Investigation On Bacterial Community Diversity From High Temperature Daqu Of Sesame-flavor Liquor

The Sesame-flavor Liquor is one of innovative liquor types in China, which has been universally popular for its technology characteristics combined with multifold-flavor liquors and elegant sesame flavor. High temperature Daqu is a sacchariferous starter and it can provide large amounts of microorganisms, enzymes, flavor components and its precursors for brewing sesame-flavor liquor. So its quality is directly relevant to the output rate and quality of liquor. Due to the unique features of traditional fermentation technology and the limitation of research methods in this field, the knowledge of microbes community structure occurred in high temperature Daqu is still imperfect. Molecular biological and molecular ecological technologies were firstly used to reveal the bacterial community diversity from high temperature Daqu of Sesame-flavor liquor manufactured by Bandaojing Co. Ltd. Further, the results can also supply scientific data for study of the functional microbes’mechanism.Culture-independent analysis of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and16S rDNA V5-V8region clone library construction combined with culture-dependent analysis of screening and identification technologies were applied to investigate dynamic bacterial community diversity of high temperature making processes, including environment of Daqu room, Muqu, raw material, prophase fermentation, middle fermentation, late fermentation and new Daqu. And the high temperature functional bacterial also were isolated and identified in this study. The results indicated that abundant bacterial community mainly originated from Daqu room envrionment, Muqu and raw material. The bacterial community structure based high temperature bacteria were formed with the increase of fermentation temperature. The first dominant bacteria (abundant%) Bacillus sp.(21.67%) was detected at the stage of prophase, middle and late fermentation, and the abundant were13.7%,12.32%and23.15%separately. The first dominant bacteria (abundant%) Pantoea sp.(73.68%) in raw material was only found a few amount at prophase fermentation, the second dominant bacteria (abundant%) Weissella sp.(16.99%) and the third dominant bacteria (abundant%) Lactobacillus sp.(4.57%) were detected separately3.22%and11.29%at the prophase fermentation,6.89%and25.11%at middle fermentation,3.74%and7.47%at late fermentation. The first dominant bacteria (abundant%) Thermoactinomyces sp.(47.57%) at prophase fermentation was also detected at the middle fermentation, late fermentation and new Daqu as abundant of4.93%,15.68%and4.35%. The predominant bacteria (abundant%) in new Daqu was Rubellimicrobium sp.(31.52%).85thennophilic bacteria anti-high-temperature strains were selected according to plate screening and conical flask screening, we obtained a strain of higher neutral protease producing capability, whose enzyme activity reached to96.06U/g. This strain was identified to be Bacillus subtilis subsp. subtilis by16S rDNA and gryA sequencing. ZM60, the dominant thennophilic bacteria, was identified as Thennoactinomyces vulgaris by polyphasic identification. It growth at55℃and not growth at37℃, with white aerial mycelium and spore-forming. Zm60had enzyme activity of Alkaline phosphatase, Esterase, Lipid esterase and Naphthol-AS-BI-phosphate hydrolase. As the dominant bacteria in process of high temperature Daqu, the role of Thermoactinomyces vulgaris during the formation of particularity flavor of sesame-flavor liquor has not been reported before.This study not only found the dominant bacteria as Bacillus sp., Weissella sp. and Lactobacillus sp. reported before, but also detected predominant bacteria as Pantoea sp., Halomonas sp., Thermoactinomyces sp., Kroppenstedtia sp., Laceyella sp., Pediococcus sp. and Shigella sp. havn’t been reported before. Among these genera, Pantoea sp. was common endophytic bacteria, Thennoactinomyces sp. and Laceyella sp. were thennoactinomyces belong to family Bacillus, Kroppenstedtia sp. was new genus published in recent years and Shigella sp. was common pathogenic bacteria. The more research need to be applied in order to reveal the functional role of these dominant bacteria in the process of high temperature Daqu making.