Bacillus thiaminolyticus in Chinese liquor and its applications

Abstract

The invention discloses a new strain of Bacillus thiaminolyticus for white liquor and its application. The strain is isolated from white liquor fermented grains and is a new strain of Bacillus thiaminolyticus for white liquor( Aneurinibacillus baijiui ), and its preservation number is GDMCC No. 65698. Tests have proved that the strain can produce white liquor flavor substances and can be used for fermenting and brewing liquor.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms and relates to Bacillus thiaminolyticus for Baijiu and its applications. Background Art

[0002] Baijiu is produced by a unique fermentation process and has a long history, being deeply loved by consumers. The production of Baijiu goes through a series of operations such as raw material cooking, saccharification, fermentation, and distillation. The production process of Baijiu is inseparable from brewing microorganisms. The types, quantities, and community structures of brewing microorganisms have important impacts on the flavor and quality of Baijiu. Brewing microorganisms mainly come from environments such as koji, pit mud, and fermented grains. These brewing microorganisms play very important roles in processes such as starch saccharification, alcohol fermentation, and the formation of flavor substances, and affect the generation of Baijiu flavors through complex microbial interactions. Baijiu contains various flavor substances, including various esters such as ethyl acetate, ethyl lactate, ethyl butyrate, and ethyl caproate, as well as various organic acids such as butyric acid and caproic acid. The formation of these flavor substances is related to brewing microorganisms, reflecting the key role of brewing microorganisms in the Baijiu fermentation process.

[0003] Baijiu fermented grains are solid materials to be distilled after the raw materials are crushed, cooked, and added with saccharifying and fermenting agents and fully fermented. The fermented grains have high microbial diversity, and there are interactions among microorganisms, which play important roles in the quality of the fermented grains and the generation of Baijiu flavor substances. There are a large number of microorganisms in a difficult-to-culture state in the fermented grains. Therefore, screening and developing new resources of brewing microorganisms in the fermented grains and studying the flavor substances they produce play important roles in enriching brewing microbial resources and improving the flavor of Baijiu. Summary of the Invention

[0004] A new species of Bacillus thiaminolyticus for Baijiu, REN35, was isolated from fermented grains in the present invention. Through identification at the morphological, physiological and biochemical, cytochemical, and molecular biological levels of the pure culture of this strain, it was found that the strain belongs to a new species of the genus Bacillus thiaminolyticus. This strain can produce various Baijiu flavor substances such as ethyl formate, 2,6-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-pentyl butyrate, and ethyl glycolate.

[0005] The present invention provides a Bacillus thiaminolyticus for Baijiu ( Aneurinibacillus baijiui ) with a preservation number of GDMCC NO.65698.

[0006] Specifically, it is cultured using NA medium, with shaking culture, the culture temperature is 25 - 35 °C, the pH is 6 - 8, and the sodium chloride concentration in the medium is 0.3 - 0.8%.

[0007] Preferably, the culture temperature is 30 °C, the pH is 7, and the sodium chloride concentration in the medium is 0.5%.

[0008] The present invention also provides a culture of the white wine thiamine bacillus obtained by the culture method.

[0009] The invention also provides application of the white wine thiamine bacillus in fermentation and wine making.

[0010] Specifically, it is used to produce liquor flavor substances, and the liquor flavor substances are selected from one or more of ethyl formate, 2,6-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-pentyl butyrate, and ethyl glycolate.

[0011] The present invention thus provides an additive for fermentation and winemaking, which is prepared from the white wine thiamine bacillus and is used to produce flavor substances in fermentation and winemaking.

[0012] The strain REN35 of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short) on December 31, 2024. The address of the depository unit is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the deposit number is GDMCC NO.65698, and the classification name is Baijiu Thiamine Bacillus ( Aneurinibacillus baijiui ). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Polar lipids in Bacillus thiaminiferum are shown.

[0014] Figure 2 A phylogenetic tree was constructed by selecting closely related model species based on sequence similarity. DETAILED DESCRIPTION

[0015] The basic components of the culture medium used in the examples are as follows:

[0016] Beef extract peptone medium (NA medium): 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, 15.0 g / L agar, and distilled water to volume.

[0017] Example 1: Isolation of Thiamine Bacillus in Liquor

[0018] The fermented grains used in the examples were sourced from a winery in Sichuan. 10 g of fermented grains were diluted to 10% with sterile distilled water. -5, 200 μL was taken and spread on NA medium for aerobic culture at 30 °C. After spreading, different morphological single colonies on the plate were picked every 2 - 3 days for isolation and purification. About 323 colonies were initially screened. This strain was obtained after aerobic culture at 30 °C for 48 h, and was purified 3 times by three - zone streaking to obtain purified cultured bacteria. The DNA of the purified cultured bacteria was extracted, and the 16S rRNA sequence was amplified using PCR technology. Through sequence alignment, the strain was determined to belong to the genus Thiaminobacillus. The pure culture was stored on a plate / slope of a test tube and by glycerol freezing preservation.

[0019] Example 2: Identification of Thiaminobacillus in Baijiu

[0020] The pure culture was identified at the morphological, physiological and biochemical, cytochemical, and molecular biological levels.

[0021] 1. Morphological characteristics

[0022] The pure culture was inoculated onto NA medium and the colony morphological characteristics were observed after 48 h of culture. It was observed that the colony surface was smooth, round, and milky white. Microscopic observation showed that the strain was rod - shaped and Gram - positive.

[0023] 2. Physiological and biochemical characteristics

[0024] The optimum growth temperature of Thiaminobacillus in Baijiu is 30 °C, the optimum growth pH is 7, and the optimum sodium chloride concentration is 0.5%. The strain can hydrolyze Tween 20 and gelatin. The strain is positive for catalase, oxidase, lipase, trypsin, α - galactosidase, β - galactosidase, β - glucuronidase, β - glucosidase, N - acetyl - glucosaminidase, and α - mannosidase.

[0025] 3. Cytochemical characteristics

[0026] The cytochemical components such as fatty acids, quinones, and polar lipids of Thiaminobacillus in Baijiu were detected by GC gas chromatography, HPLC liquid chromatography, and TLC thin - layer chromatography.

[0027] (1) The main fatty acid components of Thiaminobacillus in Baijiu are iso - C 15:0 , iso - C 16:0 , C 16:1 ω11c, C 16:0 , iso - C 17:0 , iso - C 16 : 1 H, C 16 : 1 ω5c, C 14:0 and C 16 : 1 ω7c alcohol. The cell fatty acid components are shown in Table 1.

[0028] Table 1 Main fatty acid components of Thiaminobacillus in Baijiu

[0029]

[0030] (2)The types and quantities of quinone compounds in microorganisms are important indicators of the diversity of the microbial community composition. After detection, the quinone compound of Bacillus thiaminolyticus in Baijiu is MK-7.

[0031] (3)There are 5 kinds of polar lipids in Bacillus thiaminolyticus in Baijiu (as Figure 1 shown), namely diphosphatidylglycerol, Dphosphatidylglycerol (DPG); phosphatidylethanolamine, phosphatidylethanolamine (PE); phosphatidylglycerol, phosphatidylglycerol (PG); unidentified phospholipids (PL1-3), phosphatidylcholine, phosphatidylcholine (PC).

[0032] 4. Molecular biology level

[0033] (1)Extraction of genomic DNA of Bacillus thiaminolyticus in Baijiu

[0034] The pure culture was inoculated into NA medium and cultured for 48 h, then the bacterial cells were picked and the genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit.

[0035] (2)Alignment of 16S rRNA sequence and construction of phylogenetic tree of Bacillus thiaminolyticus in Baijiu

[0036] The 16S rRNA sequence of the strain was amplified and sequenced using the universal primers 27F (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ IDNO.3: 5'-TACGGCTACCTTGTTACGACTT-3'). The 16S rRNA sequence of Bacillus thiaminolyticus in Baijiu was obtained ( Aneurinibacillus baijiui

[0037] The 16S rRNA sequence was aligned on NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The alignment results showed that the Bacillus thiaminolyticus of the present invention was Aneurinibacillus migulanus DSM2895 T and Aneurinibacillus aneurinilyticus ATCC 12856 T had the closest genetic relationship, both being 97.6%. According to the sequence similarity, the type species with a relatively close genetic relationship was selected for the construction of the phylogenetic tree. The phylogenetic tree is as shown in Figure 2 , indicating that the strain had the closest genetic relationship with the genus Bacillus thiaminolyticus and formed an independent branch, belonging to the genus Bacillus thiaminolyticus.

[0038] (3) Whole-genome sequencing of Bacillus thiaminolyticus in Baijiu

[0039] The whole genome of Bacillus thiaminolyticus in Baijiu was sequenced to obtain the whole-genome sequence. The whole-genome sequence was aligned with the most similar strains Aneurinibacillus migulanus DSM 2895 T and Aneurinibacillus aneurinilyticus ATCC 12856 T (http: / / ggdc.dsmz.de / home.php). The results showed that the genomic homology (DDH) of the Bacillus thiaminolyticus of the present invention with Aneurinibacillus migulanus DSM2895 T and Aneurinibacillus aneurinilyticus ATCC 12856 T was 19.7% and 19.5% respectively, both less than the threshold of 70%. Therefore, the new strain was initially identified as a new species of the genus Bacillus thiaminolyticus according to the generally recognized standard.

[0040] Example 3: Analysis of flavor substances produced by Bacillus thiaminolyticus in Baijiu

[0041] Bacillus thiaminolyticus in Baijiu was inoculated onto NA liquid medium and cultured with shaking for 48 hours. 10 mL of the culture solution was taken and transferred to a 50 mL centrifuge tube. After centrifugation, 5.0 mL of the supernatant was transferred to a gas chromatograph-mass spectrometer (GC-MS) vial, 2 g of sodium chloride and 20.0 μL of the internal standard 2-octanol at 99.07 mg / L were added, and the sample bottle was tightened. After the extraction head was aged at 250 °C for 30 min, it was inserted into the sample bottle, equilibrated at 40 °C for 30 min, extracted for 40 min, and the extraction head was desorbed at 250 °C in the GC injection port for 10 min.

[0042] HS-SPME / GC-MS running program: GC conditions: injection port temperature is 250 °C, the chromatographic column specification is DB-WAX (60 m × 250 μm, 0.25 μm, Thermo, USA), helium is used as the carrier gas, the flow rate is 1.0 mL / min, and splitless injection is performed; the temperature programming is as follows: hold at 40 °C for 3 min, increase to 150 °C at a rate of 4 °C / min, hold for 3 min, increase to 240 °C at a rate of 9 °C / min, and hold for 9 min. MS conditions: electron impact ionization source; electron impact energy 70 eV; transfer line temperature 230 °C; quadrupole temperature 150 °C; ion source temperature 230 °C; mass scan range m / z 35 - 600.

[0043] Through GC-MS flavor analysis, it was found that Bacillus thiaminolyticus in Baijiu can produce a large number of flavor substances (Table 2), including ethyl formate, 2,6-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-pentyl butyrate, ethyl glycolate, etc.

[0044] Table 2 Statistical table of flavor substances produced by Bacillus thiaminolyticus in Baijiu

[0045] 。

Claims

1. Aneurinibacillus baijiui, characterized in that, Its preservation number is GDMCC NO: 65698.

2. The culture method of Bacillus thiaminolyticus for Chinese liquor according to claim 1, characterized in that, It is cultured using NA medium, the culture temperature is 25 - 35 °C, the pH is 6 - 8, and the sodium chloride concentration in the medium is 0.3 - 0.8%.

3. The culture method of Bacillus thiaminolyticus in Chinese liquor according to claim 2, characterized in that, The culture temperature is 30 °C, the pH is 7, and the sodium chloride concentration in the medium is 0.5%.

4. The culture of Bacillus thiaminolyticus for baijiu obtained by the culture method according to claim 2 or 3.

5. The application of Bacillus thiaminolyticus for baijiu according to claim 1 in fermenting baijiu.

6. The application according to claim 5, characterized in that The Bacillus thiaminolyticus for baijiu is used to produce baijiu flavor substances, and the baijiu flavor substances are selected from one or more of ethyl formate, 2,6 - dimethylpyrazine, 2 - ethyl - 3,5 - dimethylpyrazine, 2 - pentyl butyrate, and ethyl glycolate.

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