Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation

Journal article, 2025

This study investigated the effect of bovine casein (CN) phosphorylation degree on micelle reassembly, stability, calcium-binding, and acid-induced gelation or precipitation, with the aim of assessing the feasibility of utilising microbial CNs in future dairy production. The four CNs (αS1-, αS2-, β-, and κ-CN) were purified from bovine milk and subjected to enzymatic dephosphorylation, resulting in three pools – fully and partially phosphorylated and fully dephosphorylated. Nine reassembled CN micelle solutions (RCMS) were investigated, consisting of three systems (two of four-CN, and one of β/κ-CN) and three phosphorylation degrees of the corresponding CNs. The distribution of different components (protein, calcium, and phosphorus) into micellar, serum, and insoluble fractions after two centrifugation steps was studied. The pH stability relative to acid-gelation or precipitation of the formed RCMS was further investigated. Results showed that the micelle reassembly ability of RCMS containing all four CNs was proportional to the phosphorylation degree; CNs with higher phosphorylation degrees contained a higher proportion of micelles and exhibited greater calcium-binding ability, whereas fully dephosphorylated CNs hardly formed CN micelle structures. The gelation pH of RCMS increased with decreasing phosphorylation degree, whereas the fully dephosphorylated CNs completely failed in gelation but precipitated when reaching their isoelectric point at pH 5.5. Moreover, RCMS containing only β/κ-CN at their native full phosphorylation were unstable at the salt concentrations applied, with more than half of the CNs self-associating to flocculate. Our study confirms the essential role of phosphorylation degree in micelle reassembly and stability, providing important information for potential future applications of microbial CNs that are expected to exhibit non-native phosphorylation levels.