4.1 Introduction
In bovine milk, β-lactoglobulin is the main source of free thiol groups that can initiate thiol- disulphide exchange reactions upon heating (Zittle et al., 1962; Sawyer et al., 1963; Lowe et al., 2004). In many published research findings, an excess level of thiol blocking reagent (e.g. NEM, iodoacetamide and hydrogen peroxide) was used to block all the free thiol groups of β- lactoglobulin, hence upon heating, formation of new intermolecular disulphide bonds is eliminated (Zittle et al., 1962; Sawyer et al., 1963; Purkayastha et al., 1967; Sawyer, 1967; Özer et al., 2003). NEM has been used extensively in many studies involving β-lactoglobulin because it reacts readily with the free thiol groups of proteins at pH 6.5 – 7.0 (Gregory, 1955). The interaction between thiol groups and NEM results in a thioether containing a strong C-S bond and is therefore irreversible (Figure 4.1). For these reasons, in the studies reported in this thesis, NEM will be used as the thiol-blocking reagent.
Figure 4.1: The interaction between the thiol group on a protein with the thiol blocking reagent N-ethylmaleimide (NEM). The newly formed bond in a thioether is a strong C-S bond (red bond). There are numerous studies investigating the effect of NEM on the interactions between whey protein or β-lactoglobulin in solution (Shimada & Cheftel, 1988; Matsudomi et al., 1991; Xiong et al., 1993; Hoffmann & van Mil, 1997; Havea et al., 2009) and a few studies on the effect of NEM on the interactions between proteins in milk and milk products (Mckenzie et al., 1971; Hashizume & Sato, 1988; Goddard, 1996; Vasbinder et al., 2003; Lakemond & van Vliet, 2008a). While most of the previous studies used NEM concentrations higher than the concentration of free thiol groups in milk, Hashizume and Sato (1988) and Goddard (1996) worked with NEM concentrations ≤ 1 mM and found that the hardness (measured by a penetration test) of the acid-heat-induced gels decreased as the concentrations of NEM increased up to 0.2 mM
Protein Protein
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(Goddard, 1996) or 1 mM (Hashizume & Sato, 1988). The work reported in this thesis will work with a range of NEM concentrations ≤ 0.8 mM, aiming to cover a ratio of NEM to free thiol groups from below to above 1.
Previous studies investigated the effects of blocking the thiol groups of β-lactoglobulin on the properties of proteins or the properties of acid-heat-induced gels of systems that had been heated in the presence of NEM (Hashizume & Sato, 1988; Shimada & Cheftel, 1988; Matsudomi et al., 1991; Xiong et al., 1993; Goddard, 1996; Hoffmann & van Mil, 1997; Havea et al., 2009). However, there have been no reports on the effects of blocking the thiol groups of β-lactoglobulin in unheated milks on the properties of the milks and the resulting milk products. One can indeed suppose that adding NEM to unheated milks does not affect their acid gelation properties since thiol groups of β-lactoglobulin are buried in the native structure (Brownlow et al., 1997), hence disulphide interactions between β-lactoglobulin and other proteins cannot occur. However non-covalent interactions may be possible since NEM has some effects on the secondary structure of pure β-lactoglobulin (Wada, Fujita & Kitabatake, 2006).
Despite the anticipated results, the experiments in this chapter involved investigating the effect of NEM on the protein interactions in unheated skim milk and unheated WPE skim milk. The acid gels were prepared from the treated milks and their properties were examined using rheological measurements and confocal microscopy. The aim of these experiments was to confirm that blocking the thiol groups of native β-lactoglobulin did not have any effects on the protein interactions and the properties of the resulting acid gels. In addition, the results of this study will provide detailed information on the protein status in milks containing NEM before heat treatment, as control samples for later comparison with milk heated in the presence of NEM (Chapter 5).
4.2 Materials and methods
Skim milk and whey-protein-enriched (WPE) skim milk were prepared as described in Section 3.1.1.
Diluted NEM (1% w/v) was added to give NEM concentrations of 0.08, 0.24, 0.4 and 0.6 mM in skim milk and 0.24, 0.4, 0.6 and 0.8 mM in WPE skim milk. The ratio of NEM to thiol groups of β- lactoglobulin in skim milk and WPE skim milk was calculated based on the average composition of skim milk (of which ~ 0.35% was β-lactoglobulin) and whey protein isolate (of which 69% was β-lactoglobulin). Hence skim milk had 0.35% and WPE skim milk had 1.14% β-lactoglobulin. Table 4.1 summarised the concentrations of NEM and their corresponding ratio of NEM to free thiol groups.
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Table 4.1: The ratio of NEM to free thiol groups (-SH) of β-lactoglobulin in skim milk and WPE skim milk corresponding to the concentration of NEM used.
Concentration of NEM (mM)
Ratio of NEM to -SH in skim milk
Ratio of NEM to -SH in WPE skim milk
0.08 0.4 0.1
0.24 1.3 0.4
0.4 2.1 0.6
0.6 3.1 1.0
0.8 4.2 1.3
The treated milk was shaken on a vortex mixer for 10 s and then left to react at 20 ± 0.5 °C in a thermostatically controlled water bath for one hour. The lids of the containers containing the milks were closed tightly to minimise any contact with oxygen.
In order to determine the appropriate holding time of reaction between NEM and the milk proteins, the extent of inhibition of the irreversible heat-denaturation of α-lactalbumin and β- lactoglobulin was assayed at different incubation times, until a plateau was found. NEM (0.6 mM) was added to skim milk that was then held for different times up to 6 h at 20 ± 1 °C before heating. After heating, the percentage of α-lactalbumin and β-lactoglobulin remaining as native protein (i.e. soluble at pH 4.6) was determined. Figure 4.2 showed that the percentage of native α-lactalbumin and β-lactoglobulin in skim milk heated in the presence of NEM was significantly higher than that in control heated skim milk (p < 0.05). However the holding time did not have a significant effect on the percentage of native whey proteins (p > 0.05). This indicated that the reaction between free thiol groups and β-lactoglobulin occurred rapidly.
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Figure 4.2: Effects of reaction time between 0.6 mM NEM and skim milk on the proportion of native proteins remaining after heat treatment (80 °C for 30 min). ●, α-lactalbumin; ▼,
β-lactoglobulin. Each data point is the average of two replicates. Error bars represent the standard deviation.
Many previous studies used one hour as a reaction time between β-lactoglobulin and NEM (Morr & Josephson, 1968; Xiong et al., 1993; Goddard, 1996; Hoffmann & van Mil, 1997; Anema & Li, 2000; Havea et al., 2009). It was also reported that NEM is able to interact with other amino acids (e.g. amine groups) when the holding time exceeds 2 h (Smyth, Blumenfeld & Konigsberg, 1964; Hansen & Winther, 2009). Throughout this thesis, when NEM was added to milk, the milk was held for 1 h at 20 ± 1 °C before further analysis or treatments.
After addition of NEM to the milks, the level of proteins participating in disulphide bonds and the proportion of proteins in the serum were investigated using SDS-PAGE (as described in Section 3.6). The size of the casein micelles in the milks were also examined using light scattering (as described in Section 3.8).
The NEM-treated milks were acidified to form acid gels of which the rheological properties and microstructure were investigated using rheometry and confocal microscopy (as described in Sections 3.10.2 and 3.10.3).
Holding Time (h)
0 1 2 3 4 5 6 No NEM%
N
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e P
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0 20 40 90 1004-71