Water kefir multi-omics reveals functional redundancies despite taxonomic differences and the underappreciated contribution of yeast

Water kefir (WK) is a fermented beverage produced by a complex symbiotic community of microbes, including yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB). Here, we combined shotgun metagenomics, NMR metabolomics, GC-MS volatile organic compound (VOC) analysis, and metaproteomics to investigate microbial succession, functional dynamics, and the roles of yeasts and Zymomonas in WK fermentations representative of two WK types, i.e., one dominated by yeast-LAB-AAB and another by Zymomonas. Metagenomic profiling revealed that yeast-LAB-AAB communities exhibited dynamic microbial succession, whereas Zymomonas-dominated communities remained stable. Despite differing microbial compositions, both fermentations maintained consistent global metabolic functions, although specialized metabolic pathways and VOC profiles diverged. Metaproteomic analysis revealed a strong underappreciation of yeast contributions in metagenomic datasets, with yeasts representing a larger fraction of the proteome than predicted by DNA-based abundance. Lentilactobacillus hilgardii was enriched on WK grains, suggesting a specialized niche role. Our findings highlight the value of integrating multi-omics approaches to uncover microbial activity and community function in fermented foods and offer insights for the design of tailored WK starter cultures.