Impact of micellar calcium phosphate concentration on the casein micelle structure, stability and functionality

Caseins, the major group of milk proteins, are individually disordered proteins with a great degree of hydrophobicity and low stability in an aqueous medium, thus they combine in a very complex structure termed the casein micelle. Many properties of milk depend on the state of the casein micelles, and the presence of calcium phosphate clusters in these casein micelles has a vital impact on the structure of the protein particles as well as their technological functionality. Calcium distribution between the colloidal and soluble phases is influenced by environmental conditions like temperature, pH, or the addition of chelators, all of which can modify the amount of micellar calcium phosphate (MCP). Changes in the micellar calcium phosphate (MCP) level within the casein micelle influences the structure and properties of casein micelles, potentially expanding the application range of milk ingredients in various domains such as food, cosmetics, and medicine. Thus, the primary aim of the study was to examine the influence of varied micellar calcium phosphate concentrations on the structure, stability, and integrity of the casein micelle. To achieve this, the micellar calcium phosphate (MCP) content of the casein micelles was modified through pH adjustment followed by dialysis. The study assessed the turbidity, zeta potential, and particle size distribution of casein micelles, along with the partitioning of calcium and milk proteins between the colloidal and soluble phases of the obtained milk. Additionally, changes in the secondary structure of proteins resulting from the adjustment of MCP content were examined using Fourier Transform Infrared (FTIR) spectroscopy. Furthermore, protein structure was characterized through 1H NMR, while organic and inorganic phosphorus were investigated using 31P NMR. Results showed that MCP adjustment had a notable effect on the calcium concentration, with its amount being significantly reduced by lowering the pH of initial adjustment below 6.7 or enriched by increasing the pH above 6.7. Smallest particle size was observed in the sample with the greatest reduction in MCP content. Concentration of individual caseins in the soluble phase gradually increased with decreasing MCP levels, indicating casein micelle disintegration during adjustment. At ~60% MCP removal, FTIR revealed a critical stage of structural rearrangement, and 31P NMR exhibited an increase in signal intensity for calcium-free Ser-P, further intensifying with decreasing MCP concentration. This study underscored the significance of MCP in preserving micellar structure and its influence on the integrity of the casein micelle. In the subsequent phase, the thermal stability of MCP-adjusted skim milk samples was studied, given that heat treatment is a common practice in the dairy industry and the thermal stability of milk proteins holds significant importance in these processes. Micellar calcium phosphate plays a crucial role in maintaining the stability of milk proteins. In this regard, four MCP-adjusted samples, comprising 67%, 96%, 100%, and 113% of the original MCP content, were subjected to heat treatment (90 °C for 10 min) at different pH values (6.3, 6.6, 6.9, and 7.2), with subsequent analysis of particle size, turbidity, protein distribution, and structure. The results revealed a substantial influence of MCP level and pH on heat-induced alterations in milk properties, with MCP67 samples demonstrating the highest thermal stability. Precisely, a 33% reduction in MCP content (MCP67) exhibited a less pronounced increase in non-sedimentable κ-casein and a reduced decrease in αs2-casein concentrations after heating, in contrast to other samples. The lower MCP content contributed to a moderate increase in average particle size and turbidity, accompanied by lower loading of the β-turn structural component after heating at low pH (6.3). In contrast, MCP113 exhibited instability during heating, evidenced by an increase in particle size, turbidity, notable changes in protein concentrations, and a slight elevation in non-sedimentable κ-casein concentration. FTIR results revealed higher loading of intermolecular β-sheet, β-turn, and random coil structures, along with lower loading of α-helix and β-sheet structures in MCP-enhanced skim milk samples. These observations imply substantial alterations in the secondary structure of milk proteins and an enhanced formation of larger aggregates. This thermal stability is vital in diverse dairy applications, especially in procedures such as ultra-high temperature (UHT) treatment and other sterilization processes. The application of intense temperatures in sterilization (115–120°C for 5–15 min) or UHT (135–150°C for 1–10 s) induces various structural alterations and can pose challenges and concerns in the industry, including protein denaturation, the Maillard reaction, vitamin degradation, and sediment formation on surfaces. Considering the intricate relationship between heat stability and micellar calcium phosphate (MCP) content, the present study investigated the thermal stability of MCP-adjusted skim milk samples including MCP67 (33% MCP-depleted), MCP113 (13% MCP-enriched), and the control (MCP100) at 120°C for 5 second and 140°C for 1 second across pH levels 6.3-7.2. MCP67 exhibited the least decrease in non-sedimentable individual casein levels, displaying lowest particle size and turbidity after heating, indicative of exceptional thermal stability. Conversely, MCP113 displayed the highest decrease in these casein levels, with the highest particle size and turbidity, implying comparatively lower thermal stability. Heating at both 120°C and 140°C at pH6.3, MCP-enhanced and native skim milk samples demonstrated coagulation, while MCP-depleted skim milk remained stable. The results reveal the substantial influence of MCP and pH level on heat-induced alterations in UHT milk. Moreover, the acid-induced gelation process in milk is a crucial step extensively employed in the production of yogurt and various other fermented dairy products. The amount of micellar calcium phosphate has a substantial impact on the sensory and functional properties of acid-induced gels. Therefore, this study explored how the concentration of micellar calcium phosphate (MCP) and the extent of heating at different pH levels influence the properties of acid-induced gels. Three MCP-adjusted samples, ranging from 67 to 113% of the original MCP content, heated at two temperatures (80 or 90°C) for 10 minutes at various pH values (6.3, 6.6, 6.9, or 7.2). Gelation was initiated by adding GDL at 30°C and stopped when the pH reached 4.5. The distribution of calcium and proteins between colloidal and soluble phases was examined, and small amplitude oscillatory rheology was employed to determine viscoelastic behavior. In skim milk samples depleted of micellar calcium phosphate (MCP), the concentrations of non-sedimentable caseins and whey proteins were found to be higher compared to both control and MCP-enhanced skim milk samples. The influence of MCP adjustment on gelation was contingent upon pH variations. Notably, a 33% MCP-depleted sample exhibited the greatest G' at low pH among all samples, while at other pH levels, MCP100 resulted in the maximum G' surge. Furthermore, the readjusted pH of skim milk played a significant role in gel properties after heating. At alkaline pH values (6.9, 7.2), higher temperatures led to a notable reduction in gelation time. Conversely, at lower pH values (6.3, 6.6), the heating temperature had no observable impact on either gelation time or gelation pH. In summary, this study underscores the substantial impact of MCP adjustment on acid gelation, highlighting a pronounced dependency of the MCP adjustment effect on pH variations. Overall, this project has established that the combination of FTIR, 1H NMR, and 31P NMR spectroscopies testing holds significant potential as useful tools for achieving a comprehensive understanding of the conformational changes occurring due to micellar calcium phosphate (MCP) adjustment. These changes impact the structure, integrity, and stability of casein micelles in skim milk. Additionally, the study contributes to a better understanding of the role of MCP in various milk processing methods, including heating, ultra-high temperature treatment, and acid-induced gelation. This newfound knowledge can be particularly beneficial in the dairy industry.