β-lactoglobulin from precision fermentation for improved heat stability

Dairy proteins have long been appreciated for their techno-functional and nutritional properties. With precision fermentation already established as a viable technology for ingredient production, there is growing interest in producing recombinant analogues of dairy proteins. This approach allows for the development of isolates containing specific protein variants, which can be bioequivalent or engineered for enhanced functionality. The functionality of such recombinant dairy protein isolate is then driven by the protein’s molecular characteristics and isolate purity. This study aims to compare the heat stability and aggregation behaviour of precision fermentation-derived β-lactoglobulin (fBLG) to milk-derived β-lactoglobulin (mBLG) and commercial whey protein isolate (WPI). Using a multilength scale approach, we investigated how molecular and physicochemical characteristics influence heat stability and aggregation behaviour, amongst others under UHT-mimicking conditions. At the molecular level, although small differences were detected in the primary structure between fBLG and mBLG, structural equivalence was confirmed across secondary, tertiary and quaternary structural levels. On a mesoscale, fBLG demonstrated comparable heat stability to mBLG and WPI under acidic pH conditions, while exhibiting superior heat stability at neutral pH. This enhanced stability was related to the molecular characteristics of fBLG and further supported by electrophoretic mobility measurements. The aggregate characteristics and viscosity contribution after heating were also evaluated. It was found that the heated fBLG had a lower viscosity contribution and specific volume compared to mBLG. Finally, the heat stability of fBLG was evaluated in mineral-fortified model food systems.  Our findings suggest that even minor variations in the primary structure can significantly impact ingredient functionality in food applications. These insights could encourage further research into precision fermentation as a tool for developing protein variants tailored to specific functionalities.