One factor contributing to the differences in antioxidant activity of the bovine and caprine caseins in algae oil-in-water emulsions is the different profile of caseins (Mora-Gutierrez et al., 2010). Bovine and caprine caseins both contain the four main casein classes: αs1-, αs2 -, β- and κ-casein, but the level of αs1-casein in caprine caseins isolated from the milks of American-bred, French-Alpine goats ranges from high to low (Mora-Gutierrez et al., 1991). The bovine and caprine caseins used in this study had different compositions in terms of αs1-, αs2 -, β- and κ-casein (Table 1). Because of the compositional differences, these milk proteins exhibit major differences (P < 0.05) in terms of the content of potential antioxidative amino acids histidine, methionine, phenylalanine, proline, tryptophan and tyrosine (Fig. 1); curiously, these differences favour the bovine casein, which in previous studies was less effective in suppressing lipid oxidation (Mora-Gutierrez et al., 2010). It was expected that a high content of antioxidative amino acids in the caseins would lead to a decrease in lipid oxidation. Instead, a high content of the β-casein fraction was correlated with improved oxidative stability of algae oil-in-water emulsions (Mora-Gutierrez et al., 2010). The results by Mora-Gutierrez et al. (2010) were largely explained on the basis of interfacial phenomena having a significant effect on oxidative stability of lipid systems. The mechanism of this process may be related to the affinity of β-casein towards the oil-in-water interfaces in emulsions, that is, β-casein was adsorbed and formed a viscoelastic film at the oil-in-water interface of the emulsion. As caprine casein has a much higher content of the β-casein fraction than bovine casein (Table 1), it would be strongly absorbed and form a film at the interface. Thus, in the algae oil-in-water emulsions, the caprine caseins high and low in αs1-casein were more protective against oxidation (Mora-Gutierrez et al., 2010). In addition, there were no significant differences (P < 0.05) in the amino acids targeted by mTG: glutamate (the sum of the target glutamine and glutamate) and lysine (Fig. 1). These bovine and caprine caseins had similar compositions in terms of protein, moisture, lactose and ash contents (Table 1). To evaluate alternative sources of milk proteins (bovine caseins) cross-linked with mTG, the caprine caseins with high and low levels of αs1-casein (Table 1) were compared in the algae oil-in-water emulsion system.
The efficient production of high-quality emulsions depends on understanding the relationship between the bulk physicochemical characteristics and their colloidal properties. Emulsifiers, that is, casein and caseinates, have the ability to form and stabilise emulsions by being absorbed to the oil-in-water interface during homogenisation, reducing the interfacial tension by an appreciable amount, thus preventing droplet coalescence from occurring during homogenisation (Dickinson, 2003a). The minimum amount of an emulsifier required to produce a stable emulsion, its ability to produce small droplets during homogenisation and its ability to prevent small droplets from aggregating over time are the prerequisites for successful microencapsulation of fats and oils.
The caseins are adsorbed to an interface so that the predominantly nonpolar regions on the surface of the molecule face the oil phase, while the predominantly polar regions face the aqueous phase. The bovine and caprine caseins tend to have a particular orientation at an interface that depends strongly on their β-casein content (Mora-Gutierrez et al., 2010). Once absorbed to an interface, the bovine and caprine caseins may or may not undergo structural rearrangements, so that they can maximise the number of contacts between nonpolar groups and oil phase. Because of their hydrophobicity, high flexibility and their degree of absorption, the caseins are excellent emulsifying agents (Dickinson, 2003a). Consequently, the bovine and caprine casein aggregates, when inserted into the lipid droplets, become more compact as evidenced by 31P-NMR (Mora-Gutierrez et al., 2010). This ‘compactness’ was particularly more evident for the caprine casein aggregates. As mentioned earlier, the content of the phosphoprotein β-casein is high in caprine caseins (Table 1). Β-Casein has been found to form a dense interfacial layer surrounding oil droplets (Berton et al., 2011) as a result of inserting its hydrophobic site compactly into the oil droplets and presenting its hydrophilic end on the surface (Mora-Gutierrez et al., 2010). The 31P-NMR line widths and relaxation times of bovine and caprine caseins in algae oil-in-water emulsions (Mora-Gutierrez et al., 2010) also led us to the conclusion that at 0.5% (w/v), the bovine and caprine caseins are highly absorbed to the lipid droplets and that the maximal oil content is 5% with a protein ratio of 1:10. However, it has been found recently that in oil-in-water emulsions prepared with low amounts of proteins, that is, β-lactoglobulin, β-casein and bovine serum albumin (BSA) in the aqueous phase (0.5% w/v), the proteins adsorbed at the interface do not efficiently protect lipids against oxidation, in comparison with surfactants, that is, Tween-20, Tween-80 and CITREM (Berton et al., 2011). The protein-stabilised interfaces seem to be more heterogeneous and porous, while the surfactant-stabilised emulsions are more homogeneous and compact (Murray & Dickinson, 1996; Bos & van Vliet, 2001; Wilde et al., 2004). Nevertheless, a thick interfacial layer surrounding oil droplets has been shown to lower oxidation rates in emulsions emulsified with 0.2% (w/v) whey proteins (Hu et al., 2003a); whey proteins are a mixture of β-lactoglobulin, α-lactalbumin, lactoferrin, BSA and immunoglobulins. In a preliminary antioxidant efficacy testing of various concentrations of bovine and caprine caseins added to algae oil-in-water emulsions, it was found that 0.5% (w/v) casein provided antioxidant activity mainly by producing thick interfacial layers (Mora-Gutierrez et al., 2010). At these later concentrations, the bovine and caprine caseins (0.5% w/v) and the whey proteins (0.2% w/v) tend to form polymers (protein aggregates), which favour the interfacial phenomena, that is, the formation of a thick interfacial film around oil droplets, and may thus have contributed to the increase in oil stability (Hu et al., 2003a; Mora-Gutierrez et al., 2010). Moreover, in oil-in-water emulsions prepared with excess emulsifier (≥1.0% w/v), casein has been shown to be more effective in protecting against lipid oxidation than other proteins (Hu et al., 2003b; Faraji et al., 2004; Clausen et al., 2009; Frisenfeldt Horn et al., 2011). It is thought that the observed antioxidant property of caseins is related to the ability of the casein molecules to chelate metal ions (Faraji et al., 2004; Villiere et al., 2005; Sugiarto et al., 2010); to scavenge free radicals (Clausen et al., 2009); or to form thick interfacial layers (Fang & Dalgleish, 1993).
When the casein present in the two phases (lipid phase and aqueous phase) was separated by SDS-PAGE using MOPS running buffer and stained with Coomassie Brilliant Blue (Fig. 2), a similar electrophoretic pattern was shown in the lipid and aqueous phases of algae oil-in-water emulsions containing bovine casein or caprine casein high and low in αs1-casein (5%, w/v) after 48 h of incubation. On the other hand, when algae oil-in-water emulsions contained only 0.5% (w/v) casein (data not shown), very little protein was present in the aqueous phase; most of the protein was absorbed onto the lipid phase during incubation. No differences in the electrophoretic migration and/or casein distribution of the lipid and aqueous phases were shown in algae oil-in-water emulsions prepared with a higher concentration of bovine and caprine caseins characterised by high and low levels of αs1-casein.