CRÍTICA AL MERCADO COMO MECANISMO

Denaturation levels of whey proteins increase with increasing pressure or duration of treatment ( L6pez-Fandifio et aI., 1 996; L6pez-Fandifio & Olano, 1 998a, 1 998b; Huppertz et aI., 2004a). I n the case of heat treatment of milk, the minor whey proteins ( L F, B SA and Igs) are most labile; however, j3-LG is the most pressure-sensitive whey protein, denaturing at pressures as low as 1 00- 1 50 MPa. I n contrast, a-LA and BSA are stable to pressures up to about 400-500 MPa (L6pez-Fandifio et aI ., 1 996; L6pez­ Fandifio & Olano, 1 998a, 1 998b; Huppertz et aI., 2002). It has been reported that, compared with j3-LG, a-LA ( L6pez-Fandifio et aI., 1 996; Felipe et aI ., 1 997; L6pez­ Fandi fio & Olano, 1 998a; Garcia-Risco et aI., 2000; Needs et aI., 2000a; Scollard et aI. , 2000; H uppertz et aI., 2002; Huppertz et aI., 2004b) o r BSA ( Hayakawa e t aI., 1 992; Hinrichs et aI ., 1 996b) is more resistant to pressure denaturation in milk and that different whey proteins have different sensitivities to HP treatment.

Several reports suggested that treatment of raw milk at up to 1 00 MPa does not denature j3-LG. However, 70-80% denaturation of j3-LG occurs at 400 MPa ( L6pez-Fandifio et aI., 1 996; L6pez-Fandifio & Olano, 1 998a; Arias et aI., 2000; Garcia-Risco et aI., 2000; Scollard et aI ., 2000). Relatively l ittle further denaturation of j3-LG occurs at 400-800 MPa (Scollard et aI ., 2000). Various studies have reported different extents of denaturation of j3-LG fol lowing HP treatment at 600 MPa of pasteurised milk (Needs et aI ., 2000a) or reconstituted skim milk powder (Gaucheron et aI., 1 997); this may be attributed to the level of denaturation caused by treatments before pressurisation, which may influence the amount of denaturation measured afterwards. The reaction order of H P-induced denaturation of j3-LG is 2.5 (Hinrichs et aI. , 1 996b), indicating that the denaturation process is concentration dependent and that a lower initial concentration of native j3-LO should reduce the extent of denaturation of j3-LG under pressure.

a-LA is very resistant to pressure at ambient temperature and denaturation of a-LA was noted at pressures > 400 MPa for long treatment times ( L6pez-Fandifio et aI., 1 996; Hinrichs et aI ., 1 996b; Hinrichs et aI., 1 996a, 1 996b; Arias et aI., 2000; Needs et aI., 2000a, Scollard et aI., 2000; Huppertz et aI., 2002, 2004a). Various studies using raw milk ( L6pez-Fandifio et aI ., 1 996; L6pez-Fandino & Olano, 1 998a; Garcia-Risco et aI ., 2000), reconstituted skim milk ( Gaucheron et aI., 1 997) and pasteurised skim milk

(Needs et aI., 2000a) have shown that a-LA is resistant to denaturation at pressures up to 500 MPa.

Several reasons have been proposed for the differences in the sensitivity of these proteins. Differences in the pressure stabil ity of a-LA and p-LG may be linked to the more rigid molecular structure of the former ( L6pez-Fandino et aI., 1 996; Gaucheron et aI., 1 997), caused partially by the different numbers of intramolecular disulphide bonds in the two proteins (Hinrichs et aI., 1 996a, 1 996b; Gaucheron et aI ., 1 997), or the lack of a free sulphydryl group in a-LA ( L6pez-Fandino et aI ., 1 996; Funtenberger et aI. , 1 997), or probably due to differences in the secondary structures of these whey proteins. It has also been commented that the molecular structure of a-LA is more stable than that of p­ LO, and that oligomerisation takes place only if, during unfolding, free SH groups are available from other molecules ( Hinrichs et aI., 1 996b; L6pez-F andino et aI., 1 996; Oaucheron et aI., 1 997; Jegouic et aI., 1 997). This difference in pressure sensitivity can also be explained by the types of bonds stabilising the conformational structures of p­ LO and a-LA (Hinrichs et aI ., 1 996b; Messens et aI., 1 997).

BSA was also found to be resistant to pressures up to 400 MPa in raw milk ( L6pez­ Fandino et aI ., 1 996) or 600 MPa (Hayakawa et aI., 1 992). The high stability of BSA could be explained by the fact that BSA carries one SH group and 1 7 disulphide bonds. The energy received under pressure treatment was too small to disrupt all the disulphide bonds and to change the molecular structure of BSA. Also, greater resistance of p-LG and a-LA in whey than in milk may be attributed to the absence of casein micel les and CCP in whey ( Huppertz et aI., 2004b).

2.6.5 H P-induced interactions of caseins and whey proteins

It was reported that, when mixtures of K-CN and P-LO were pressure treated at 400 MPa, the presence of p-LG reduced the susceptibil ity of K-CN to subsequent hydrolysis by chymosin, indicating interactions between the proteins ( L6pez-Fandino et aI., 1 997). SDS-PAGE studies of pressure-treated and untreated milks or solutions containing K­ CN or P-LO or both i n the presence or absence of denaturing agents showed evidence for the formation of aggregates linked by intermolecular disulphide bonds ( L6pez­ Fandino et aI., 1 997). On HP treatment of milk at 300-600 M Pa, p-LG may form small

Chapter

Literature Review

aggregates (Felipe et al., 1 997) or may interact with the casein micelles (Needs et al., 2000a; Scol lard et aI., 2000). However, the exact mechanism for these processes needs further clarification. L i ke heat treatment, no study suggesting pressure-induced interactions of Us2-CN with whey proteins has been reported. Also, no studies suggesting pressure-induced interactions of other whey proteins with the casein micel les have been reported.

It has also been shown that the heat-induced interactions of the denatured whey proteins with the casein micelles are strongly pH dependent (e.g. Singh & Fox, 1 987a, 1 987b; Anema & K lostermeyer, 1 997; Anema & Li, 2003a, 2003b). Some these reports suggested that high level ( about 80% of the total) of denatured whey protein associate with the casein micelles at pH 6.5, and this level decreases with i ncreasing pH, so that about 30% of the total is associated with the casein micel les at pH 6.7, and even lower levels are observed at higher pH. In contrast to the numerous studies on the distribution of whey proteins between the colloidal and serum phases in heated mi lk, few studies have examined this distribution in pressure-treated milk. Huppertz et al. (2004a) suggested that the majority of the denatured whey protein was sedimentable, and presumably associated with the casein micelles, under more severe centrifugation conditions than used in the more recent heating experiments. Arias et al. (2000) examined the effect of the pH of the mil k at pressurisation (400 MPa for 1 5 min). However, concl usions on the association of the casein micelles with the whey proteins or the distribution of the whey proteins between the colloidal and serum phases in pressure-treated milk could not be made.

2.7 Conclusion s

H P processmg I S a rapidly growmg preservation technology that, even if i n the development stage, presents the potential for making products that are microbiologically safe and with improved functional properties (Chefiel, 1 992). Although HP processing is currently a focus of major interest (Johnston et aI., 1 992a, 1 992b; Shibauchi et al., 1 992; Desobry-Banon et aI ., 1 994), many of the effects of pressure on the components of milk are sti l l not known.

Many studies on HP treatment of dairy systems have focused on the effects of H P on the denaturation and aggregation of the individual whey proteins, particularly p-LG, in pure protein solutions, whey or milk. Also there have been many studies on microstructural or rheological aspects of whey protein gels or effects of H P on protein particle size, casein micelle dissociation, appearance of milk etc., but there is limited information identifying the pressure-induced aggregation products in whey protein solutions or interaction products of casein with whey proteins in pressure-treated milk. Although there is a clear understanding of the effects of heat treatments on the proteins i n dairy systems, there is a need for a comparable level of understanding of the effects of pressure treatments on the milk/whey system. A detailed study of the differences between heat- and pressure-induced changes in denaturation, aggregation and interactions of different whey proteins will help to find new applications for H P processing i n the food industry.

It is expected that pressure-induced whey protein gels may have different properties from those i nduced by heat. However, there is limited information on the pathways of pressure-induced whey protein gel formation and on how the protein concentration, pressurising temperature and holding time will influence the denaturation and aggregation of whey proteins. Further fundamental studies are required to understand the pressure-induced aggregation and gelation pathways of whey proteins in detail .

Most of the prevIOUS studies concermng interactions of milk proteins have been conducted in model systems ( mainly p-LG) and very little has been reported on the pressure-induced changes and interactions that take place during processing in the actual milk/whey system. In addition, various heating methods have been used, including heating samples in glass tubes, capillary tubes immersed in water/oi l baths and laboratory-scale heat exchangers. Only a few studies (e.g. Oldfield et aI ., 2000) on the heat-induced interactions of milk proteins using typical commerc ial process conditions are available. It is l ikely that proteins respond differently depending on how the sample is heated, i.e. on the time required to reach the desired temperature, flow conditions, and cooling times and rates etc. Hence, the process of denaturation of the whey proteins on heating milk under i ndustrial conditions is likely to be different from that observed in mi lks heated in the laboratory.

Chapter 2: Literature Review 47