Properties of milk system during concentration and subsequent heating

Production of concentrated milk encounters several instabilities in regards to the quality of the final product. Main problems that concern industry are aggregation and gelation of milk during heat treatment, age-gelation and thickening during storage. Some shelf life improvements have been achieved by pre-warming/pre-heating of milk prior concentration process intended to denature whey proteins and prevent their further involvement in aggregations with casein micelle. Nevertheless, precise knowledge describing behaviour of these proteins at the molecular level is lacking. Moreover, multitude of factors needs to be considered as they all have an impact on concentrated systems during water removal and heat treatment, including mineral content, protein rearrangements and pH. Hence, the present study aimed at evaluating observations of physiochemical changes in skim milk in regards to reduce water content, sequential heat treatment and the effect of altered pH during heating, all observations performed under laboratory conditions intended to imitate common industrial applications. During the first study, physiochemical changes of proteins in skim milk were investigated after evaporation process at 55ºC starting from 9% up to two solids concentration, 17 and 25%. Moreover, after reaching desirable concentration level, samples were analysed for protein portioning between the serum and colloidal phases, and how this affected electric dabble layer and particle size distribution, then variations of minerals (calcium, phosphate, magnesium and sodium) and changes in the secondary structure of proteins. Results shown that concentration indeed affected the overall stability of the system, which was dependent of total solids (TS) content. Thus, reaching 17% TS αs- and β- casein dissociated from the micelle and became more prominent in the serum. However, greater total solid concentration (25%) affected the level of κ- casein in the serum by slight increase only, on the other hand, β-LG concentration in the serum declined as it apparently interacted with the casein micelle. These modifications were confirmed with rearrangement of the secondary structure of the proteins advocated by slight shifting of minerals in the micellar state. In the second study, it was established how the proteins were affected during heating by determining changes at 75, 85, 95, 100, or 110ºC and prolonged heating of 2.6 minutes at 121ºC. All three concentration level (9, 17 and 25% TS) were examined. Treated samples were analysed for changes in the particle diameter, zeta potential, changes in protein level between the serum and colloidal phase, protein rearrangement in the secondary structure and variations in the mineral content (calcium, magnesium, phosphate and sodium) between the serum and micellar state. According to the results applied temperature induced formation of aggregates, which intensity was dependant of concentration level. Moreover, whey proteins denaturation was delayed in increased solids contented, which moved to higher temperatures as TS increased. However, after reaching specific temperature they were involved in intense aggregation among themselves through thiol-sulfhydryl interchange reactions and with caseins by disulfide interactions forming particles with larger diameter. In skim milk with 9% TS this delay of denaturation of milk proteins was not present and aggregation resulted only in more soluble particles which did not affect the average size distribution. Other important detail was large variations in the casein level in the serum, thus involving intense dissociation of specific caseins at certain temperatures. Observed changes were supported with rearrangement of the secondary structure of the micelle that was variable in regards to concentration level and applied temperature. The final study investigated the effect of pH adjustment on protein stability during heat treatment of skim milk with different solids content. Concentrated samples with 17 and 25% TS were altered to pH 6.7, which was native pH of skim milk prior to concentration intending to return them to their native environment in the system. Other set of experiment was performed with all samples including unconcentrated milk which included alkali addition to pH 7.5. Properties of the samples were studied in regards to concentration levels and heat treatment under this condition. SDS Polyacrylamide gel electrophoresis and FTIR were used for protein portioning between the serum and micellar phase and modifications in proteins conformational rearrangement, respectively. For each observed concentration intensive aggregation occurred with formation of small and large aggregates composed of whey proteins, dissociated caseins and casein micelle. The intensity of aggregation increased as pH and TS were moved to higher level. The results in this study resulted in many variations in the casein levels between the serum and micellar phase promoting intense dissociation when concentration and temperature increased. However, conditions created by adjusting pH to 6.7 promoted transfer of dissociated αs- and β- caseins from the serum into the micelle. This transfer occurred at lower heating point for 25% TS in comparison to 17%TS, although this occurrence seized for both at 110ºC, indicating stabilization of the casein micelle to a certain extent or almost restoration of its native state. Adjusting pH to 7.5 had destabilizing effect on all observed proteins during heat treatment resulting in intense dissociation of caseins, covalent bonding and aggregations. In regards to whey protein changes it was observed that heating samples with pH > 6.5 and temperatures up to 110ºC, whey proteins formed soluble aggregated complexes among themselves or with the serum caseins. On the other hand, when temperature was raised above 110ºC in addition to formation of soluble aggregates these proteins were involved in aggregation with the casein micelle. The present study revealed the findings that any alteration of solids content from the natural value in the skim milk would promote rearrangement of all proteins in the system indicating their destabilization due to increased particles number. Further heat treatment would induce more intense destabilization in the system by disintegration of the casein micelle, denaturation of whey proteins and formation of aggregates, which size increased when temperature of heating was higher. Moreover, these observations concluded the need of pre-treatment or stabilization of proteins up to point before concentration step that would produce samples with higher heat stability. However, when it comes to the effect of pH it is interesting to point out that slight alteration of pH up to 6.7 in concentrated samples may have a positive effect on stabilization of the micelle up to certain temperature that can be taken in consideration when it comes to heat stability of concentrated milk.