In previous studies, NEM was added to whey protein solutions and milks after heat treatment and before acid gelation. The Gʹ values and the hardness of the gels decreased with the increase in NEM concentrations (Lucey et al., 1998; Alting et al., 2000; Alting et al., 2003; Vasbinder et al., 2003). It was also found that once the acid gels were solubilised in SDS solution without disulphide reducing agent, the aggregates in acid gels without NEM were larger than those in acid gels with NEM. Therefore, thiol-disulphide exchange reactions were proposed to occur during acid gelation, leading to formation of intermolecular disulphide bonds that increased the firmness (Gʹ) and hardness (measured by penetration tests) of the acid gels (Alting et al., 2000; Alting et al., 2003; Vasbinder et al., 2003).
In this study, the addition of NEM to heated skim milk did not significantly affect the final Gʹ values of the resulting acid gels prepared from the heated skim milk (Figure 6.11A), which is contradictory to the previous findings of Vasbinder et al. (2003) and Lucey et al. (1998). Even though the acid gels prepared from heated skim milk with added NEM had a yield stress ~ 13% lower than those made from the control heated milk, they still had a markedly higher yield stress value than those made from unheated skim milk (which was 59 ± 2 Pa as shown in Chapter 4). This result was also in contrast with those from the study of Vasbinder et al. (2003) who reported that acid gels made from heated milk containing NEM had yield properties comparable to gels prepared from unheated milks.
In heated WPE skim milk, the final Gʹ values of acid gels decreased (by ~20%) when acid gels were prepared in the presence of NEM, regardless of the concentration of NEM added (Figure 6.11B). The NEM-containing acid gels broke at lower stress and strain values than gels without NEM (Figure 6.12B). The yield stress values of acid gels with NEM were not as low as those from
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acid gels prepared from unheated WPE skim milk (46 ± 2 Pa, as shown in Table 4.2). Although this effect on the yield properties was similar to what was observed in gels made from heated skim milk, the decrease in gel yield properties was much greater in the heated WPE skim milk than in heated skim milk (Table 6.1).
The contrasting findings between previous studies and this study may be due to the conditions of gelation. In previous studies, the gelation process occurred over a period of 15 to 24 h at 20 °C (Lucey et al., 1998; Alting et al., 2000; Alting et al., 2003; Vasbinder et al., 2003) whereas in this study, the acid gelation took place at 30 °C, over 3 h. In addition, lower GDL concentrations were used in previous studies than in this current study. This affected the rate of pH decreasing during gelation.
A test was carried out to monitor the change of the pH under the gelation conditions of previous studies. The pH was found to decrease at a much slower rate compared with this study and reached ~ pH 5.9 after 3 h. It was found that thiol-disulphide exchange reactions between whey proteins were slower at acidic pH (Shimada & Cheftel, 1989; Hoffmann & van Mil, 1999). The reason was that the thiol group was protonated and this reduced its reactivity compared with the un-protonated form found under neutral or alkaline conditions. Therefore, thiol-disulphide exchange reactions during acidification were likely to happen to a greater extent in previous studies than in this study because the acidification process occurred over a markedly longer period of time and the pH of the system remained relatively high for a longer time in previous studies than in this study.
Despite the different magnitude of effects in this study compared with the previous ones, the rheological analyses in this study did show that blocking the thiol groups in heated skim milk and WPE skim milk caused the yield stress (and yield strain) values of acid gels to decrease. This suggested that blocking thiol groups prevented thiol-disulphide exchange reactions between the proteins during acid gelation. Consequently, new disulphide bonds may not be formed between the particles that participated in the gelation. Since disulphide bonds are stronger than non-covalent bonds, the prevention of the formation of new disulphide bonds between particles may cause the resulting acid gels to be easily broken, as summarised in Table 6.1.
Overall, in this study, it can be understood that low levels of thiol-disulphide exchange reactions between proteins can occur during acidification and using NEM to prevent these exchange reactions had a bigger effect on yield properties (where the bonds need to be broken) than on the storage modulus (i.e. the low amplitude properties where bonds are not broken) of the resulting acid gels. Furthermore, adding NEM to skim milk and WPE skim milk after heat
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treatment (this Chapter) had little effect on the rheological properties of acid gels when compared to adding NEM to milk before heating (Chapter 5). This indicated that the thiol- disulphide exchange reactions during acidification may play only a minor role in the acid gel firmness and may have a bigger influence on the yield properties of the gels.
The effects of preventing the formation of new disulphide bonds during gelation on the final Gʹ values and the yield stress values were more pronounced in gels formed from heated WPE skim milk than those from heated skim milk. In WPE skim milk, due to the high protein concentration, the proteins can be “physically” closer together than in skim milk where the protein can be more scattered. Hence, thiol-disulphide exchange reactions can occur more readily during gelation of WPE skim milk compared to skim milk. Indeed, Alting et al. (2000; 2003) found that disulphide bonds were formed only during acid gelation of whey protein solutions consisting > 4.5% protein concentrations. When the thiol-disulphide exchange reactions were prevented during gelation, the number of newly formed disulphide bonds may decrease more drastically in WPE skim milk gels than in skim milk only gels. As a result, the effect of NEM on the acid gel properties was more pronounced in WPE skim milk gels than in skim milk only gels.
6.5 Conclusions
In heated skim milk and WPE skim milk, interaction of NEM with the thiol groups may prevent the thiol-disulphide exchange reactions between the proteins during acid gelation. Consequently, disulphide bonds between neighbouring particles may not be formed. The formation of new disulphide bonds may not greatly influence the Gʹ values of the resulting acid gels but definitely contributed to the yield properties of the acid gels. Therefore, the acid gels made from heated skim milk and WPE skim milk with added NEM had lower yield properties than those made from control heated milks.
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