms of the percentage of the whey proteins that precipitate at pH 4.6 (Dannenberg and Kessler, 1988; Resmini et al., 1989). However, in practice, as the level of whey proteins before heat treatment is seldom

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known, the percentage denaturation cannot be determined. Consequently, the concentration of whey proteins that remain soluble at pH 4.6, that is, acid-soluble whey proteins, is usually measured. These proteins are determined by a range of methods with HPLC and electrophoresis-densitometry being the most widely used. Some authors have measured the concentration of whey proteins remaining in the supernatant after ultracentrifugation to remove the casein micelles (Oldfield et al., 1998; Anema, 2017). These whey proteins include native and native-like whey proteins and those complexed to κ-casein, which reassociate with the casein micelles and precipitate when milk is acidified to pH 4.6 (Anema and Li, 2015). The extent of denaturation can be determined from the concentration of the native and native-like whey proteins in the supernatant, which can be analyzed by nonreducing polyacrylamide gel electrophoresis (Oldfield et al., 1998). An alternative method for separating the casein from the whey proteins to assess the extent of denaturation is to precipitate the casein by saturating the milk with salt. This is the basis of the rapid method for determining the extent of whey protein denaturation in the whey protein nitrogen index (WPNI) test. This test was developed by the American Dairy Products Institute (2009) for grading skim milk powders into low-, medium-, and high-heat powders. The native whey proteins, which remain in the supernatant after removal of the casein, can be quantified by a protein analytical method such as the dye-binding method (Sanderson, 1970). Unlike the HPLC and electrophoresis methods, the total amount of undenatured whey proteins, not the amounts of the individual whey proteins, is measured. The test has not generally been applied to UHT milk; however, Oliveira et al. (2015) used the WPNI as a heat treatment indicator for UHT milk in Brazil. On the basis of the test, they rated 90% of 60 commercial UHT samples analyzed to be equivalent to medium heat and 10% to be high heat. Role in fouling of heat exchangers The denaturation of whey proteins, particularly β-Lg, during UHT processing plays a major role in the formation of fouling deposits in the first stages of heating; deposits formed in the later stages of heating, that is, in high-temperature sections, are largely mineral in nature (Burton, 1988). Several authors have reported good correlations between fouling deposit formation and β-Lg denaturation during the thermal processing of milk and cream (Hiddink et al., 1986; de Jong et al., 1992). For example, Grandison (1988) found a significant correlation (r ¼ 0.792) between plant run times and the weight of the deposit formed in the regenerative section of a UHT plant operating at 80–110°C. Interestingly, the deposit in the high-heat section was not correlated with run time. Several models of fouling have been developed, and many are based on the kinetics of the denaturation of β-Lg (de Jong et al., 2002; Grijspeerdt et al., 2004; Schutyser et al., 2008). de Jong et al. (1993) reported that such a model was valid up to 115°C. Others have reported the models to be valid up to 90°C (Grijspeerdt et al., 2004). The denatured/unfolded monomer of β-Lg appears to be largely responsible for the deposit formation ( Jeurnink et al., 1996; Mounsey and O’Kennedy, 2007). This is because this molten-state form of β-Lg is unable to associate with casein micelles or other whey proteins before the protein moves toward, and deposits on, the wall of the heat exchanger. Therefore, the longer is the time for which the unfolded state of β-Lg is present in the UHT plant, the greater is the deposit formation, or conversely, the shorter is the time for which the unfolded form is present, the longer is the run time of the plant (Grijspeerdt et al., 2004). This suggests

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that the unfolded form is “sticky” and readily attaches to the walls of the stainless steel tube or plate and forms a base for further deposit formation. For this reason, it has been proposed that the preheat conditions in a UHT plant should be designed to minimize the time spent by this nonaggregated, “sticky” form of β-Lg in the UHT plant. This accords with the reports of a reduction in fouling when relatively severe preheating conditions are used (Prakash et al., 2015). Role in flavor production in UHT milk Volatile sulfur-containing compounds have long been associated with the cooked flavor of UHT milk. The major compounds responsible are hydrogen sulfide, methane thiol, and dimethyl disulfide, the last being considered to be an oxidation product of methane thiol. The major source of these compounds in UHT milk is the denaturation of β-Lg although, in milk containing fat globules, the proteins of the milk fat globule also contribute. Higher levels of hydrogen sulfide and methane thiol occur in UHT whole milk than in UHT skim milk soon after processing (Al-Attabi et al., 2014). The mechanisms for the formation of these sulfur volatiles have been discussed by several authors including Walstra and Jenness (1984), Al-Attabi et al. (2009), and Zabbia et al. (2012). A major source of hydrogen sulfide is β-elimination of cysteine with the formation of dehydroalanine (Walstra and Jenness, 1984) and of methane thiol from a Strecker degradation of methionine via methional (Zabbia et al., 2012). The dehydroalanine formed from cysteine can take part in protein cross-linking. In relation to the role of the denaturation of β-Lg in cooked flavor production, Gaafar (1987) concluded that the threshold of cooked flavor corresponds to about 60% denaturation. This was reported to correspond to a hydrogen sulfide concentration of 3.4 μg/L and a reactive sulfhydryl concentration of 0.037 mmol/L. Reducing the effects of whey protein denaturation The addition of potassium iodate at 10–20 mg/L reduces fouling and increases UHT run times and also reduces cooked flavor development (Skudder et al., 1981). The potassium iodate oxidizes the heat-activated sulfhydryl groups of β-Lg and reduces or prevents the interaction of the “sticky” intermediate form with other proteins and the heated surfaces of heat exchangers. The use of iodate is not a practical solution to the fouling and flavor problems; the processed milk develops a bitter flavor on storage because the plasmin is not inactivated. A similar effect to that of iodate can be achieved with hydrogen peroxide. Al-Attabi (2009) found that hydrogen peroxide at 50 mg/L reduced fouling and increased the run time during the UHT processing of concentrated skim milk (15% total solids). Furthermore, low concentrations (10 or 50 mg/L) of hydrogen peroxide significantly reduced the level of sulfur volatiles in UHT milk. When hydrogen peroxide was added at 50 mg/L before or after processing, the UHT milk contained no detectable hydrogen sulfide or methane thiol by the second day after manufacture (Al-Attabi, 2009). Without the peroxide addition, these compounds are detectable for up to 10 days. The denaturation of whey proteins and the consequent fouling make the UHT processing of drinks containing high levels of whey proteins difficult. However, this problem can be alleviated by the addition of sodium caseinate, because of the chaperoning effect of the caseins on the whey proteins (O’Kennedy and Mounsey, 2006; Mounsey and O’Kennedy, 2010). In the author’s laboratory, the processing of a 2% whey protein concentrate (WPC) solution

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caused severe fouling, but the addition of 2% sodium caseinate to the 2% WPC enabled it to be successfully UHT processed on a laboratory UHT plant. Furthermore, sodium caseinate + WPC mixtures up to 5% + 5% were successfully processed. Another approach to heat processing whey proteins is to reduce the pH, as whey proteins are stable at low pH ( 3) (Kelly, 2018b). Hence, it is possible to produce shelf-stable, highprotein acidic beverages based on whey protein isolate (WPI) at these pH values. (WPI is better to use than WPC because, as the lipid content is very low, clear products can be obtained.) These products, which are popular with athletes and health-conscious consumers, are shelf stable for up to 6 months with processing at 120°C for 20 s being reported to provide longterm stability (Villumsen et al., 2015a). These conditions prevented the formation of visible aggregates during storage, which are largely caused by the presence of caseinomacropeptide (CMP) in WPI made from cheese whey (Villumsen et al., 2015b). A further approach to stabilizing whey protein is through microparticulation (Kelly, 2018a). This has been utilized in the production of a commercial shelf-stable acidic whey protein drink, Upbeat, containing 8% protein. Ultrasonication has also been used to increase the heat stability of whey proteins (Ashokkumar et al., 2009; Gordon and Pilosof, 2010). After a whey protein solution (4%– 15% protein) had been partially denatured at  90°C and then ultrasonicated for 5 s at low frequency (20 kHz), it was stable to heating at 130°C (Huppertz et al., 2018). The acoustic cavitation caused by the ultrasonication treatment disrupted aggregates and thereby reduced the particle size.

Proteolysis Proteolysis occurs in many UHT products; however, with good control of the quality of the raw milk and judicious selection of the processing conditions, it can be minimized or prevented. Proteolysis is mostly caused by proteases although some nonenzymatic proteolysis can occur during the heat treatment of milk. Enzymatic proteolysis can be caused by indigenous or exogenous proteases. The alkaline protease, plasmin, is the most important indigenous protease, but others such as cathepsins (Gaucher et al., 2009) have been implicated. Of the exogenous enzymes, the heat-resistant proteases produced in raw milk by psychrotrophic bacteria, particularly Pseudomonas species, are the most significant, but proteases produced by spore-forming bacteria such as Bacillus and Bacillus-like species can also be involved. Plasmin The plasmin system

The plasmin system in milk is a complex system that includes plasmin and its precursor plasminogen, as well as a plasmin inhibitor, a plasminogen activator, and a plasminogen activator inhibitor (Ismail and Nielsen, 2010). Plasmin, plasminogen, and plasminogen activator are associated with the casein micelles, whereas plasmin inhibitor and plasminogen activator inhibitor are present in the serum. This distribution is of relevance when products such as milk protein concentrate and micellar casein, which contain plasmin, plasminogen, and

396

10. The effect of UHT processing and storage on milk proteins

plasminogen activator but no inhibitors, are used in long shelf life products such as UHT products. The components of the plasmin system have different heat stabilities, which makes the interpretation of the effect of heat treatments interesting. For example, heating at  75°C for 15 s completely inactivates plasminogen activator inhibitor and causes 36% inactivation of plasmin inhibitor. The sensitivity of the plasmin inhibitor is exemplified in the work of Newstead et al. (2006), in which the plasmin activity was greater in UHT-processed (direct steam injection to 140°C for 4 s) reconstituted skim milk when a preheat treatment of 80°C for 30 s was used than when a preheat treatment of 75°C for 15 s was used. Plasminogen activator has a similar or higher heat stability than plasmin and plasminogen, which explains why plasminogen is converted to plasmin during the storage of UHT milk if some remains in the milk after processing (Kelly and Foley, 1997; Prado et al., 2007). In fresh unheated milk, plasminogen is normally in higher concentration than plasmin. The plasminogen to plasmin ratio has been reported to be between 50:1 and 2:1. For example, Richardson (1983) reported up to nine times more plasminogen than plasmin in unheated milk, with a total level of plasmin of  0.3 mg/L. As plasminogen is converted to plasmin during the storage of heat-treated milks by the plasminogen activator, its level in milk and its heat stability are highly significant. Plasminogen, which can be activated to plasmin by plasminogen activator through the cleavage of a single peptide bond, is more heat stable than plasmin. Manji and Kakuda (1988) reported that 19% plasmin activity and 37% plasminogen remained in directly (steam infusion) processed (142°C for 5 s) milk, whereas no residual plasmin activity and 19% of the original plasminogen remained in indirectly (plate heat exchanger) processed milk (145°C for 3 s). Similarly, Cauvin et al. (1999) found that commercial UHT milks had virtually no remaining plasmin and attributed the significant proteolysis during storage to activated plasminogen. Plasminogen activators are even more heat stable than plasmin and plasminogen. Therefore, to prevent plasmin-induced proteolysis during the storage of UHT milk, plasminogen should be inactivated in the heat process. Milk with a high somatic cell count (SCC), from cows with mastitis, has an elevated level of plasmin (Bastian and Brown, 1996). In fact, Bikker et al. (2014) proposed the use of a plasmin test based on specific substrates as a potential diagnostic tool for detecting mastitic milk. As the SCC increases from <250,000 to > 1,000,000/mL, the concentrations of both plasmin and plasminogen approximately double (Politis et al., 1989). The stage of lactation also affects the level of plasmin in milk; late-lactation milk tends to have a higher level than early- and midlactation milks (Auldist et al., 1996). The order of susceptibility of the caseins to hydrolysis by plasmin is β ¼ αs2 > αs1 > κ. The peptides formed are largely gamma-caseins (γ1 and γ2). The specificity of plasmin and the peptides it releases differ from those of bacterial proteases; this is useful for diagnostic purposes (Datta and Deeth, 2003). Proteolysis induced by plasmin and its effects in UHT milk

Plasmin-induced proteolysis in UHT milk has been reported by several authors (Kelly and Foley, 1997; Cauvin et al., 1999; Huijs et al., 2004; Newstead et al., 2006; Rauh et al., 2014a,b; Anema, 2017; Malmgren et al., 2017). From these reports, it is now clear that such proteolysis occurs only if the heat treatment conditions are insufficient to inactivate plasmin. In addition, it is much more likely to occur in directly processed UHT milk than in indirectly processed

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UHT milk. The following examples illustrate the importance of appropriate heating conditions. • Newstead et al. (2006) used direct steam injection heating at 140°C for 4 s with preheat conditions ranging from 75°C for 15 s to 90°C for 60 s. With the lower intensity heat treatments (75°C for 15 s and 80°C for 15 and 30 s), extensive proteolysis occurred on storage, but, with preheat treatments of 90°C for 30 and 60 s, proteolysis was minimal during storage. • Rauh et al. (2014b) used preheating at 72°C for 180 s and direct steam infusion heating to > 150°C for < 0.2 s and found extensive proteolysis on storage; adjustment of the preheat conditions to 95°C for 180 s prevented proteolysis. • Anema (2017) processed reconstituted skim milk using preheating at 75°C for 15 s, or 80°C for 30 s, and direct steam injection heating to 143°C for 3 s and found extensive proteolysis on storage; adjustment of the preheat conditions to 90°C for 60 s prevented proteolysis. Furthermore, adjusting the high-heat conditions to 143°C for 30 s with a preheat of 75°C for 3 s also prevented subsequent proteolysis; however, heating at 143°C for 30 s is excessive as it is equivalent to a B* of 17 and an F0 of 78 for a direct process. • An interesting case was reported by Huijs et al. (2004). UHT milk was produced with innovative steam injection heating at 150–180°C for < 0.1 s. Although this process had an excellent bactericidal effect, it did not inactivate the plasmin, and the milk became bitter. To overcome this problem, a pretreatment of 80°C for 300 s was devised, which inactivated the plasmin and prevented proteolysis in the UHT milk (van Asselt et al., 2008). Because of the higher level of plasmin in mastitic milk, UHT milk made from high SCC milk shows more proteolysis than that made from low SCC milk (Auldist et al., 1996). Furthermore, it has been reported that indirectly processed UHT milk made from raw milk with high and low SCCs exhibited very low residual plasmin activity; however, when plasminogen was added, the high SCC milk showed the greatest tendency to form a plasmin-induced gel. This was attributed to an elevated level of plasminogen activator in the high SCC milks (Kelly and Foley, 1997) converting residual plasminogen to plasmin during storage. The major physical effects associated with plasmin-induced proteolysis are bitterness and age gelation, but its roles in sedimentation and fat separation have also been suggested. Evidence for the role of plasmin in age gelation is strong. Numerous papers have linked the level of residual plasmin in UHT milk and the tendency to gel during storage (e.g., Rauh et al., 2014a,b; Malmgren et al., 2017). Furthermore, the addition of plasmin or plasminogen has been shown to lead to gelation (Kohlmann et al., 1991). Proteolysis is considered to be a major factor in the development of age gelation in UHT milks. In some early reports of proteolysis-induced gelation, it is not entirely clear whether the proteolysis was due to plasmin or bacterial proteases. Therefore, it is essential to be able to eliminate the possibility of bacterial proteolysis in describing the gelation caused by plasmin. Several reports in which this condition is satisfied are available. • Newstead et al. (2006) found a strong correlation between plasmin-induced proteolysis and age gelation. No gelation occurred in milk samples that had undergone a preheat treatment of 90°C for 30 or 60 s. • Another example is that reported by Rauh et al. (2014b), in which the milk used was 1 day old, which was unlikely to contain bacterial proteases. The milk in which active plasmin

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10. The effect of UHT processing and storage on milk proteins

remained after manufacture was processed with a preheat of 72°C for 180 s and high heat, using steam infusion, at > 150°C for < 0.2 s. It was stored at 20°C. The residual plasmin and plasminogen activities were 30.9% and 14%, respectively, of their activities in the original milk. Several points in this paper are typical of plasmin-induced proteolysis. Plasminogen was converted to plasmin during storage of the milk, resulting in an increase in both plasmin activity and the rate of release of peptides. Bitterness occurred after 7 weeks and was intense after 8 weeks. Viscosity and opacity increased from 10 weeks, at which time a gel commenced to form on the bottom of the container. The gel increased in thickness and floating gel particles were visible at 11 weeks. The gel occupied the whole of the container at 16 weeks. It was soft and fragile, and syneresis was observed. Extensive proteolysis of β- and αs1-casein occurred. A creamy layer formed on top of the gelled milk at 11 weeks. Although such fat separation is seldom linked to proteolysis, there are now several reports indicating a relationship (e.g., Kohlmann et al., 1991; Hardham, 1998). This is consistent with a report by Zhang et al. (2007) that the inactivation of proteases reduces fat separation. Along with this milk, which gelled, a corresponding milk in which the preheat conditions were changed to 95°C for 180 s was processed. This milk showed no active plasmin and did not gel or develop proteolysis or bitterness during storage (up to 16 weeks). • A report by Malmgren et al. (2017) also concerned UHT milk in which plasmin was active and no bacterial proteases were present. The processing conditions were preheating at 74°C for 20 s and high-temperature heating using steam infusion or injection, at 140°C for 4 s. The UHT milks were stored for 6 months at 5°C, 22°C, 30°C, and 40°C. Virtually, no proteolysis was observed at 5°C as plasmin has very low activity at this temperature (Grufferty and Fox, 1988), but proteolysis, particularly of β-casein and, to a lesser extent, αs1-casein, was observed at higher temperatures. Gelation occurred in milks stored at 22°C after 5 months and in milks stored at 30°C after 4 months. No gelation occurred in milks stored at 5°C or 40°C, a result that was consistent with previous reports (Kocak and Zadow, 1985a; Kohlmann et al., 1988). Heat inactivation of plasmin

It is evident from this discussion that proteolysis can cause major quality defects in UHT milk, including bitterness, gelation, and fat separation. All these defects can be overcome by selecting appropriate heating conditions during UHT processing. This was mentioned for the denaturation of β-Lg because, during its denaturation, it interacts with plasmin via disulfide bonds and causes loss of proteolytic activity. Therefore, it is possible to inactive plasmin in the preheat section under certain conditions. Conditions to achieve this are discussed in the section on The plasmin system; a summary of the conditions that have been found to inactivate plasmin and to prevent plasmin-induced proteolysis is as follows: 90–95°C for 30–60 s (Newstead et al., 2006), 80°C for 300 s (van Asselt et al., 2008), 95°C for 180 s (Rauh et al., 2014b), and 90°C for 60 s (Anema, 2017). Bacterial proteases The interest in bacterial proteases is largely due to their heat stability and their ability to survive, at least partially, UHT heat processes. Given that UHT milk is a product with a long shelf life (up to 12 months) at ambient temperature, which can be > 30°C, only trace amounts

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of active protease can cause considerable proteolysis in UHT milk during storage. The major two effects of this proteolysis, as for plasmin-induced proteolysis, are age gelation and bitter flavor production; bacterial proteases can also cause casein micelle aggregation and sedimentation. The major proteases of interest are those produced by psychrotrophic bacteria, particularly Pseudomonas species. The nature of these proteases and their heat stabilities have been previously reviewed (Griffiths et al., 1981; Fairbairn and Law, 1986; Sørhaug and Stepaniak, 1997; Datta and Deeth, 2001) and are not covered in detail here. The main relevance of these enzymes is the changes they make to the proteins in UHT milk and the related changes to the properties of the milk. Production and characteristics of proteases of psychrotrophic bacteria

Extracellular proteases are produced by psychrotrophic bacteria such as Pseudomonads during the late log phase of growth. Most are alkaline metalloproteases of the AprX family with molecular weights of around 50,000 Da (Bagliniere et al., 2013; Mat
eos et al., 2015; Zhang et al., 2018). It is generally considered that they are produced after the total bacterial count reaches  105 cfu/mL although some strains have been reported to produce protease at 104 cfu/mL (Adams et al., 1976). However, as many authors have reported, there is considerable variation in the counts at which the production of protease occurs. Therefore, the total bacterial count is not always a good guide to the presence of extracellular enzymes as different bacteria have different propensities to produce these enzymes. Haryani et al. (2003) showed that significant quantities of enzymes were produced in milk with total bacterial counts of 105 cfu/mL, whereas some milks with a total plate count of
107 cfu/mL did not contain significant quantities. As a rule of thumb, raw milk for UHT processing should have a total bacterial count of less than 106 cfu/mL and preferably less than 105 cfu/mL. The specificity of the bacterial proteases toward milk proteins differs from that of plasmin, which has a strong preference for β-casein. In general, the proteases of psychrotrophic bacteria such as Pseudomonads have a preference for κ-casein, followed by β-casein, followed by αs1-casein. Most show little activity toward whey proteins but may hydrolyze them with prolonged exposure to high concentrations of the enzymes. The hydrolysis of κ-casein by these enzymes is similar to that of rennin, in that the first break is around the Phe105Met106 bond to produce a glycomacropeptide (GMP), sometimes referred to as caseinomacropeptide (CMP). However, the specificity of the bacterial proteases on κ-casein is not as defined as that of rennin. Miralles et al. (2003) reported that peptides released by Pseudomonas fluorescens protease included fragments 1–103, 1–104, 1–106, and 1–107. However, different specificities of bacterial proteases have been reported. This may have been due to the different bacterial species and strains tested, the presence of some active plasmin, or the different experimental conditions used (Nicodeme et al., 2005). Some authors have reported that β-casein was the most preferred: for example, Bagliniere et al. (2012) reported the order of preference of proteases from five strains of P. fluorescens to be β- > αs1- > κ- > αs2-casein. Proteolysis induced by bacterial proteases and its effects in UHT milk

Knowledge of the effects of bacterial proteases has been acquired through two main experimental approaches: studies of the effects on UHT milk of the growth of normal psychrotrophic bacteria in raw milk before processing; addition of cultures of proteolytic

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10. The effect of UHT processing and storage on milk proteins

bacteria previously isolated from milk or of proteases purified from such cultures. Some examples of the former are as follows: • Law et al. (1977) grew a proteolytic strain of P. fluorescens (B11) in raw milk until the total plate count (TPC) reached three different levels (8  105, 8  106, and 5  107 cfu/mL), and then, UHT processed the milks at 140°C for 3.5 s. The UHT samples produced from milks with 8  106 and 5  107 cfu/mL gelled after 8–10 weeks and 10–14 days, respectively. These samples showed extensive hydrolysis of κ-casein with the formation of “para-κcasein,” considerable hydrolysis of β-casein, and some hydrolysis of αs1-casein. The UHT sample from the raw milk with 8  105 cfu/mL did not gel but showed some degradation of κ-casein; neither β-casein nor αs1-casein was affected in this milk. • Richardson and Newstead (1979) added crude preparations of heat-stable proteases produced by P. fluorescens isolates B12 and B52 to raw milk. Culture supernatants were diluted from 1 in 1000 to 1 in 100,000 in the raw milk before UHT processing on an indirect plant at 138–141°C for 2.8 s. After 3 months of storage, proteolysis and bitterness were detected in all treatment milks, even those with the lowest concentration of added protease. It was concluded that, for UHT milk to have a shelf life of more than 3 months, it should contain no more than 1–2 ng protease/mL. • Snoeren et al. (1979) and Snoeren and Both (1981) UHT processed “good” milk (TPC ¼ 2.7  103 cfu/mL) and “bad” milk (TPC ¼ 2.07  106 cfu/mL) on direct and indirect UHT plants with holding tube conditions of 142°C for 4 s. The results, summarized in Table 10.3, show the much higher level of proteolysis in the “bad” milk than in the “good” milk and the resultant effects on the time to gelation. They further illustrate that indirect processing reduces the proteolysis and increases the time to gelation by increasing inactivation of the proteases present. There is also clear evidence in these data of plasmin-induced proteolysis, particularly in the directly processed milk. • Button (2007) stored raw milk at 4°C for 4 days (control) and 7 days (treatment) before UHT processing on an indirect plant at 140°C for 4 s. The TPCs of the control and treatment milks were 1.0  107 and 8.0  107 cfu/mL, respectively. Rapid proteolysis occurred in the treatment milk and it gelled at 15–19 days, whereas the control milk showed no proteolysis or gelation. It should be noted that this is an example of the TPC not giving a true indication of the proteolysis potential; it would normally be expected that milk with a TPC of 1.0  107 cfu/mL would contain significant protease activity.

TABLE 10.3 Effects of raw milk quality and processing mode on proteolysis and gelation in UHT milk Total proteolysis in 90 days at 28°C (increase in nonprotein nitrogen, mg/100 g)

Days to gelation at 28°C

Milk quality (TPC)

Direct

Indirect

Direct

Indirect

Good (2.7  10 cfu/mL)

34

7

91

>>91

Bad (2.07  10 cfu/mL)

40 (in only 20 days)

30

20

91

3

6

Data from Snoeren, T.H.M., van Riel, J.A.M., Both, P., 1979. Proteolysis during storage of UHT-sterilised whole milk. 1. Experiments with milk heated by the indirect system for 4 seconds at 142°C. Neth. Milk Dairy J. 33, 31–39; Snoeren, T.H.M., Both, P., 1981. Proteolysis during storage of UHT-sterilised whole milk. 2. Experiments with milk heated by the direct system for 4 seconds at 142°C. Neth. Milk Dairy J. 35, 113–119.

Protein changes during processing and storage

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• Bagliniere et al. (2012) grew nine Pseudomonas isolates “selected because of their differences in terms of proteolytic activity quantified using azocasein as substrate” in raw milk to 2.5  106 cfu/mL. These milks and an uninoculated control were UHT processed on an indirect plant at 140°C for 4 s. The control milk and four of the inoculated milks showed no sign of destabilization, whereas the other five inoculated milks were destabilized with the production of a “white sediment.” Particle size analysis showed that all stable milks had a monomodal size distribution with an average size of  0.2 μm (which corresponded to unmodified casein micelles), whereas the unstable milks showed populations of particles with sizes up to 700 μm, which the authors assumed were aggregates of casein micelles. The unstable milks also showed low phosphate test results; a similar finding was made by Gaucher et al. (2011). • Pinto et al. (2016) produced UHT milk from raw milk containing 3.0  106 cfu/mL of psychrotrophic bacteria and 8.0  105 cfu/mL of proteolytic psychrotrophs. Although the UHT treatment reduced the protease activity by 93.2%, proteolysis and associated sedimentation still occurred during storage of the UHT milk. • Stoeckel et al. (2016) UHT processed raw milk to which aliquots of milk cultures of one of three Pseudomonas species were added to give appropriate protease levels (apparent protease activities in the milks were from 0.01 to 0.35 pkat/mL, which relate to activities measured against the substrate azocasein). The UHT processing conditions used were indirect (tubular) heating with preheating at 90°C for 40 s followed by heating to 140°C and holding for 8.4 s (as discussed earlier, such conditions are excellent for ensuring that there is no residual plasmin in the final products; unfortunately, this has not been the case for several trials and hence the role of plasmin in some of those trials cannot be ruled out). The UHT milk samples were stored at 20°C for up to 4 months. The authors summarized their results by stating that the defects in the milks “were detected in the order: bitterness > formation of particles > creaming > sediment (> 5%) > gelation”; the order was chronological not severity. The control milk samples, which contained no added Pseudomonas cultures, showed no negative product changes. Some examples of the second approach are as follows: • Gebre-Egziabher et al. (1980) added various dilutions of a filter-sterilized supernatant of a Pseudomonas culture to commercial UHT milk. At an addition level of 2 protease units/mL, bitterness developed within 7 days at 21°C (1 protease unit was defined as the amount of enzyme producing 1 μg of acid-soluble tyrosine/mL of enzyme solution per 24 h at 40°C). Addition of higher enzyme concentrations resulted in the development of an unclean flavor and bitterness, followed by coagulation, within 3 days at 21°C. • Mitchell and Ewings (1985) aseptically added eight purified proteases, six of P. fluorescens and two of Serratia marcesens, to UHT milk at concentrations from 0.3 to 30 ng/mL, and studied the effects on proteolysis, flavor, and gelation. A control milk with no added protease, held under the same conditions as the trial milk samples, showed no protein breakdown at up to 22 weeks of storage. All milks with added protease showed proteolysis and gelled. The time to gelation varied from 2 to 21 weeks. Bitterness developed before gelation in all cases; however, the flavor progressed through the following stages: lacks freshness, slightly stale, unclean, mild off-flavor, slightly bitter, and bitter. There was no statistical relationship between time of onset of gelation and proteolysis, as measured by

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10. The effect of UHT processing and storage on milk proteins

nonprotein nitrogen or noncasein nitrogen; however, there was a (negative) relationship between the time to gelation and the protease activity in the milk. A threshold activity value of 30 U/mL, below which UHT milk could be expected to be stable at 23°C for 5 months, was proposed. Such levels of protease activity in milk are beyond the sensitivity level of existing assay methods. All eight proteases showed a distinct preference for κ-casein, followed by β-casein, and then αs-caseins in milk. • Button et al. (2011) added a cell-free culture of a proteolytic P. fluorescens to UHT milk at from 0.0003% to 0.006% and stored the milk at 25°C for 12 months. The level of proteolysis, as measured by the concentration of free amino groups, increased in all samples during storage. The authors determined that a concentration of free amino groups of at least 12 mM Leu-Gly equivalents was required before the onset of age gelation. In this trial, this level was reached in the milk sample containing the lowest level (0.0003%) of added crude protease after 5 months of storage. Distinguishing between proteolysis induced by plasmin and by bacterial proteases Plasmin and bacterial proteases cause similar effects in milk. Both cause age gelation and bitterness, and there is growing evidence that both can be associated with sediment formation and creaming or fat separation. However, to be able to remedy problems associated with proteolysis, it is necessary to be able to diagnose the cause. If the cause is plasmin, then adjusting the processing conditions, as discussed earlier, is essential; however, if the cause is bacterial protease, then the quality of the raw milk needs to be addressed. An alternative for the latter is increasing the severity of the sterilization conditions, particularly increasing the holding time, that is, the length of the holding tube; however, such heating conditions exacerbate the level of cooked flavor. In terms of gelation, plasmin gels are soft and voluminous, whereas gels initiated by bacterial proteases tend be firm and more compact, and resemble rennet gels (Zhang et al., 2018). However, it is sometimes difficult to distinguish between the causes when the milk presents with some gel-like material or sediment in the bottom of the container and possibly some creaming. Such changes could be the beginning of more complete gelation caused by either plasmin or bacterial protease. In addition, it is quite possible to have both active plasmin and bacterial proteases present; this can occur if the raw milk quality is poor and the UHT processing conditions are insufficient to inactivate plasmin. Laboratory-based tests for distinguishing between proteolysis by plasmin and by bacterial proteases are now available (Lo´pez Fandin˜o et al., 1993; Datta and Deeth, 2003). They are based on the fact that plasmin preferentially acts on β-casein, whereas bacterial proteases act primarily on κ-casein. These tests have proved to be valuable in determining the cause of proteolysis in practice (e.g., Topc¸u et al., 2006). This is despite some reports indicating that some bacterial proteases preferentially attack β-casein (Gebre-Egziabher et al., 1980; Bagliniere et al., 2012). UHT milk containing such proteases would give misleading results by the tests described here. The tests are based on the properties of the peptides produced. Namely, plasmin tends to produce large hydrophobic peptides and bacterial proteases tend to produce small hydrophilic peptides. Therefore, by RP-HPLC, the peptides produced by bacterial protease will elute before those produced by plasmin. Furthermore, the peptides from bacterial proteases

403

Protein changes during processing and storage

TABLE 10.4 Interpretation of peptide analyses by RP-HPLC and total amino groupsa for diagnosing the cause of proteolysis in UHT milk 4% Trichloroacetic acid filtrate

pH 4.6 filtrate

Peptide peaks by RP-HPLC

Proteolysis— amino groups

Peptide peaks by RP-HPLC

Proteolysis— amino groups

Interpretation

Virtually no peaks after the initial solvent peak

Little or none

Virtually no peaks after the initial solvent peak

Low

Good quality milk

Few peaks after the initial solvent peak

Little or none

Significant late peaks

Significant

Plasmin action only

Significant early peaks but few late peaksb

Significant

Significant early peaks but few late peaks

Significant

Bacterial proteinase action only

Significant early peaks but few late peaks

Significant

Significant early and late peaks

Significant

Both bacterial proteinase and plasmin action

a

Determined by a method such as fluorescamine. In the original paper (Datta and Deeth, 2003), early and late peaks were defined as eluting before and after 20 min, respectively; however, elution times are dependent on the conditions of analysis and hence specific times have not been included here.

b

are soluble in both pH 4.6 filtrate and 4% trichloroacetic acid, whereas those produced by plasmin are soluble in pH 4.6 filtrate but insoluble in 4% trichloroacetic acid. The peptides in the pH 4.6 filtrate and the 4% trichloroacetic acid can be quantified by methods such as fluorescamine and ortho-phthalaldehyde (OPA), which detect total amino groups (Vaghela et al., 2018). Table 10.4 outlines the possible results of peptide analyses by RP-HPLC and total amino group analysis and their interpretation for diagnosing the cause of proteolysis in UHT milk. Reducing enzymatic proteolysis in UHT milk In summary, plasmin-induced proteolysis can be greatly reduced, if not prevented, by the selection of appropriate preheat UHT conditions. Bacterial proteolysis can be prevented by using raw milk with low bacterial counts. If bacterial proteases are present in the raw milk, their effect can be minimized by the use of quite severe sterilization conditions. A further method to reduce the effect of proteases is to employ what is known as low temperature inactivation (LTI). This has been shown by several authors to be effective in reducing protease action (Hill, 1988; Reddy et al., 1991). Interestingly, Zhang et al. (2007) found that LTI of proteases reduced fat separation, a further indication of the role of proteolysis in the fat separation or creaming of UHT milk during storage. LTI involves a mild heat treatment, which can be performed before or after UHT processing. Typical heating conditions for LTI are  55°C for 30–60 min (Barach et al., 1976) although different proteases may require different conditions for optimum effect (Kocak and Zadow, 1985c). The mechanism of LTI is unclear but has been suggested to be due to autodigestion of the enzyme or changes in the conformation of the enzyme through interaction with milk proteins (Schokker and van Boekel, 1999).

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10. The effect of UHT processing and storage on milk proteins

Proteolysis in UHT hydrolyzed-lactose milk Several authors have reported higher levels of proteolysis in UHT hydrolyzed-lactose milk than in unmodified milk (Tossavainen and Kallioinen, 2007; Jansson et al., 2014). The proteolysis has been attributed to the proteolytic side activity in the β-galactosidase preparations. Jansson et al. (2014) reported that β-casein and αs1-casein are the most susceptible milk proteins to this proteolysis. Heat-induced proteolysis Several peptides are produced from β- and αs1-caseins when milk is heated and some have been proposed as markers for distinguishing UHT milk from other heat-treated milk. For example, Dalabasmaz et al. (2017) found 12 peptides that increased with time of heating (at 99°C) and could be used to distinguish UHT milk from mildly heated milk (pasteurized and extended shelf life). However, the peptide with the highest discriminatory power was found to be pyroGln-β-casein 194–209 with an m/z of 1710. It originated from cleavage at Tyr193-Gln194 at the C-terminal region of β-casein and the concomitant formation of pyroGln. Formation of the pyroGln modification was associated with a mass reduction of 17 Da, because of the loss of ammonia, compared with the parent peptide with an m/z of 1718.0 Da. Interestingly, this latter peptide is also formed by cleavage at Tyr193-Gln194 by cathepsin B, which can be active in UHT milk. However, Dalabasmaz et al. (2017) believed that cathepsin-B-induced formation of β-casein 194–209 could occur in parallel with the heat-induced formation of pyroGln-β-casein 194–209. Interestingly, Gaucher et al. (2008) identified eight peptides from αs1-casein in UHT milk that they concluded could have been produced by heat treatment or cathepsin B; many of these were not detected in freshly processed UHT milk, but only after storage, which casts doubt on their origin being from heat treatment. The authors suggested that long-term storage could have a similar effect to heat treatment. Ebner et al. (2016) identified nine peptides that increased with time of heating (at 120°C) and had higher intensity in UHT milk than in high-temperature-short-time (HTST)-pasteurized milk when analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry. Of these, the peptide β-casein 196–209 (m/z 1460.9 Da) was most influenced by the intensity of the heat treatment and was suggested by these authors to be a possible heat treatment marker. Practical effects of heat-induced proteolysis have not been reported. However, it may contribute to destabilization of the casein micelle, which leads to sedimentation and/or gelation.

Age gelation Age gelation is one of the major causes of a reduction in the shelf life of UHT milk. It is now well established that proteolysis by plasmin or bacterial proteases is the major initiator of gelation. However, gelation has also been observed in milk in which proteolysis did not appear to be a factor; the cause of this type of gelation has been designated to be physicochemical (Datta and Deeth, 2001; Nieuwenhuijse and van Boekel, 2003; Deeth and Lewis, 2016, 2017; Anema, 2017). Physicochemical gelation tends to occur in UHT milk with high solids content, that is, concentrated milks, although cases involving nonconcentrated milk have also

Protein changes during processing and storage

405

been reported (Auldist et al., 1996; Anema, 2017). According to Anema (2017), it is more likely to occur in milk after long storage. As indicated earlier, several studies have indicated the involvement of proteolysis in age gelation. Proteolysis caused by both plasmin and bacterial proteases has been proved to be involved. However, curiously, many authors have not been able to find a good correlation between the time to onset of gelation and the extent of proteolysis. There has been more success in relating the protease activity to the onset of gelation (e.g., Mitchell and Ewings, 1985). However, most protease assays are not sensitive enough to quantify the levels that can cause gelation after storage of the milk. Assays involving long incubation times have been shown to detect low protease activities (Button et al., 2011). However, scientists in Australia have developed a biosensor-based method, the Cybertongue method, that can detect “proteases in milk at concentrations relevant to industry within a few minutes” (Anon, 2014). The biosensor uses a Bioluminescence Resonance Energy Transfer (BRET) transduction system. Factors that influence the time of onset of age gelation in UHT milk Temperature of storage

Several authors have shown that the susceptibility of UHT milk to age gelation is low at low temperatures, reaches a maximum in the 25–28°C range, and then decreases at temperatures above 30°C (Kocak and Zadow, 1985a; Manji et al., 1986). The reason for this is not entirely clear. However, one hypothesis is that, at the high temperatures, proteolysis proceeds at such a rate as to prevent the formation of the ordered protein network that is required for a gel. A second hypothesis is that the higher temperature favors protein cross-linking, which stabilizes the casein micelles and prevents release of the β-Lg-κ-casein complex into the milk serum, which would otherwise form into a gel (McMahon, 1996). The cross-linking can occur either through dehydroalanine forming linkages between lysine and alanine or histidine on adjacent molecules (Al-Saadi and Deeth, 2008; Holland et al., 2011), or via advanced Maillard reaction products such as the dicarbonyls glyoxal and methylglyoxal (Le et al., 2013). Other reactions that may retard gelation at temperatures >30°C are lactosylation of lysine residues and deamidation of asparagine and glutamine residues of proteins. The involvement of lactosylation was indicated by Malmgren et al. (2017), who showed that high degrees of lactosylation were associated with the absence of gelation and long tendrils. Deamidation alters the charge on proteins and can cause changes in their physical properties. An implication of the phenomenon of decreased gelation at higher temperatures of storage is that accelerated shelf life trials to assess the susceptibility of a product to age gelation at temperatures higher than  30°C will provide misleading results (Grewal et al., 2017a). However, such trials can provide valuable information on the susceptibility of the product to proteolysis (Deeth and Lewis, 2017). They have also been shown to be useful for assessing susceptibility to sedimentation (Grewal et al., 2017c). Temperature-time conditions of heat treatment

The importance of selecting preheat conditions that will inactivate plasmin and prevent plasmin-related proteolysis and gelation was mentioned in the section on Heat inactivation of plasmin. However, the temperature and time in the sterilization holding tube can also be manipulated to minimize proteolysis and age gelation. For example, Topc¸u et al. (2006)

406

10. The effect of UHT processing and storage on milk proteins

reported that increasing the conditions from 145°C for 4 s to 150°C for 4 s substantially reduced proteolysis and bitterness in UHT milk. However, increasing the severity of the high-heat step has a major effect on the flavor of the product. In other words, there is a trade-off between flavor retention and physical stability of the product. Mechanism of age gelation As indicated earlier, proteolysis by either plasmin or bacterial proteases is accepted as the major cause of age gelation, but it can also occur by physicochemical means in the absence of proteolysis. Proteolysis-induced gelation is generally accepted to occur via a three-stage process. The first stage occurs during heat treatment and involves the interaction of β-Lg with κ-casein on the micelle surface and with κ-casein in the serum phase, which is released from the micelle during heating to form a β-Lg-κ-casein complex (Hillbrick et al., 1999; Anema, 2017). Electron microscopy has been used to show tendrils of β-Lg-κ-casein complex attached to casein micelles (McMahon, 1996; Malmgren et al., 2017). The tendrils have been shown to grow in length prior to gelation; when prevented from growing because of, for example, extensive lactosylation, gelation does not occur (Malmgren et al., 2017). The second stage is proteolysis of the caseins within the casein micelle, which cleaves the peptide bonds that anchor the κ-casein to the casein micelle, facilitating the release of the β-Lg-κ-casein complex tendrils into the serum. The third stage is the formation of a gel from the β-Lg-κ-casein complexes in the serum when their concentration exceeds a critical value (McMahon, 1996). For the physicochemical type of age gelation, Anema (2017) proposed a mechanism involving the aggregation of the κ-casein-depleted casein micelles that are formed during heat treatment. The aggregation was proposed to be mediated by calcium bridging. Retardation of age gelation with polyphosphate The most common additive that is used commercially to delay age gelation is sodium hexametaphosphate (SHMP), sometimes referred to as “polyphosphate.” Some UHT milk manufacturers add it routinely but this may not be allowed in some jurisdictions. When added to milk at  0.1% (w/w) before UHT treatment, it extends the time to gelation (Kocak and Zadow, 1985b). In terms of its mode of action, SHMP does not prevent proteolysis but appears to prevent the subsequent aggregation of the milk proteins and the formation of the gel. In fact, milk with added SHMP may undergo considerable proteolysis without gelling. The effect of SHMP is probably due to its association with casein, thereby altering the net charge on the casein micelles (increasing their negative charge). The addition of a low level of SHMP would facilitate bridging between ionized groups of casein micelles, which would not otherwise form an ionic bond. This would hold the κ-casein more tightly to the micelle and delay release of the β-Lg-κ-casein complex, retarding gelation of the UHT milk during storage. Another possible role of SHMP is to bind ionic calcium and reduce its activity in the milk. However, this role seems to be unlikely as EDTA and citrate, which also bind ionic calcium, accelerate rather than retard gelation (Kocak and Zadow, 1985b). Fat separation or creaming related to age gelation Some creaming or fat separation occurs in almost all fat-containing UHT milks. Some of this is undoubtedly related to poor homogenizer performance and maintenance, but there

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is increasing evidence that it is related to age gelation and the often-associated proteolysis, because the defects tend to occur at the same time (Kohlmann et al., 1991; Hardham, 1998; Stoeckel et al., 2016). As homogenization causes the formation of a casein-dominant membrane around the fat globules (Lee and Sherbon, 2002), bacterial proteases can hydrolyze the casein in this secondary milk fat globule membrane during the storage of UHT milk and lead to aggregation of the fat globules in a similar manner to the formation of a proteolysis-induced gel.

Sedimentation Sedimentation is one of the major defects that develop in UHT milk. It is defined as the material that settles to the bottom of the container in a fairly compact manner. Although there is some confusion in the literature between a gel and a sediment, it is generally accepted that a gel is softer and more voluminous than a sediment. The confusion sometimes arises when gelation commences in the bottom of the container (as mentioned in the section on The plasmin system) before it spreads to the rest of the container. Furthermore, as noted in that section, the formation of a sediment can be followed by gel formation (Malmgren et al., 2017). Most UHT milk contains some sediment. Lewis et al. (2011) reported 0.29% sediment in UHT cows’ milk. In general, the amount of sediment resulting from the UHT processing of good quality cows’ milk should be < 0.5% (On-Nom et al., 2012). The amount of sediment increases with the time of storage (Blanc and Odet, 1981; Ramsey and Swartzel, 1984; Stoeckel et al., 2016; Malmgren et al., 2017). In most cases, the sediment is composed of milk proteins, specifically caseins. Gaur et al. (2018) reported that the sediment consisted largely (>  85%) of aggregated κ-casein-depleted casein micelles with small amounts of whey proteins (β-Lg and α-La) and κ-casein; a similar predominance of β- and αs-caseins in the sediment of directly processed UHT milk was reported by Malmgren et al. (2017). An exception to the dominance of protein in the sediment is the presence of fat in the sediment of fat-containing milk. For example, Grewal et al. (2017b) reported changes to Fourier transform infrared (FTIR) spectra that were associated with the conformation of the fat in whole milk samples stored for 14 days at 40°C and 50°C. They indicated the formation of an intermolecular beta sheet of proteins, which was attributed to protein-lipid interactions and aggregation. The sediment from these samples contained fat, which confirmed the involvement of protein-lipid interaction in the sedimentation. Boumpa et al. (2008) showed that the sediment from whole goats’ milk that was processed indirectly at 140°C for 2 s with upstream homogenization contained  40% fat. Another exception is the sediment in some UHT mineral-fortified milks such as those fortified with insoluble calcium salts, for example, calcium carbonate or calcium citrate. These sediments contain a high proportion of mineral salts. In contrast, Boumpa et al. (2008) reported <5% mineral in the sediment of UHT goats’ milk. A further exception is UHT chocolate milk drinks, in which the cocoa particles often form a sediment on standing despite the drink being stabilized by gums such as carrageenan. There appears to be more than one cause of sedimentation. However, the root cause is destabilization of the casein component such that it can no longer remain in suspension. Dalgleish (1992) calculated theoretically that the rate at which the largest natural casein

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10. The effect of UHT processing and storage on milk proteins

micelles would sediment was 1.3 mm/week. This indicates that, in all UHT milk, a significant number of these large micelles, together with any casein aggregates, would be expected to sediment during the storage of the milk for several months. Smaller micelles would sediment at a much slower rate. Furthermore, the rate of sedimentation would be expected to be faster at higher temperature because of the reduction in viscosity. This is borne out by reports on the sedimentation in cows’ milk stored at different temperatures (Ramsey and Swartzel, 1984; Hawran et al., 1985; Vesconsi et al., 2012; Malmgren et al., 2017), although a report on the sedimentation in buffalo milk indicated more sedimentation during storage at 15°C than during storage at 30°C (Sharma and Prasad, 1990). It has been suggested that the temperature of storage may be significant only for milks with poor heat stability. Frozen storage of UHT milk results in considerable sediment when the milk is thawed because of destabilization of the caseins (Deeth and Lewis, 2017, pp. 304–307). The type of UHT process has been shown to influence the amount of sedimentation in UHT milk. Several authors have reported more sediment in directly processed UHT milk than in indirectly processed UHT milk (e.g., Perkin et al., 1973; Ramsey and Swartzel, 1984; Gaur et al., 2018). This may be related to the greater heat input resulting in chemical change in indirect processing compared with direct processing for the same bactericidal effect; several authors have reported that higher processing temperatures lead to more sedimentation (Swartzel, 1983; Ramsey and Swartzel, 1984; Boumpa et al., 2008). Furthermore, Malmgren et al. (2017) observed that more sediment was formed in UHT milks produced by steam injection than in those produced by steam infusion. One reason advanced as to why more sediment forms in directly processed milks is that the sedimented material is material that has not had an opportunity to attach to the walls of the heat exchanger as a fouling deposit (Burton, 1968; Swartzel, 1983). However, Gaur et al. (2018) considered this to be unlikely as fouling deposits are enriched in whey proteins, particularly β-Lg, and sediments are largely composed of caseins. However, the relationship between fouling deposit and sediment may be different for indirectly processed milk. Hawran et al. (1985) found that the amount of initial sediment in indirectly processed UHT milk (measured at 1 week) increased linearly with processing run time, as did fouling of a tubular heat exchanger that heated milk from 74°C to 114°C. They concluded that the sediment was largely due to erosion of deposit from that heat exchanger. They also showed that there were two rates of sediment formation during storage: a fast rate that resulted in sediment forming before 6 days and a slower rate that resulted in sediment forming after 6 days. It was suggested that the eroded fouling deposit sedimented rapidly as the initial sediment, presumably because of the larger aggregated particle size. The relationship between fouling deposits and sedimentation is most apparent with milk that has low heat stability. One such type of milk is goats’ milk, which is unstable to UHT processing. Boumpa et al. (2008) reported  5 g sediment/100 mL (dry weight basis), that is, > 40% of the total solids in the milk, in UHT goats’ milk. The heat instability of goats’ milk is attributable to a high ionic calcium level. Hence to enable goats’ milk to be processed without excessive fouling and sedimentation, the ionic calcium level has to be reduced by stabilizers, such as phosphate and citrate salts, or to be removed by, for example, a cation exchange resin (Prakash et al., 2007). Boumpa et al. (2008) used three added stabilizers, disodium hydrogen phosphate, trisodium citrate, and sodium dihydrogen

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phosphate, to reduce ionic calcium and found that, for a given reduced ionic calcium content, the amount of sediment following UHT processing was greatest for sodium dihydrogen phosphate and least for disodium hydrogen phosphate. Sediment levels as low as 0.13 g/100 mL were achieved in UHT goats’ milk when stabilizers were added. As well as ionic calcium, pH has a great effect on the heat stability of milk and hence its propensity to cause fouling and sedimentation (Lewis et al., 2011). Either a decrease in pH or an increase in ionic calcium decreases the heat stability of milk and reduces its ability to be successfully UHT processed and produce minimal sediment. Both of these changes reduce the ethanol stability, an indication of its suitability for UHT processing; the ethanol stability should be
74% for UHT processing (Deeth and Lewis, 2017, pp. 220–222). Gaur et al. (2018) attributed sedimentation in New Zealand UHT milks to high ionic calcium (
1.5 mM) and/ or low pH ( 6.65) and showed that sedimentation could be reduced by reducing the ionic calcium using chelators or by increasing the pH with alkali. Adding trisodium citrate could affect both of these modifications. Although such an addition may reduce sedimentation, it may have an adverse effect on age gelation. There have been numerous indications in the literature that sedimentation in UHT milk is linked with proteolysis. Newstead et al. (2006) observed a strong correlation between plasmin-induced proteolysis in directly processed UHT milk and sedimentation. Pinto et al. (2016) found a good correlation between sediment formation and proteolysis during the storage of UHT milk at 37°C and concluded that sedimentation could be prevented by the use of raw milk containing low levels of proteolytic psychrotrophic bacteria. Similarly, Bagliniere et al. (2012, 2017) reported that residual proteolytic activity of strains of P. fluorescens and Serratia liquifaciens L53 led to the destabilization of UHT milk, with sedimentation and the formation of aggregates. Likewise, Gaucher et al. (2009), Mat
eos et al. (2015), and Stoeckel et al. (2016) reported that casein micelles are destabilized by the action of Pseudomonas peptidases and that this results in the development of sediment. However, some reports suggest that there is not always a strong link between proteolysis and sediment formation. Richardson and Newstead (1979) added cultures of two proteolytic P. fluorescens strains, B12 and B52, to raw milk before UHT processing and found that, on storage of the UHT milk, one strain, B12, formed a “granular precipitate” (a sediment), whereas the other, B52, formed a “gel similar to that obtained by rennin action.” In a 16-week storage study by Rauh et al. (2014b) of UHT milk containing active plasmin, proteolysis but no sedimentation was observed. Malmgren et al. (2017) considered the link between proteolysis and sedimentation to be unlikely as, in their study, sediment occurred in milk stored at 5°C, but virtually no proteolysis occurred at this temperature.

Maillard reaction The following quote from Fennema (1996) indicates the importance of the Maillard reaction in food science: “Among the various processing-induced chemical reactions in proteins, the Maillard reaction (nonenzymic browning) has the greatest impact on sensory and nutritional properties.” Aspects of the Maillard reaction have been reviewed by O’Brien (1995), van Boekel (1998), and Mehta and Deeth (2016).

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10. The effect of UHT processing and storage on milk proteins

Lactosylation The Maillard reaction commences with a reaction between a reducing sugar and a protein. In milk, this initial reaction is usually lactosylation, that is, the reaction between lactose and the ε-amino group of lysine and, to lesser extent, arginine, methionine, tryptophan, and histidine. The first major product formed with lysine is the protein-bound Schiff’s base lactosyl lysine, but this readily undergoes an Amadori rearrangement to produce the more stable protein-bound lactulosyl lysine (ε-N-deoxylactulosyl-L-lysine). This rearrangement is highly significant because the lysine in the Amadori product is biologically unavailable; the lactosylated lysine hinders proteolysis of the protein by digestive proteases. This is often referred to as lysine blockage (Mehta and Deeth, 2016). Therefore, the greater is the extent of lactosylation, the greater is the negative effect on the nutritional quality of the milk. However, the reduction in nutritive value in this manner is not considered to be a significant issue in UHT milk (Dehn-M€ uller et al., 1991). It is sometimes raised as a concern with infant formulas, including UHT-processed formulas, where up to one-third of the lysine can be blocked (Mehta and Deeth, 2016). However, infant formulas generally have a high ratio of whey proteins to casein (Fenelon et al., 2018), and whey proteins are rich in lysine ( 12%); therefore, the product will still contain an abundance of available lysine. The Maillard reaction in UHT milk is initiated during heat treatment but continues during storage. It is very dependent on temperature, and hence, the more severe is the heating process and the higher is the temperature of storage, the more the reaction will progress. Because of this, the extent of lactosylation is sometimes used as an index of heat treatment. It is usually measured indirectly as furosine (furosine does not occur in milk), which is formed in the analysis by acid digestion. However, because lactosylation continues during storage, often to a greater extent than during heating, furosine is a good indicator of thermal treatment only in freshly processed UHT milk. Lactosylation can also be observed by methods based on mass spectrometry (Holland et al., 2011; Siciliano et al., 2013). They have been used to observe the various lactosylated forms of whey proteins and caseins as each lactose adds 324 Da to the molecular weight. These forms can be observed not only in the mass spectrographs but also in two-dimensional electrophoresis gels, in which they appear as a series of parallel spots for each protein; they are most easily seen for β-Lg and α-La (Holland et al., 2011). More molecules of lactose attach to the milk proteins with increased severity of heat treatment and time and temperature of storage. Holland et al. (2011) showed five parallel spots for β-Lg in UHT milk that had been stored at 40°C for 2 months; the five bands represented β-Lg with zero, one, two, three, and four attached lactose molecules. By analyzing tryptic digests of lactosylated proteins, it is possible to locate the positions of the lactosylated lysines. Interestingly, the lactose in a monolactosylated molecule may be attached to different lysines in different molecules. Fogliano et al. (1998) reported that the lactosylation of the caseins in UHT milks seemed to be nonspecific, with 7 of the 14 lysines in αs1-casein and 5 of the 11 lysines in β-casein being lactosylated. In monolactosylated forms of β-Lg, Lys100 was preferentially lactosylated. Lactosylation of the milk proteins during storage is one reason why new peaks for the proteins appear in HPLC chromatograms of the proteins in stored UHT milk. Furthermore, it is also a reason for existing peaks to broaden and for the chromatograms to contain an increasing number of protein species (Gaucher et al., 2008).

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Protein changes during processing and storage

Browning A brown discoloration of UHT milk is an indication of the final stage of the Maillard reaction. This is due to the formation of melanoidins, a complex mixture of largely highmolecular-weight brown pigments. The melanoidins formed by reaction with amino acids bound to proteins have a protein skeleton (Wang et al., 2011). The heating conditions in UHT processing are normally insufficient to cause a noticeable brown coloration in milk. For example, the conditions represented by the temperature-time profiles in Fig. 10.1A and B are equivalent to 106 and 38 s at 121°C (based on z ¼ 26.3°C; Browning et al., 2001); according to Fink and Kessler (1988), a brown coloration is noticeable at the equivalent of
400 s at 121°C. Although little browning occurs in unmodified UHT milk during heating, it occurs during storage, particularly if the storage temperature is
30°C. This is particularly significant for UHT hydrolyzed-lactose milk, in which both the products of the hydrolysis, glucose and galactose, are more Maillard reactive than lactose. The data in Table 10.5 illustrate the increase in color in UHT whole milk, skim milk, and hydrolyzed-lactose milk after storage for 4 months at 35°C and 50°C; greater color development occurred in all milks at 50°C than at 35°C and more occurred in hydrolyzed-lactose milk than in unmodified milks (data derived from Deeth and Lewis, 2017, p. 295). The ΔE value for visual perception of a brown color in milk has been reported to be 3.8 (Pagliarini et al., 1990). Thus, all milk such as those shown in Table 10.5 would be noticeably brown. When Maillard browning occurs in UHT milk, there is also a significant decrease in pH. This is largely due to the concomitant formation of formic and acetic acids, which are also products of the Maillard reaction (Adhikari and Singhal, 1991; van Boekel, 1998).

Deamidation Deamidation is the hydrolysis of the amide groups in asparagine (Asn) and glutamine (Gln) residues to aspartic acid (Asp) and glutamic acid (Glu), respectively, and ammonia. Nonenzymatic deamidation occurs to only a small degree during normal UHT processing but occurs significantly during the storage of UHT milk (Holland et al., 2011). The level of deamidation increases with temperature and time of storage. Asparagine residues adjacent to glycines show the highest rates of deamidation, but asparagine residues adjacent to serines TABLE 10.5 Browning, as measured by color difference (ΔE) values, for UHT whole milk, skim milk, and hydrolyzed-lactose milk stored at 35°C and 50°C for 4 months; color comparisons were made with the same product stored at 4°C Product

Storage at 35°C

Storage at 50°C

Whole milk (average of 3)

4.8

21.1

Skim milk (average of 2)

10.6

33.7

Hydrolyzed-lactose milk

20.3

43

Data from Deeth, H.C., Lewis, M.J., 2017. High Temperature Processing of Milk and Milk Products, Wiley Blackwell, Oxford, UK, p. 295.

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10. The effect of UHT processing and storage on milk proteins

and histidines are also deamidated. Deamidation of asparagine occurs more readily than that of glutamine (van Boekel, 1999). Holland et al. (2012) examined deamidation of αs1-casein. Two-dimensional electrophoresis showed predominantly one spot for this casein at manufacture; however, during storage, this spot split into multiple spots with approximately the same molecular weights. Each new spot represented the loss of ammonia and one negative charge. Up to four new spots appeared for samples stored at 40°C for 2 months. Each spot could represent more than one molecular species with the same charge. Several sites of deamidation were identified. These included Gln24, Asn32, Asn129, and Asn205. Both Holland et al. (2011) and Le et al. (2016) reported that Asn63 was the site for deamidation of β-Lg A and B. Although deamidation can be clearly detected by two-dimensional electrophoresis (Holland et al., 2011, 2012; Le et al., 2016), it can also be detected by measuring the ammonia released (van Boekel, 1999). Gaucheron and Le Graet (2000) reported a sensitive method (to < 0.5 mg/kg) based on separation of the ammonium ion by cation exchange chromatography and detection by suppressed conductivity. The effects of deamidation during storage on the functional and sensory qualities of UHT milk have not been determined. However, Le et al. (2016) showed that the proteins in a shelfstable acidic whey protein beverage produced by heating at 120°C for 20 s were deamidated during 12 months of storage. They suggested that deamidation, in which an Asn is converted to an Asp, may increase the susceptibility of proteins and peptides to acid hydrolysis by making an extra hydrolysis site available. Acid hydrolysis during storage of these beverages was beneficial as it reduced the formation of CMP-rich aggregates. Furthermore, it is also now known that the deamidation of proteins can increase their heat stability, reduce the extent of fouling deposit formation, preserve their nutritional value through reducing blockage of lysines, and improve some physical functional properties (Miwa et al., 2010; Timmer-Keetels et al., 2011). Miwa et al. (2010) have shown that enzymatic deamidation using protein glutaminase improves the solubility, viscosity, and emulsification capacity of skim milk solutions.

Cross-linking Cross-linking is a major change that occurs in the proteins in UHT milk. The nature of the cross-linking includes disulfide bonding and linking via dehydroalanine and Maillard reaction products. Other forms of cross-linking such as via lipid oxidation products may also be involved (Singh, 1991; Grewal et al., 2017a). Disulfide bonding occurs mainly during the heating process and occurs when the whey proteins are denatured, as discussed in the section on whey protein denaturation. Nondisulfide cross-linking can occur during thermal processing but occurs mainly during storage of the product. Nondisulfide cross-linking can be distinguished from disulfide cross-linking, as the latter links are broken by reduction with sulfhydryl reagents such as 2-mercaptoethanol and dithiothreitol. Therefore, high-molecular-weight bands remaining in reducing electrophoresis gels are due to nondisulfide cross-linking. These were clearly shown in two-dimensional electrophoresis gels by Holland et al. (2011). The extent of cross-linking increases with both temperature and duration of storage. Reports of the extent of cross-linking occurring during UHT processing have varied. By electrophoresis, little if any was detectable (Al-Saadi and Deeth, 2008; Holland et al., 2011).

Protein changes during processing and storage

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In contrast, by high-performance gel permeation chromatography, 5% of the protein in indirectly processed (138°C for 2 min) UHT milk was found to be cross-linked (Zin El-Din et al., 1991); by size-exclusion chromatography under denaturing reducing conditions, up to 14% cross-linking was found in freshly processed milk (Andrews, 1975; Lauber et al., 2001). However, much higher percentages of cross-linked protein occur in stored UHT milk, particularly if the milk is stored at elevated temperatures. Andrews (1975) reported that more than 50% of the casein was polymerized in UHT milk after storage at 37°C for 9 months; 23% was polymerized after storage at 4°C for 9 months. Al-Saadi and Deeth (2008) reported that the storage of UHT milk at 45°C for 12 weeks caused a 36% increase in high-molecular-weight proteins, but little increase occurred during storage at 5°C and 20°C. This cross-linking is a major cause of the marked change in the HPLC chromatograms of the proteins that is observed during the storage of UHT milk (Al-Saadi and Deeth, 2008; Gaucher et al., 2008). Using a proteomic approach, Holland et al. (2011) showed that the high-molecular-weight bands on two-dimensional electrophoresis gels of stored UHT milk contained mostly αs1-casein together with some β-casein and αS2-casein. Interestingly, this is consistent with the suggestion by Andrews (1975) that αs1-casein was most involved and that β-casein was involved to a lesser extent. A major pathway for nondisulfide cross-linking is via (protein-linked) dehydroalanine, which is a reactive intermediate formed by the elimination of phosphate from O-phosphorylserine, of a saccharide from O-glycosylserine, or of hydrogen sulfide from cysteine. It can react with lysine, histidine, or cysteine to produce lysinoalanine (LAL), histidinoalanine, and lanthionine cross-links (Friedman, 1999). These isodipeptides can be detected by amino acid analysis, gas chromatography/mass spectrometry, HPLC, and other methods after digestion with acid or enzymatic proteolysis, as the bonding between the amino acids is not a classical amide peptide bond. LAL is the most commonly measured isodipeptide. Fritsch et al. (1983) reported up to 50 mg LAL/kg protein in indirectly processed UHT milk, which was much less than in in-container sterilized milk, that is, 110–710 mg LAL/kg protein. Al-Saadi and Deeth (2008) found indirectly processed UHT milk to contain 62 mg LAL/kg protein immediately after manufacture. After storage at 5°C and 20°C, the LAL had increased to 64 and 70 mg/kg protein, respectively; after storage at 37°C and 45°C, it had increased to 130 and 170 mg/kg protein, respectively. Protein cross-linking can also occur via several Maillard reaction products. These include formaldehyde and dicarbonyls such as glyoxal and methylglyoxal. Such cross-linking in UHT milk was first proposed by Andrews and Cheeseman (1971). Le et al. (2013) demonstrated the involvement of Maillard reaction products in cross-linking by incubating milk proteins with methylglyoxal and producing similar polymerized proteins to those observed in UHT milk during storage. In milk products containing reducing sugars such as lactose and the lactose hydrolysis products, glucose and galactose, cross-linking via these reactive Maillard products can occur concomitantly with cross-linking via dehydroalanine (Al-Saadi et al., 2013). Protein cross-linking in milk may have some practical consequences. It can reduce the digestibility of the proteins and decrease the availability of some essential amino acids such as lysine (Friedman et al., 1981). However, it may have some advantages. As discussed in the section on the effect of storage temperature on age gelation, McMahon (1996) suggested that intramicellar cross-linking of caseins may be the reason for the decreased susceptibility of UHT milk to gelation when stored at temperatures >30°C.

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10. The effect of UHT processing and storage on milk proteins

Conclusions Several chemical and physical changes to the proteins occur during the processing and storage of UHT milk. Many of the changes can be minimized by storage at low temperature, but this negates a major advantage of UHT milk, that is, it does not require refrigeration. Therefore, other strategies are required to minimize the changes. At this stage, some of the changes, such as deamidation, are mainly of academic interest but others have major practical and ultimately economic implications. Although the causes of some defects are not completely understood, other defects such as those caused by proteolysis are now well understood. Current knowledge enables defects caused by proteolysis by plasmin to be effectively prevented and those caused by bacterial proteases to be identified and managed through attention to raw milk quality.

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