Daqu Fermentation Selects for Heat-Resistant Enterobacteriaceae and Bacilli

摘要

Daqu is a spontaneous solid-state cereal fermentation used as saccharification and starter culture in Chinese vinegar and liquor production. The evolution of microbiota in this spontaneous fermentation is controlled by the temperature profile, which reaches temperatures from 50 to 65 degrees C for several days. Despite these high temperatures, mesophilic Enterobacteriaceae (including Crono-bacter) and bacilli are present throughout Daqu fermentation. This study aimed to determine whether Daqu spontaneous solid-state fermentation selects for heat-resistant variants of these organisms. Heat resistance in Enterobacteriaceae is mediated by the locus of heat resistance (LHR). One LHR-positive strain of Kosakonia cowanii was identified in Daqu, and it exhibited higher heat resistance than the LHR-negative K. cowanii isolated from malted oats. Heat resistance in Bacillus endospores is mediated by the spoVA(2mob) operon. Out of 10 Daqu isolates of the species Bacillus licheniformis, Brevibacillus parabrevis, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velezensis, 5 did not contain spoVA(2mob), 3 contained one copy, and 2 contained two copies. The presence and copy number of the spoVA(2mob) operon increased the resistance of spores to treatment with 110 degrees C. To confirm the selection of LHR-and spoVA(2mob)-positive strains during Daqu fermentation, the copy numbers of these genetic elements in Daqu samples were quantified by quantitative PCR (qPCR). The abundance of LHR and the spoVA(2mob) operon in community DNA relative to that of total bacterial 16S rRNA genes increased 3-fold and 5-fold, respectively, during processing. In conclusion, culture-dependent and culture-independent analyses suggest that Daqu fermentation selects for heat-resistant Enterobacteriaceae and bacilli.