Microbial profiling of goat milk kefir and kefir grains by dual-marker amplicon sequencing (16S rRNA and ITS)

This study aimed to characterize and compare the bacterial and fungal communities associated with goat milk kefir (GMK) and kefir grains (KG) using a dual-marker amplicon sequencing approach targeting the full-length 16S rRNA and internal transcribed spacer (ITS) regions. Based on 16S rRNA and ITS analyses, a total of 137 and 356 bacterial operational taxonomic units (OTUs) and 16 and 71 fungal OTUs were detected in GMK and KG, respectively. Kefir grains exhibited a more diverse and taxonomically complex microbial community than goat milk kefir, as reflected by higher observed OTU richness and greater variability in Shannon diversity indices. At the phylum level, bacterial communities in both substrates were strongly dominated by Bacillota, accounting for nearly all bacterial sequences in GMK and approximately 98% in KG, although marked differences in community composition were observed at the genus level. Putative species-level assignments based on reference databases indicated that sequences affiliated with Lactobacillus kefiranofaciens in GMK and Lactobacillus delbrueckii in KG collectively represented approximately 45.30–88% of the total microbial abundance across samples. In contrast, minor taxa such as Pichia fermentans were detected at low relative abundance (approximately 2%), highlighting the compositional complexity of kefir-associated microbiota. Overall, these findings emphasize kefir grains as a stable multispecies microbial ecosystem and provide a molecular basis for the development of substrate-specific and functional kefir starter cultures derived from local microbial resources.