Discrimination and Identification of Urea-Degrading Bacteria in the Fermented Grains of Chinese Nong-xiang Baijiu

Urea is the main precursor substance of the carcinogenic ethyl carbamate in the production of Baijiu. In this study, urea-degrading microbiota was revealed in the fermented grains from different fermentation cellars of the same brewing enterprise, by Illumina NovaSeq high-throughput sequencing combined with bioinformatics and the correlation analysis between the microbiota and urea. The results showed that the urea content of six fermented grains ranged from 60.89 to 72.29 mg/kg, and significant negative relations between urea content and Brevibacterium (rho=−0.7591), Streptococcus (rho=−0.7969), Veillonella (rho=−0.8522), and Pseudomonas (rho=−0.8023) were observed by Pearson correlation analysis. Furthermore, the function of bacterial microbiota in these fermented grains was mainly reflected in Metabolism, which occupied relative abundance of 80.59% by PICRUSt2 analysis. Notably, the relative abundances of carbohydrate metabolism and amino acid metabolism associated with urea degradation and urea cycle were 15.95% and 14.70%, respectively. Meanwhile, above four genera were positively correlated with urease (EC: 3.5.1.5), and the key enzymes including argininosuccinate synthetase (EC: 6.3.4.5), argininosuccinate lyase (EC: 4.3.2.1), and ornithine transcarbamylase (EC: 2.1.3.3) related to the urea cycle were negatively correlated with these genera by Pearson correlation analysis, which showed potential of these genera for urea degradation. Based on this, a strain capable of effectively degrading urea was isolated from the fermented grains, and preliminarily identified as Brevibacterium linens by molecular biology, and the preserved strain has been registered in the China Typical Culture Repository. Taken together, unveiling the urea-degrading bacteria in fermented grains could provide a theoretical foundation for urea and ethyl carbamate reduction in Baijiu fermentation by bioaugmentation.