Abstract
Natural butter possesses a mild aroma that can be enhanced through biotransformation into cheese-flavored bases. This study systematically optimized hydrolysis and fermentation conditions using four proteases, lipase, and Lactococcus lactis. Initial single-factor experiments suggested that the lipase-to-protease ratio, hydrolysis temperature and duration, and starter culture inoculum level significantly affected flavor development. Response surface methodology (RSM) was used to further refine the enzymatic hydrolysis and fermentation conditions for the four treatment groups. GC-MS analysis identified 218 volatile metabolites across all samples, with esters, acids, and ketones being the predominant volatile classes in enzymatically hydrolyzed and fermented butter. Notably, distinct flavor profiles were observed for each of the four processes studied. Metabolic pathway analysis confirmed that these characteristic flavors originated from the synergistic interaction of proteases, lipase, and L. lactis. These findings provide a theoretical basis for the development and application of natural cheese-flavored essences in the food industry.