Milk composition shapes structural and microbial dynamics of kefir grain formation: Linking microbiota, metabolites, and grain architecture

The objective of this study was to investigate the mechanistic effects of different milk-derived substrates, cow milk, goat milk, and whey protein concentrate (WPC), on the structural development, microbial succession, and metabolite production of kefir grains during 28 d of continuous subculturing. Kefir grain morphology, microbial community dynamics, and substrate-driven metabolic shifts were analyzed using scanning electron microscopy, culture- and sequencing-based microbial profiling, and untargeted metabolomics. Despite initial differences among substrates, Lactobacillus kefiranofaciens became the dominant bacterium across all treatments, contributing to the structural and metabolic foundation of the grains. Its abundance was greatest in goat milk, intermediate in cow milk, and lowest in WPC, corresponding to differences in substrate-derived CN, peptides, and free AA. Fermentation resulted in a decrease in primary nutrients, such as lactose and palmitic acid, and an increase in secondary metabolites, including short-chain fatty acids, glycine, hydroxykynurenine, and 2',4'-dihydroxyacetophenone. These metabolites acted as cross-feeding substrates and ecological modulators, facilitating competitive and cooperative interactions among yeasts (Kazachstania turicensis, Kluyveromyces marxianus) and lactic acid bacteria (Lactobacillus kefiri, Leuconostoc mesenteroides). Substrate-specific microbial-metabolite networks influenced final kefir grain morphology: goat milk promoted filamentous, extracellular polysaccharide-rich grains; cow milk supported compact and stable grains; and WPC produced fragmented structures with altered metabolite profiles. These findings provide insights into microbial-metabolite interdependencies in kefir grain development and suggest strategies for substrate optimization and targeted starter culture design in functional dairy fermentation.