Spatio-temporal-functional assembly of Daqu microbiome: Mechanistic insights and quality improvement

Daqu is the fermentation starter of traditional Chinese vinegar production. However, its quality is location-specific, experience-dependent, and batch-differential. This study aims to unveil the physiochemical and microbial dynamics of Daqu during fermentation and improve the qualities of Daqu as well as vinegar through engineering the microbiota. Layered and mixed Daqu samples were collected at six fermentation stages and subjected to physiochemical and microbiota analysis. Correlation analysis revealed that the Daqu microbiota-driving factors changed from daily average temperature and contents of water and soluble protein to accumulated temperature, pH, and reducing sugar content, resulting in the higher activities of acidic protease, amylase, cellulase, and xylanase at early stages and higher glucoamylase, pectinase, and esterase activities at late stages. Null-model inference based on the beta-nearest taxon index (βNTI) and the Bray-Curtis-based Raup-Crick metric (RCbray) revealed different assembly patterns in Daqu communities, with stronger deterministic assembly of bacterial communities and ecological drift in fungal communities. The core microbiota consisted of molds (Aspergillus, Thermoascus, Rhizopus, Rhizomucor, and Rasamsonia), yeast (Saccharomycopsis), actinomycete (Saccharopolyspora), and bacilli (Bacillus, Lactilactobacillus, Leuconostoc, and Weissella). Using the culturomics strategy, 443 distinct strains confined into 40 genera and 74 species were isolated, and 59 of them showed significant protease and/or carbohydrase activities. A synthetic microbial consoria (SynCom) of five strains (Aspergillus oryzea, Saccharomycopsis fibuligera, Lichtheimia ramosa, Bacillus velezensis, and Lactilactobacillus plantarum) was prepared referring to the core microbiota composition and hydrolytic performance, which supplementation significantly improved the Daqu quality as well as the fermentation efficacy for vinegar brewing, including higher activities of glucoamylase and protease, higher yields of alcohol, organic acids, and amino acid nitrogen, and better sensory properties. The SynCom-modified microbiome with low microecological vulnerability and upregulated metabolism of carbohydrates, amino acids, and lipids might be the underlying reason. This study advances the Daqu microbiome research and offers valuable ideas to engineer the Daqu microbiota for improved qualities of Daqu and vinegar.