Microbial Dynamics of Yogurts with Different Starter Cultures Under an In Vitro Gastrointestinal System Using 16S rRNA Sequencing

Abstract

Background: The aim of this study was to compare microbial diversity and compositional changes under digestive stress in yogurts produced using different culture strategies in a dynamic in vitro gastrointestinal system. Methods: Yogurts produced with probiotic starter cultures, standard yogurt cultures, and probiotic sachet supplementation were evaluated using a dynamic in vitro gastrointestinal system including mouth, stomach, and small intestinal phases under controlled pH, temperature, and digestion conditions. Microbial diversity and compositional changes before and after the in vitro gastrointestinal simulation were determined using a 16S rRNA amplicon-based Oxford Nanopore sequencing approach, and the resulting data were analyzed using bioinformatics, alpha-diversity, and beta-diversity metrics. Results: Probiotic sachet addition significantly increased microbial species richness, alpha diversity, and community balance compared to starter culture yogurts (Shannon and Inverse Simpson, p < 0.05). In vitro gastrointestinal system led to a reduction to a Firmicutes-dominant core microbiota in all samples; in contrast, beta diversity and PCoA analyses showed that the post-digestive microbial structure of sachet-supplemented yogurts was significantly different (PERMANOVA, p = 0.001). Conclusions: Consequently, it was demonstrated that adding probiotic sachets to yogurt increases the diversity and resilience of beneficial bacteria throughout the digestive tract, making the product a more robust and functional food for health.