pH-Dependent behaviour of soluble protein aggregates formed during heat-treatment of milk at pH 6·5 or 7·2

Abstract

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.

Role of protein aggregation in heat-induced heat stability during milk powder manufacture

“…A band representing the largest casein, α S2 , is present on SDS-PAGE in some of the samples, notably 1.1 and 1.4, and in 2.1 and 2.4. The qualitative compositional analyses of the soluble aggregate fractions are generally in line with previous reports [17,18,22,32]. These studies consider that the primary constituents of the aggregates are β-lactoglobulin, α-lactalbumin and κ-casein.…”

“…Unfortunately many different techniques have been used to separate the micellar and serum phases. Centrifugation is the most common but many different conditions have been used, ranging from 175 000× g/60 min to 19 200× g/240 min [2,4,11,17,18,22,29,32,33,[35][36][37]41]. Other researchers have rejected the use of centrifugation on the basis of being too harsh and likely to cause micellar disintegration and have used treatment with rennet to precipitate the micelle fraction instead [38].…”

“…More recent investigations have included works by Newstead et al [26] and Darling [15]. There has also been a growing interest in the formation and structure of milk protein aggregates in heated milks [17,18,22,32,33,38,39]. These investigations have generally been directed towards understanding the importance of these aggregates on the formation of acid induced gels and although Corredig and Dalgleish [11] hinted that variation in the composition of heat induced soluble aggregates may be related to the effects of preheating, their influence on heat stability has not yet been described.…”

“…These investigations have generally been directed towards understanding the importance of these aggregates on the formation of acid induced gels and although Corredig and Dalgleish [11] hinted that variation in the composition of heat induced soluble aggregates may be related to the effects of preheating, their influence on heat stability has not yet been described. Estimates of the size of these aggregates range between 10 nm and 40 nm in diameter [18,32] and that could out-number the micelles by at least 10 to 1. They are potentially very significant contributors to the physical properties of milk.…”

Effect of heat treatment at alkaline pH on the rennet coagulation properties of skim milk

“…Upon heating at pH > 6.7, heat-induced milk protein aggregates are reported to be smaller, more numerous [24] and with a higher proportion of disulphide bonds compared with hydrophobic interactions [12,13]. Upon heating milk at pH > 6.9, the proportion of heat-induced whey protein/κ-casein aggregates found in the serum phase is increased, while casein micelles are depleted in κ-casein as a result of their interaction with denatured whey protein and dissociation [2-5, 26-28, 32].…”