The effects of heat treatment and limited kappa-casein hydrolysis on the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates were investigated as a possible explanation for the gelation properties of combined rennet and acid gels. Reconstituted skim milk was submitted to combinations of 0-67% hydrolysis of the kappa-casein at 5 degrees C and heat treatment at 90 degrees C for 10 min. The protein composition of the ultracentrifugal fractions was obtained by reverse-phase high-performance liquid chromatography (RP-HPLC). The aggregates contained in each phase were isolated by size-exclusion chromatography and analyzed by RP-HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon heating only, 20-30% of the total kappa-casein dissociated, while 20-30% of the total whey protein attached to the micelles. When heated milk was renneted, little changes were observed in the distribution and composition of the aggregates. Conversely, the heat treatment of partially renneted milk induced the formation of essentially micelle-bound aggregates. The results were discussed in terms of the preferred interaction between hydrophobic para-kappa-casein and denatured whey proteins.
The pH-dependent behaviour of soluble protein aggregates produced by the pre-heating of reconstituted skim milk at 90 degrees C for 10 min was studied, in order to understand the role of these aggregates in acid gelation of heated milk. The following milk samples were prepared: (1) control (unheated reconstituted milk, pH 6.5); (2) milk heat-treated at pH 6.5 (mHtd6.5) and (3) milk heat-treated at pH 7.2 (mHtd7.2). They were centrifuged and the supernatants (SPNT 1) pH-adjusted to yield a series of pH values ranging from 6.5 or 7.2 to 4.6 using HCl at 20 degrees C or GDL at 20 and 38 degrees C. pH-Adjusted SPNTs 1 were re-centrifuged. The resulting supernatants (SPNTs 2) were analysed by OD (at 600 and 280 nm) and SDS-PAGE in order to characterise proteins still soluble as a function of pH. Particle size in SPNTs 1 was analysed by Steric Exclusion Chromatography. The OD600 nm revealed that during acidification soluble casein in both control and heat-treated samples exhibits variations in its optical properties or size as previously shown with micellar casein. In heat-treated samples, soluble casein and heat-induced covalent soluble aggregates precipitate at the same pH value. A progressive acidification of the soluble phase did not separate them. Increasing the temperature of acidification from 20 to 38 degrees C resulted in an increase in the precipitation pH of the proteins. However choice of acidifier did not have a significant effect on OD profiles. The soluble covalent aggregates from mHtd7.2 were smaller, more numerous, and had a higher content of kappa-casein than mHtd6.5. Both types of aggregates began to precipitate at the same pH value but precipitation occurred over a narrower pH-range for soluble aggregates prepared from mHtd7.2. This may explain the higher gelation pH of mHtd7.2 compared with mHtd6.5.
Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.
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