David S. Horne

Factors affecting the ethanol stability of bovine milk: II. The origin of the pH transition

Dialysing milk against phosphate-free sera showed that the transition in the ethanol (EtOH) stability/pH profile was associated with the soluble phosphate component of the milk serum. Sigmoidal behaviour similar to that of milk was reproduced when the EtOH stability of artificial mixtures of casein, Ca and phosphate was measured as a function of pH. A mechanism for the s coagulation of skim-milk is discussed.

Factors affecting the ethanol stability of bovine skim-milk

Changes in the ethanol (EtOH) stability/pH profile of skim-milk induced by forewarming have been investigated as a function of temperature and duration of forewarming and of milk pH prior to heating. Forewarming increased the EtOH stability of a milk in the mid-pH range, shifting the profile laterally to more acid pH values, with little effect being observed on the maximum and minimum EtOH stabilities. No significant changes in forewarming effects were obtained on adding the sulphydryl-blocking agent, N -ethylmaleimide, indicating the lack of involvement of serum protein interactions in the stabilizing effects of forewarming. It is suggested that the observed effects of forewarming are due to a heat-induced precipitation of colloidal calcium phosphate. This suggestion is supported by measurements of Ca 2+ levels in forewarmed and untreated skim-milk as a function of pH and is in agreement with the previously suggested mechanism for the EtOH-induced coagulation of skim-milk. The effects of permitted additives (citrate, phosphate and Ca) on the EtOH stability of forewarmed milk paralleled their effect on untreated milk. A comparison is made of the effects of forewarming on the heat and EtOH stabilities of skim-milk.

Calcium-induced precipitation of α s1 -casein: effect of inclusion of citrate or phosphate

The calcium-induced aggregation of α s1 -casein at pH 7·0 has been studied in the presence of citrate and phosphate anions. The effects of the citrate can be reconciled with the suggestion that the citrate, acting as a Ca chelator, reduces the Ca available for the aggregation reaction, but in no other way interferes with the mechanism of that process. In the presence of phosphate, precipitation is induced below the normal critical Ca concentration. This reaction proceeds to completion at low and high concentrations of phosphate. There is, however, an intermediate range of phosphate concentrations, whose limits depend on the protein concentration, where a colloidally stable precipitate is produced. The significance of these observations is discussed in terms of the currently held theories of micellar structure and formation.

Ethanol stability of casein micelles – a hypothesis concerning the role of calcium phosphate

The ethanol (EtOH) stability of skim milk and the stability towards aggregation of casein micelles diluted into ethanolic buffer solutions were compared using data obtained from previously published experiments. Differences in absolute stability and in relative response were observed when Ca 2+ level and pH were adjusted, the buffer system results lying below those from skim milk in both cases. Increasing the ionic strength of skim milk adjusted to pH 7·0 lowered its EtOH stability whereas increasing the ionic strength of the diluting buffer increased the stability of the casein micelles. The hypothesis is put forward that the differences are due to the simultaneous precipitation of Ca phosphate when EtOH is added to skim milk. This draws calcium from the caseinate sites of the micelle, counteracting the destabilizing effects of the EtOH towards the micelle. Such removal and the consequent restructuring are kinetically controlled and micellar precipitation in skim milk finally occurs when the micellar coagulation time falls within the time scale of the restructuring reactions.

Calcium-induced precipitation of αs1-casein: effect of modification of lysine residues

Abstract Turbidity measurements have been used to study the calcium-induced precipitation of α31-casein whose amino groups have been modified by reaction with dansyl chloride and fluorescamine. Provided account is taken of the change in charge wrought by these modifications, the precipitation behaviour of these modified caseins is shown to be no different from that of the native αs1-casein protein. The results provide further support for the previously suggested isoelectric precipitation model for this reaction.

The Kinetics of the Precipitation of Chemically Modified αs1-Casein by Calcium

: The aggregation of alpha S1-casein by Ca is viewed as resulting from a reduction in the net negative charge by the binding of positively charged Ca2+. This reduction in charge leads to the equivalent of isoelectric precipitation. A quantitative relationship between the rate of aggregation of the casein and the monomer net charge is predicted in this isoelectric precipitation model. Precipitation results are presented for alpha S1-casein labelled with dansyl chloride. Providing the changes in charge brought about by the modification are taken into account, the precipitation behaviour of these caseins can be reconciled with that of native alpha S1-casein. This emphasises the role of electrostatic repulsion in this process and lend further support to the isoelectric precipitation model.

Relationship between ethanol stability of bovine milk and natural variations in milk composition

Several ionic components of ultrafiltrate were measured in bulk and individual cow milks and an assessment was made of their relationship with the parameters of the corresponding ethanol (EtOH) stability/pH profiles. From linear regression analysis the strongest relationships (P i +Cit) or as the ratio to (P i +Cit)] and pK (correlation coefficient, γ ∼ 0·82) or S max , the maximum stability at high pH (γ ∼–0·72), and between P i and pK(γ = –0·84)or S max (γ = –0·61). These relationships agree with the view that the parameters of the EtOH stability/pH profile are determined by pH-induced changes in concentration of divalent cations. Natural variations in these parameters may be attributed to variations in relative concentrations of divalent cations and their chelators. EtOH stabilities at the natural pH of bulk milks from winter/spring- and autumn-calving animals were lowest in early and late lactation. The most important contributory factors appeared to be a high salt balance ratio in late lactation and a low natural milk pH in early lactation. The main component responsible for variable salt balance ratio was usually soluble P i . Decrease in EtOH stability at the natural pH of late lactation milks reflected a more general change in the characteristics of the EtOH stability/pH profile, seen as an increase in pK and, in extreme cases, a decrease in S max and profile gradient.

Application of fractal concepts to the study of caseinate aggregation phenomena

The concepts of fractals and fractal dimension are introduced and applied to the description of the disordered structures of colloidal aggregates. It is demonstrated that the structure of the aggregates produced by ethanol de-stabilization of casein micelles can be quantitatively characterized by a fractal dimension. The values measured are compared to literature predictions from various computer studies simulating different models of the aggregation process.

Factors affecting the ethanol stability of bovine milk: V. Effects of chemical modification of milk protein

The effect of chemical modification of milk protein on the ethanol (EtOH) stability of skim-milk has been investigated. Modification of lysine residues by reaction with anhydrides increased the EtOH stability whereas amidation of carboxyl groups destabilized the milk. Lysine modification also counteracted destabilization induced by Ca 2+ addition. By using multiple linear regression techniques these latter effects can be accounted for in terms of the changes they bring about in micellar charge. These results provide a further verification and an extension of the previously proposed mechanism for EtOH-induced coagulation of skim-milk.

Factors affecting the ethanol stability of bovine skim-milk: VI. Effect of concentration

The ethanol (EtOH) stability characteristics of skim-milk concentrates were found to be governed by their chloride ion content. This effect appeared to be largely one of ionic strength. At pH values around 7·0 and above, EtOH stability showed a linear decrease with increasing chloride content but at pH values