Synthetic microbial community mimicking kefir for investigating community dynamics and interspecies interactions

Kefir grains serve as natural dairy starter cultures, surviving in high-altitude environments with low temperatures and limited oxygen, while maintaining robust fermentation capabilities. In this study, we reconstructed a synthetic microbial community (SMC) within the kefir microbial ecosystem and explored the strategies that keep this SMC stable and functioning within the complex environment. We investigated the interactions among kefir species by comparing their symbiotic capabilities, milk acidification properties, and fermentation profiles during growth in both individual cultures and co-cultures across various media. Additionally, to deepen our understanding of system-level responses within the SMC, we integrated metabolomics with pure culture techniques to elucidate the mechanisms that enable coexistence among SMC members. The composition of the SMC in fermented milk was determined through co-cultivation assessments and flavor profile analysis, which identified the key members as Lactobacillus kefiranofaciens CZ22, Lactococcus lactis CZ19, and Saccharomyces cerevisiae Y8. The fermented milk produced by SMC shared identical volatile compound profiles with traditional kefir milk, including seven alcohols, seven aldehydes, six ketones, five esters, two carboxylic acids, two ethers, one acyl compound, and five miscellaneous volatile compounds. Our findings revealed that the coexistence mechanism among these three species is based on cross-feeding interactions. Lc. lactis CZ19 provides L. kefiranofaciens CZ22 with amino acids such as tyrosine, proline, and arginine, promoting its growth. Moreover, S. cerevisiae Y8 supplies primary metabolic products, including purines, pyrimidines, and nucleotides, to L. kefiranofaciens CZ22, facilitating the coexistence of all three species. During the fermentation process of the SMC, L. kefiranofaciens CZ22 maintained high abundance and accelerated acidification and enhanced flavor volatiles in milk. The SMC we constructed effectively maintained the core kefir species and fermentation performance of kefir starter cultures, simplified the complex fermentation system and laid the groundwork for the modernization and improvement of the production process. This study systematically elucidates the coexistence strategies employed by synthetic microbial systems in fermented milk production, while enhancing our understanding of microbial interactions in traditional fermented foods.