Matrix-associated spatial structure contributes to compositional stability of a complex multispecies starter culture under microbial perturbation: Insights from kefir grains.

Complex multispecies starter cultures can enrich the metabolic and sensory diversity of fermented foods, but industrial use is limited by compositional drift, overgrowth of dominant taxa, and vulnerability to exogenous microbial contamination. Kefir grains (KG), a matrix-encapsulated complex starter culture with stable serial propagation, provide a model for how spatial structure contributes to starter stability. We compared matrix-associated KG with free kefir-fermented milk (FKFM), representing a grain-free liquid-phase kefir microbial community, under a large-scale microbial challenge. During the acute 24 h challenge, target-taxon drift estimated from PMA-qPCR data was lower in KG than in the liquid compartments. Ten-generation serial transfer caused stronger community restructuring and Shannon diversity loss in FKFM, whereas KG showed constrained compositional adjustment and remained relatively clustered in Bray-Curtis ordination space. Species-level metagenomic tracking showed broader expansion, retention, and cumulative drift of species-level signals corresponding to exogenous challenge strains in FKFM, while more of these signals in KG followed restricted/stable trajectories. Kefir cell-free supernatant inhibited multiple challenge strains beyond the effect of low pH, indicating a contribution of diffusible inhibitory factors. SEM and live/dead staining revealed rough matrix-associated surfaces and heterogeneous cell states in KG. Together, the results support a model in which spatial structure, prior niche occupancy, and diffusible inhibition jointly contribute to the compositional stability and anti-perturbation capacity of this complex starter culture, informing the design of spatially structured synthetic starter consortia for industrial fermentation.