Diversity, Enzyme Production and Antibacterial Activity of Bacillus Resource in Sesame-Flavored Liquor Daqu

Daqu provides enzymes and precursors for liquor fermentation, and is the core of liquor fermentation. In this study, 11 strains of Bacillus were isolated from sesame-avored liquor Daqu, which can not only produce protease and amylase, but also have antagonistic effects on common pathogens Escherichia coli and Staphylococcus aureus. According to the gyrA gene phylogeny analysis, these 11 Bacillus strains belong to three species, B1, Y14, Y15, and YPDW9 belong to Bacillus mojavensis, W7, W13, YPDW6, and YPDW12 belong to Bacillus subtilis, W14, Y5 and YPDW1 belong to Bacillus velezensis. According to the results of random amplied polymorphic DNA (RAPD) typing, these 11 Bacillus strains are completely different. The specic primers were used to randomly amplify the biological control genes expressing lipopeptide antibiotics (bioA, bmyB, ituc, fend, srfAA, srfAB, yngG and yndJ), and they all expressed different expressions in these 11 Bacillus strains. This research provides new ideas for strengthening Daqu and lays a foundation for improving the quality of liquor.


Introduction
Sesame-avored liquor is an innovative form of Chinese traditional liquor and has a unique style of "fragrant, soft and mellow". High-temperature fermentation of Daqu is the key to the special avor of sesame-avored liquor. Daqu is obtained through a complex fermentation process (Fan et al. 2020), made of corn, sorghum, wheat, etc. As a saccharifying agent for liquor fermentation (Tian et al. 2020), Daqu provides raw materials such as enzymes and avor substances for liquor fermentation. The microbial community of Daqu is the key to determine the liquor avor (Wang et al. 2017). During the fermentation process, microorganisms convert the organic macromolecules (mainly starch) in the raw materials into intermediate products such as pyruvate and amino acids, and nally produce special avor substances of liquor (Wang et al. 2011). In this experiment, when using traditional methods to isolate bacteria from sesame-avored liquor Daqu, it was found that Bacillus was dominant. Most of Bacillus has the ability to produce amylase and protease. In the Daqu brewing process, the main sources of microorganisms are food and airborne microorganisms, the pollution of common pathogenic bacteria such as E. coli is inevitable. According to reports, Bacillus can produce antibacterial peptides, bacteriocins, etc (Fira et al. 2018), which have inhibitory effects on common pathogens in Daqu.
In this study, a further antagonistic experiment was performed on enzyme-producing bacteria, and it was found that 11 Bacillus strains have antibacterial activity against E. coli and Staphylococcus aureus. These strains may also be potential probiotic resources. 11 Bacillus strains were classi ed and identi ed based on 16S rDNA identi cation, the gyrA functional gene was ampli ed, and then the random ampli ed polymorphic DNA (RAPD) was used for typing. Among the 11 strains of Bacillus, including 4 strains of Bacillus mojavensis, 3 strains of Bacillus velezensis, and 4 strains of Bacillus subtilis, they are all different. Finally, through antagonism experiment and biocontrol gene ampli cation, the antagonistic activity and biocontrol mechanism of 11 strains were studied. This research has made contributions to natural biocontrol resources and probiotic resources, provides new ideas for strengthening Daqu, and lays a foundation for improving the quality of sesame-avored liquor Daqu.

Identi cation of Protease and Amylase Activity of Bacillus
The bacteria were inoculated in LB Agar solid medium containing 1% skimmed milk powder and cultured at 32℃ for 24 hours to observe whether there was a transparent circle around the colony. A transparent circle means that the bacteria can produce protease.
The bacteria were inoculated on LB agar solid medium containing 1% starch and cultured at 32°C for 24 hours. Drop iodine solution on the culture medium and observe after staining. If the Bacillus has amylase activity, a transparent circle appears around the colony, otherwise the culture medium should all turn blue.
Study on the antibacterial activity of Bacillus against E. coli and Staphylococcus aureus E. coli was cultured in LB broth medium for 24 hours, 400 µL bacterial solution was added to 400 ml LB Agar medium cooled to 50 ℃, and the mixed medium was poured on the plate. Streak Bacillus on the pathogenic plate and observe whether there is a transparent inhibition zone around the colony.
Staphylococcus aureus was cultured in LB broth medium for 24 hours, 1 ml bacterial solution was added to 400 ml LB Agar medium cooled to 50 ℃, and the mixed medium was poured on the plate. Streak Bacillus on the pathogenic plate and observe whether there is a transparent inhibition zone around the colony.

Identi cation of Bacillus by gyrA
Based on the analysis of 16S rDNA, gyrA can be more effectively used for the identi cation and differentiation of Bacillus species. The DNA of Bacillus was extracted by freeze-thaw method, and the gyrA gene was ampli ed by PCR, and then sequenced. The primers used for gyrA are BS-gyrA-F (5'-CAGTCAGGAAATGCGTACGTCTT-3'), BS-gyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3'), and the reaction process was pre-deformation at 95 ℃ for 5 minutes, 35 cycles (95 ℃ for 1 minute, 55 ℃ for 1 minute, 72 ℃ for 1 minute), and nally 72 ℃ for 10 minutes. The reaction volume was 50 µL. The gyrA gene sequences of 11 Bacillus strains retrieved from GenBank were aligned using the program CLUSTAL. The neighbor-joining method of MEGA7 was used to construct a phylogenetic tree and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Kumar et al. 2016).
Broad-spectrum resistant Bacillus antagonistic gene ampli cation.
According to reports, bioA, bmyB, ituC, fenD, srfAA, srfAB, yngG and yndJ genes are common genes in Bacillus that can produce antibacterial substances (Cochrane et al. 2016). We cloned the biocontrol genes of these 11 Bacillus species, the primers used are shown in Table 1, and The reaction volume was 20 µL. The program is pre-denaturation 95°C for 5 minutes, 33 cycles (94°C for 1 minute, annealing temperature as shown in Supplementary Table 1, 72°C for 2 minutes), 72°C for 8 minutes. After ampli cation, 10 µL PCR products were taken and analyzed by 1% agarose gel electrophoresis, and then observed and analyzed by gel imager. Identi cation of Bacillus with high enzyme activity and antibacterial activity Based on the results of previous experiments, the accession numbers of 16S rDNA of 11 Bacillus strains with high enzyme production and antibacterial activity are shown in Table 2, The phylogenetic tree is shown in Fig. 1. The identi cation based on 16S rDNA and gyrA gene showed that 11 strains of Bacillus with high enzyme activity and antibacterial activity belong to three species respectively. Among them, B1, Y14, Y15, and YPDW9 belong to Bacillus mojavensis, W7, W13, YPDW6, and YPDW12 belong to Bacillus subtilis, and W14, Y5, and YPDW1 belong to Bacillus velezensis. The phylogenetic tree of gyrA is shown in Fig. 2.

RAPD typing of 11 bacteria with high enzyme activity and antibacterial activity
The RAPD molecular typing was used to further analyze 4 strains of Bacillus mojavensis, 4 strains of Bacillus subtilis, and 3 strains of Bacillus velezensis, and found that these 11 strains were of different types. The results are shown in Fig. 3.

Discussion
The dominant bacteria in Daqu of sesame-avored liquor mainly include Kroppenstedtia, Lactobacillus, Saccharopolyspora, Bacillus, Thermoactinomyces, and Weissella (Xie et al. 2020). Bacillus is a kind of heat-resistant microorganism, commonly found in mature Daqu, which can secrete a variety of hydrolytic enzymes, including amylase, protease, lipase, cellulase, glucanase, etc., used for the hydrolysis of macromolecules, and produce avor compounds in the brewing process (He et al. 2019). As early as 2010, Anissa Haddar et al. reported that Bacillus mojavensis A21 can produce at least 6 extracellular proteases (Haddar et al. 2010); Later in 2018, A Bacillus strain capable of simultaneously producing high alkaline protease and heat-resistant amylase was identi ed as Bacillus mojavensis (Hammami et al. 2018). In 2016, an author isolated a strain of Bacillus mojavensis BmB4 with broad-spectrum antibacterial properties from plant endophytic bacteria, which has antibacterial activity against E. coli., Salmonella typhi, and Staphylococcus aureus. After identi cation by LC-MS/MS, the bacterium can synthesize lipopeptides, surface proteins, rheumatic acid, fengmycin, and other antibacterial substances.
Bacillus subtilis is a dominant microorganism not only in liquor Daqu (He et al. 2019), but also in vinegar Daqu (Li et al. 2014) and yellow Jiuqu ). The high-temperature tolerance and high enzyme activity made Bacillus subtilis are important in liquor Daqu. Bacillus Subtilis can produce avor substances such as ethyl acetate and its derivative ligustrazine (Xu et al. 2018). Shen et al. added Bacillus subtilis LBM 10019 and Bacillus vallismortis LBM 10020 to the sorghum extract to increase the content of 2-furanthiol in sesame-avored liquor to ultimately produce l-cysteine to improve the avor of sesame-avored liquor quality . It is well known that Bacillus subtilis has good biocontrol effects, but there is little about liquor. The Bacillus subtilis and Bacillus amyloliquefaciens isolated from Maotai Daqu have natural antagonistic effects on Streptomyces. Streptomyces spp. producing geosmin is the most common and most serious source of pollution in liquor (Zhi et al. 2016). Some researches have proposed that lipopeptide antibiotics are Bacillus metabolites with broadspectrum antibacterial activity encoded by fenD, bmyB, ituC, yndJ, bioA, srfAA, srfAB, and yngG (Cochrane et al. 2016;Joshi et al. 2006). As a carrier for high-protease production, Bacillus has the antibacterial activity of the spectrum, but also the function of probiotics. Marie Lefevre and others mentioned that Bacillus subtilis CU1, as a newly discovered probiotic for the immune health of the elderly, is clinically safe and well tolerated, and has no adverse effects on liver and kidney function, vital signs, etc (Lefevre et al. 2016).
There have been few reports about Bacillus velezensis in Daqu. Wang et al(2018) discovered that Bacillus velezensis is a thermostable Bacillus during Daqu fermentation. He et al.(2019) strengthened Daqu by adding Bacillus velezensis and Bacillus subtilis and found that the liquefaction power, sacchari cation power, and esteri cation power of Daqu increased signi cantly, and also the content of volatile compounds such as alcohols, the esters, and pyrazines. Bacillus velezensis exist widely in nature, its metabolites are abundant and have spectral antibacterial activity. Fan et al.(2018) reported that Bacillus velezensis FZB42 had 13 gene clusters that can produce secondary metabolites with potential antibacterial effects, including the cyclic lipopeptides surfactin, bacillomycin, fengycin (Chen et al. 2009).
These substances can not only inhibit the growth of plant pathogens and fungi, but also Can induce plant systemic resistance. Yang et al.(2020) obtained 7 Bacillus velezensis strains from corn seeds that have good antagonistic effects against pathogenic maize strains and successfully cloned the biocontrol genes of Bacillus velezensis. In recent years, with the indepth research on the genetic, biological and physiological characteristics of Bacillus velezensis, more and more researchers have sequenced the whole genome sequence of this strain to obtain its biological control mechanism.
In a conclusion, this study explored the Bacillus in sesame-avored liquor Daqu in both enzyme production and antibacterial directions. This not only provides a direction for speeding up the fermentation process of Daqu, but also provides a theoretical basis for exploring the enzymes and precursors in liquor fermentation. Besides, this study also explored the reasons why 11 Bacillus strains can inhibit E. coli and Staphylococcus aureus from a genetic point of view, which is of great signi cance to deeply explore the function of Daqu microorganisms and to strengthen Daqu and liquor quality control.   Schematic diagram of Bacillus RAPD molecular typing. A, B, C, D, and E represent 5 different random sequences. According to the ampli cation of 5 random sequences, B1, Y14, Y15, and YPDW9 were identi ed as different strains, W7, W13, YPDW6, and YPDW12 were different strains, and W14, Y5, and YPDW1 were different strains. Cloning results of 8 biocontrol genes bioA (273bp), bmyB (308bp), ituC (575bp), fenD (293bp), srfAA (395bp), srfAB (210bp), yngG (212bp) and yndJ (372bp) from Bacillus. A represents 4 Bacillus mojavensis strains; B represent 4 Bacillus subtilis strains, and C represents 3 Bacillus velezensis strains.

Declarations
In each electrophoresis result, the position of the biocontrol gene is marked with a red arrow.