Autochthonous bioaugmentation with Pediococcus pentosaceus MZ-4 enhances indigoid production by accelerating indican biotransformation and remodeling the fermentation microbiome

Indigo Naturalis, a traditional Chinese medicine for chronic myeloid leukemia, is plagued by long fermentation cycles, uncontrollable processes, and low levels of active ingredients. These inefficiencies are largely due to the undefined role of microbes in the fermentation process. Hence, this study investigated the key microbes involved in the fermentation of Indigo Naturalis and their functional characteristics and proposed an efficient fermentation strategy. Pediococcus pentosaceus MZ-4 was identified as a key strain promoting indican conversion and indigo(47.46%) and indirubin(58.19%) accumulation through β-glucosidase-mediated hydrolysis. MZ-4 inoculation markedly reshaped the microbial community by intensifying early selection and accelerating succession toward a stable lactic acid bacteria-dominated state. Pediococcus and Lactococcus became dominant and were associated with decreases in pH, dissolved oxygen, and oxidation-reduction potential, together with enhanced accumulation of indigo-type metabolites. Functional prediction indicated increased amino acid metabolism and secondary metabolite biosynthesis during fermentation. Whole-genome sequencing revealed abundant carbohydrate-active enzymes, including glycoside hydrolases, with GH1 family genes prominently represented, supporting their involvement in indican hydrolysis. Collectively, MZ-4 enhances Indigo Naturalis fermentation through coordinated microecological remodeling and glycoside hydrolysis, thereby accelerating precursor conversion and promoting indigo and indirubin biosynthesis. These findings provide a mechanistic basis for improving fermentation efficiency and standardizing Indigo Naturalis production.