Abstract: This study evaluated the effects of different doses of Ejiao hydrolysates (> 1 kDa) obtained after in vitro simulated digestion on acidification during in vitro anaerobic fermentation, short-chain fatty acid production, gut microbiota composition, and predicted microbial functions. The results showed that the addition of Ejiao hydrolysates accelerated acidification during the early stage of fermentation. Overall, acetic acid and propionic acid contents tended to increase, and butyric acid levels increased in the medium- and high-dose groups. Total short-chain fatty acid production also rose, with the most pronounced increase observed in the high-dose group. The α-diversity and β-diversity of the gut microbiota exhibited dose-dependent and differential changes in response to Ejiao hydrolysates. Principal coordinate analysis indicated a clear separation of microbial community structures between the treatment groups and the blank control group. At the phylum level, the microbial community was mainly composed of Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, and the relative abundance ratio of Firmicutes to Bacteroidetes exhibited divergent trends under different doses of Ejiao hydrolysates. At the genus level, specific taxa were enriched in the low-dose group, accompanied by a decrease in the relative abundance of Bifidobacterium, whereas the relative abundances of Bifidobacterium and Lactobacillus increased in the high-dose group. Linear discriminant analysis for effect size and functional prediction further suggested the presence of dose-specific microbial biomarkers and differences in multiple metabolism-related functional modules. In conclusion, Ejiao hydrolysates (> 1 kDa) can reshape gut microbiota composition and predicted microbial functions during in vitro anaerobic fermentation, exhibiting dose-dependent microbial response characteristics.

Key words: Ejiao; in vitro digestion and fermentation; 16S rRNA sequencing; gut microbiota; functional prediction