Michael W. Taylor

Characterization of protein components of natural and heat-treated milk fat globule membranes

The proteins associated with the milk fat globule membrane (MFGM), isolated from early, mid and late season whole milks, were characterized using one- and two-dimensional SDS-PAGE under reducing and non-reducing conditions. In some experiments, MFGM separated from fresh whole milk was suspended in simulated milk ultrafiltrate (SMUF) and heated at various temperatures and times. SDS-PAGE under reducing conditions followed by staining with Coomassie blue showed the presence of about 37 protein bands, ranging in molecular weight from 15 to 200 kDa. SDS-PAGE under non-reducing conditions showed only about 25 distinct bands and the intensity of xanthine oxidase and butyrophilin bands was much less, while the intensity of PAS 6/7 band was similar compared with the reduced SDS-PAGE. Two-dimensional SDS-PAGE showed that the protein complexes that remained at the top of non-reducing gel were resolved into mostly xanthine oxidase and butyrophilin with a small proportion of PAS 6. These results indicate that xanthine oxidase and butyrophilin may be complexed via intermolecular disulfide bonds in the natural MFGM, although it is not possible to differentiate the individual protein distributions within these aggregates. It was found that the total protein content (mg/g fat) of MFGM and the percentage of xanthine oxidase and butyrophilin in early and late season MFGM were higher than that of mid season MFGM. In heated samples (above 60°C), xanthine oxidase and butyrophilin interacted further to form higher molecular weight protein complexes, while PAS 6/7 was relatively heat stable.

Kinetics of Denaturation and Aggregation of Whey Proteins in Skim Milk Heated in an Ultra-high Temperature (UHT) Pilot Plant

Skim milk was subjected to various temperature–time (70–130°C for 3–1800 s) treatments in a pilot-scale ultra-high temperature (UHT) plant using a direct steam injection (DSI) system. The rates of denaturation and aggregation of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) were determined by quantitative polyacrylamide gel electrophoresis while that of immunoglobulin G (IgG) was determined by affinity chromatography. The order of reaction, the rate constant and the activation energy were determined for the denaturation of β-lg, α-la and IgG using non-linear regression methods. The kinetic parameters obtained for β-lg and α-la showed a marked temperature dependence. Aggregation of β-lg mainly involved the formation of intermolecular disulphide linkages at all temperatures studied, while the aggregation of α-la, particularly below 80°C, appeared to also involve hydrophobic interactions. The kinetic parameters obtained for β-lg aggregation, due to disulphide bond formation, were similar to those observed for β-lg denaturation.

Heat-induced interactions of β-lactoglobulin and α-lactalbumin with the casein micelle in pH-adjusted skim milk

Skim milks, adjusted to pH 6.48, 6.60 or 6.83, were heated for various temperature–time combinations in a pilot-scale ultra-high temperature (UHT) plant. Heat-treated samples were ultracentrifuged and their supernatants analysed by quantitative polyacrylamide gel electrophoresis in order to measure the extent of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) denaturation and their subsequent association with the casein micelle. The activation energy of β-lg denaturation decreased as the pH increased. In contrast, there was no apparent trend for α-la. The extent of β-lg and α-la association with the micelle increased with heating time and temperature. The association of both proteins with the micelle was markedly affected by the milk pH. The rate and extent of association were greatest at pH 6.48, and least at pH 6.83. α-La continued to associate with the micelle although most of the β-lg had already associated. It was possible that α-la interacted at the micelle surface with β-lg that had previously associated with the micelle. A pseudo-first-order mathematical model was used to calculate the apparent rate constant for β-lg association with the micelle.

Association of β-Lactoglobulin and β-Lactalbumin with the Casein Micelles in Skim Milk Heated in an Ultra-high Temperature Plant

Abstract Skim milk was subjected to various time–temperature treatments using a direct steam injection (DSI) system in a pilot-scale ultra-high temperature (UHT) plant. The heated samples were ultracentrifuged and the supernatants analysed using quantitative polyacrylamide gel electrophoresis to determine the extent of denaturation of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) and their association with the casein micelles. The extent of β-lg and α-la denaturation and association increased with an increase in both heating time and temperature; the rate of association was markedly less than that of denaturation. The association behaviour was affected by heating temperature; during the initial stages of heating in the range 80–130°C, mainly β-lg appeared to associate with the casein micelles, but after prolonged heating, α-la began to associate with the micelle. In contrast, below 80°C both β-lg and α-la appeared to associate simultaneously with the micelles. The maximum level of association of α-la varied with heating temperature, ∼40% of total in the range 95–130°C and ∼55% below 90°C. For β-lg, the maximum level of association was ∼55% of total regardless of the temperature. A pseudo-first-order model was used to calculate the reaction kinetics of the association of β-lg with the casein micelle.

Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration

Microfiltration and ultrafiltration were used to manufacture skim milks with an increased or reduced concentration of whey proteins, while keeping the casein and milk salts concentrations constant. The skim milks were heated on a pilot-scale UHT plant at 80, 90 and 120 degrees C. The heat-induced denaturation and aggregation of beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA) were quantified by polyacrylamide gel electrophoresis. Apparent rate constants and reaction orders were calculated for beta-lg, alpha-la and BSA denaturation. Rates of beta-lg, alpha-la and BSA denaturation increased with increasing whey protein concentration. The rate of alpha-la and BSA denaturation was affected to a greater extent than beta-lg by the change in whey protein concentration. After heating at 120 degrees C for 160 s, the concentration of beta-lg and alpha-la associated with the casein micelles increased as the initial concentration of whey proteins increased.

Interactions of fat globule surface proteins during concentration of whole milk in a pilot-scale multiple-effect evaporator.

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 degrees C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.