Successive fermentation selects for reduced microbial diversity and heterofermentative dominance in milk kefir fermentation

Successive grain reuse imposed directional selection that reduced ASV richness, promoted Lactobacillales dominance, shifted citrate metabolism toward acetate production, and altered volatile profiles. This study integrated genomic, physicochemical, and sensory analyses to evaluate the effects of repeated kefir grain reuse on milk kefir fermentation performance and product quality. Three kefir beverages were produced using the same grains after approximately 100 (K1), 150 (K2), and 200 (K3) successive fermentation cycles. Amplicon sequencing of the 16S rRNA and ITS regions revealed a progressive reduction in bacterial and fungal diversity with increasing grain reuse, particularly in K3, accompanied by dominance of members of the order Lactobacillales and the fungal family Saccharomycetaceae. These microbial shifts were associated with marked changes in fermentation metabolism, including reduced citric acid levels and increased acetic acid accumulation, indicating a shift toward heterofermentative activity. Additional differences were observed in nutritional components, including vitamins and proteins. Sensory evaluation indicated that K2 was the most accepted beverage, coinciding with higher microbial diversity and the presence of aroma-active compounds such as butanoic and hexanoic acids, nonanone, and heptanone. In contrast, K3 exhibited elevated acetic acid levels, which were associated with lower consumer acceptance. Overall, the results demonstrate a consistent relationship in which successive grain reuse reduces microbial diversity, promotes heterofermentative dominance, alters fermentation outputs, and ultimately affects sensory perception. These findings demonstrate that repeated back-slopping acts as a deterministic ecological filter in complex starter communities.