Casein micelle formation as a calcium phosphate phase separation process: Preparation of artificial casein micelles through vacuum evaporation and membrane processes

Industrial-scale production of artificial casein micelles (ACM) is required to produce dairy alternatives from recombinant casein. However, the currently common micelle preparation method of dropwise mixing casein and salt solutions is inefficient and may prove difficult to scale up. Here, we view casein micelle formation as a process driven by calcium phosphate phase separation in the presence of casein. On this basis, we developed novel routes to prepare ACM through vacuum evaporation, forward osmosis, or reverse osmosis. ACM prepared through these methods have similar properties and improved coagulation behaviour compared to those prepared through the currently common method and natural bovine casein micelles. The properties and functionality of the micelles depend on the preparation time and surface area available for micelle formation, with longer times and larger surfaces (i.e. lower fluxes) yielding smaller ACM that form firmer curds. These novel processes enable fast, efficient, and continuous production of ACM for application in future dairy alternatives. Industrial relevance: Artificial casein micelles can be used as a building block in the production of animal-free milk and cheese based on precision fermentation. The herein described novel processes to prepare artificial casein micelles are based on vacuum evaporation, forward osmosis, and reverse osmosis, which are mild, resourceefficient, and easily scalable processes. The processes require a dilute feed stream (e.g. caseins after precision fermentation), provide an elegant way to minimise local differences in the concentration of caseins and ions during micelle production, and offer the opportunity to design continuous micelle formation processes. These are all advantages over the existing methods to prepare artificial casein micelles with regard to industrial application.