Bacillus multifaciens sp. nov., a Crucial and Highly-Active Flavor and Protease Producer Isolated from the qu-Starter of Chinese Wuliangye Baijiu

In the study presented herein, an aerobic, Gram-stain-positive, spore-forming bacterium, designated as WLY-B-L8^T, was isolated from a <i>qu</i>-starter (<i>baobaoqu</i>) cultivation facility used for the production of Wuliangye baijiu in Yibin city (Sichuan province, China). The strain comprised short, rod-shape cells of 1.2-1.9 μm in width and 1.7-4.8 μm in length, arranged singly or in pairs. The isolate was able to grow at temperatures of 20-42 °C (optimum growth at 40 °C), pH 5.0-10.0 (optimum growth at pH 8.0), and in the presence of 0-2% (<i>w</i>/<i>v</i>) NaCl (optimum growth with 1% NaCl). Ribose, xylose, arabinose, mannose, glucose, and galactose constituted the major cell-wall sugars. Moreover, <i>meso</i>-diaminopimelic acid (<i>meso</i>-DAP) constituted the diagnostic amino acid. The main polar lipids of WLY-B-L8^T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified aminolipids (UAL 1-2), an unidentified aminophospholipid (UAPL), an unidentified aminoglycolipid (UAGL), and an unidentified lipid (UL). MK-7 was the predominant menaquinone and <i>iso</i>-C_15:0 (23.00%) was the major fatty acid. Comparisons of the 16S rRNA gene sequence indicated that WLY-B-L8^T was most closely related to <i>Bacillus rhizoplanae</i> JJ-63 DSM 12442^T (98.71%), <i>Bacillus pseudomycoides</i> DSM 12442^T (98.21%), and <i>Bacillus cytotoxicus</i> NVH 391-98^T (98.14%). The average nucleotide identity (ANI) values of strain WLY-B-L8^T and the three type strains mentioned above were 88.24%, 80.57%, and 78.70%. The average amino identity (AAI) values between them were 89.84%, 79.51%, and 80.41%. In addition, the digital DNA-DNA hybridization (dDDH) values between them were 36.70%, 26.10%, and 23.90%. The genomic DNA G+C content was 35.97%. Based on the evidence presented herein, WLY-B-L8^T (CICC 25210^T = JCM 36284^T) exhibits promise as the type strain of a novel species, designated as <i>Bacillus multifaciens</i> sp. nov., that can produce protease (119.38 ± 7.44 U/mL) and volatile flavor components when cultured on raw wheat, such as 2-pipendinone (21.95 ± 1.56 mg/L), phenylethyl alcohol (19.08 ± 0.82 mg/L), hydrocinnamic acid (18.60 ± 0.53 mg/L), and acetoin (7.58 ± 0.11 mg/L).