Behaviour of fat globules and membrane proteins under different processing environments as related to milk powder manufacture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology

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Massey University
The objective of the first part in this study was to gain a better understanding of the protein components of the milk fat globule membrane (MFGM). In the second part, the influence of processing factors on the fat globules and the MFGM during the manufacture of whole milk powder were examined. Relationships between the state of the MFGM in whole milk powders and their reconstitutions properties were also explored. The MFGM proteins, isolated from early-, mid- and late-season fresh whole milks, were characterized using one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. SDS-PAGE under reducing conditions showed the presence of about 40 protein bands, ranging in molecular weight from 15 to 200 kDa. The major MFGM proteins e.g., xanthan oxidase, butyrophilin, PAS 6 and PAS 7 constituted 60-70% of total MFGM proteins while 20-30% were minor proteins. Two-dimensional SDS-PAGE indicated that xanthine oxidase and butyrophilin might be complexed via intermolecular disulfide bonds in the natural MFGM. The examination of MFGM proteins heated at > 60 °C in the absence of skim milk proteins (caseins and whey proteins) showed that xanthine oxidase and butyrophilin interacted further to form very high molecular weight protein complexes, whereas PAS 6 and PAS 7 were relatively heat stable and did not form complexes. Heat treatment of fresh whole milk in the temperature range 65-95 °C caused incorporation of β-lactoglobulin (β-1g) into the MFGM. Small amounts of α- lactalbumin (α-la) and κ-casein were also observed in the MFGM material of heated milk. The amounts of β-lg and α-la that associated with the MFGM increased with an increase in temperature up to 80 °C, and then remained almost constant. The maximum values for β-lg and α-la association with the MFGM were ~1.0 mg/g fat and ~0.2 mg/g fat, respectively. Association of β-lg and α-la with the MFGM was described by a first-order reaction (65-85 °C for β-lg and 70-80 °C for α-la) in the low temperature range and by a second-order reaction in the high temperature range (85-95 °C for β-lg and 80-95 °C for α-la). Arrhenius plots showed an abrupt change in temperature dependence of the rate constants at 85 °C for β-lg and 80 °C for α-la. Of the major original MFGM proteins, xanthine oxidase and butyrophilin were not affected by the heat treatment of whole milk, whereas PAS 6 and PAS 7 decreased during heating. Interestingly, this behaviour is in contrast to that shown by these proteins in systems containing no skim milk proteins. The changes in fat globule size and MFGM proteins during the manufacture of whole milk powder were determined using light scattering, SDS-PAGE, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Heat treatment of whole milk by direct stream injection (DSI) prior to evaporation caused a decrease in the fat globule size and an increase in the MFGM protein, through the association of caseins and whey proteins with the MFGM material. Evaporation of milk by a multiple-effect falling film evaporator caused a gradual decrease in the fat globule size and an increase in the MFGM protein after each effect. Caseins dominated the total MFGM protein, indicating the adsorption of casein micelles to the newly formed surface of the fat globules during evaporation. When whole milk was preheated (95 °C for 20 s) before evaporation, the amounts of total MFGM protein were higher (~6 mg/m2 compared to ~4 mg/m2 for the non-preheated whole milk) because of association of whey proteins with the native MFGM proteins and casein micelles. The average fat globule size decreased further upon homogenisation of the concentrated milk. The amount of MFGM protein (mg/m2) of concentrated milk also increased after homogenisation, the extent of the increase being dependent upon the temperature and pressure of homogenisation. Furthermore, heat treatment of concentrated milk to 79 °C either before or after homogenisation also increased the amount of MFGM protein. However, at the same homogenisation temperature and pressure, the amounts of whey proteins in the MFGM of the concentrated milk that was heated after homogenisation were higher than the concentrated milk that was heated followed by homogenisation. The amounts of the major native MFGM proteins did not change during homogenisation, indicating that the skim milk proteins did not displace the native MFGM proteins but adsorbed onto the newly formed surface. The fat globule size of homogenized concentrated milk decreased after spray drying, while the amount of MFGM protein (mg/m2) decreased slightly. Some "uncovered fat" was observed on the surface of powder particles. It is possible that the proteins do not adsorb to all newly formed fat surfaces during spray drying. The reconstitution properties of whole milk powders produced using different processing treatments were determined. High homogenization pressure and temperature used before spray drying resulted in poor reconstitution properties of the powder, particularly when the heat treatment was carried out after homogenization. It is suggested that the proteins adsorbed at the fat globule surfaces during homogenisation of the concentrated milk and their subsequent aggregation during heat treatment play a key role in determining the reconstitution properties of whole milk powders.
Milk proteins, Milk powder, Dried milk, Milk fat