Artificial casein micelles and the road towards animal-free cheese

Summary

Caseins are the main proteins in mammalian milk and form the structural foundation of most cheeses. In milk, they naturally assemble into casein micelles, which are crucial for cheesemaking. Precision fermentation enables the production of animal-free caseins, but these proteins are produced individually rather than in micellar form. Artificially assembling recombinant caseins into micelles could offer a route to leverage traditional dairy processing techniques to produce animal-free cheese with similar quality to conventional cheese. This thesis investigates the assembly of caseins into functional artificial casein micelles, establishing guidelines for their efficient production and application in animal-free cheese. Three key objectives guide the research: (1) identifying necessary caseins and post-translational modifications for micelle formation, (2) customising micelle properties through process conditions, and (3) developing scalable methods for their production.---Chapter 2 demonstrates that artificial casein micelles formed by mixing bovine caseins with salts at 37°C over one hour, resemble natural casein micelles in composition, size, hydration, and chymosin-induced coagulation. Faster assembly leads to irregular micelles with impaired coagulation and curd plasticisation. Chapter 3 examines how assembly temperature (5–65°C) affects micelle properties, showing that intermediate temperatures optimise micelle formation and curd firmness, while high temperatures lead to weaker curds but more stable foams. These findings highlight that assembly conditions permanently influence micelle functionality.---Chapters 4 and 5 investigate compositional requirements for functional artificial micelles. Chapter 4 reveals that dephosphorylated casein does not form micelles effectively, instead stabilising calcium phosphate nanoclusters into larger, less hydrated colloidal particles with poor rennet coagulation. This underscores the necessity of phosphorylated caseins for food applications relying on micelle-based structures. Chapter 5 explores micelle formation from different bovine casein blends, demonstrating that alphas- and kappa-casein yield dense micelles and firm gels, while beta- and kappa-casein produce looser micelles and softer gels. Higher kappa-casein levels result in smaller micelles and firmer curds, whereas increased beta-casein leads to more hydrated micelles and weaker gels. Additionally, bovine kappa-casein can be substituted by dephosphorylated beta-casein and a recombinant beluga whale kappa-casein.---Chapters 6 and 7 propose alternative micelle formation methods beyond controlled mixing. Chapter 6 introduces concentration-driven processes such as vacuum evaporation, forward osmosis, and reverse osmosis to induce micelle assembly. These methods yield micelles with superior coagulation properties compared to the mixing process and offer advantages in efficiency and scalability. Chapter 7 presents an alternative approach using calcium-hexametaphosphate (HMP) complexes to cross-link caseins, forming stable micelles at a 10:1 calcium-to-HMP ratio. However, excess HMP impairs micelle formation by sequestering calcium.---In summary, artificial casein micelles form when insoluble calcium phosphate precipitates in the presence of at least one calcium-sensitive (phosphorylated) and one calcium-insensitive, amphipathic casein. Beyond conventional mixing, micelles can be produced via concentration-based processes or HMP cross-linking. Their composition and preparation conditions dictate coagulation behaviour, enabling both replication of bovine casein micelles and customisation. While the road to animal-free cheese also requires an alternative fat phase and considering sustainability and economics, advancements in precision fermentation and the creation of artificial micelles could herald a revolution in the dairy industry.