Baijiu thiamine bacillus and application thereof

Abstract

The invention discloses a novel Baijiu bacillus thiamine strain and application thereof. The bacterial strain is separated from fermented grains of Baijiu and is a new strain of Baijiu thiamine bacillus (Aneurinibacillus baijiui), and the preservation number of the bacterial strain is GDMCC (China General Microbiological Culture Collection Center) No.65698. Tests prove that the strain can generate white spirit flavor substances and can be used for fermenting and brewing wine.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to liquor thiamine bacillus and applications thereof. Background Art

[0002] Baijiu adopts a unique fermentation production process, has a long history, and is deeply loved by consumers. The production of baijiu is made 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 type, quantity and community structure of brewing microorganisms have an important influence on the flavor and quality of baijiu. Brewing microorganisms mainly come from environments such as koji, cellar mud and fermented grains. These brewing microorganisms play a very important role in the processes of starch saccharification, alcohol fermentation and the formation of flavor substances, and affect the production of baijiu aroma through complex microbial interactions. Baijiu contains a variety of flavor substances, including esters such as ethyl acetate, ethyl lactate, ethyl butyrate and ethyl caproate, as well as a variety of 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 fermentation process of baijiu.

[0003] Liquor mash is a solid material that is prepared for distillation after the raw materials are crushed, boiled, and saccharified and fermented. The mash has a high diversity of microorganisms and there are interactions between the microorganisms, which plays an important role in the quality of the mash and the production of liquor flavor substances. The mash contains a large number of microorganisms that are difficult to culture. Therefore, screening and developing new resources of mash brewing microorganisms and studying the flavor substances they produce are important for enriching the brewing microbial resources and improving the flavor of liquor. Summary of the invention

[0004] The present invention separates a new species of white wine thiamine Bacillus REN35 from fermented grains. The pure culture of the strain is identified at the morphological, physiological and biochemical, cytochemical and molecular biological levels, and it is known that the strain belongs to a new species of the genus thiamine Bacillus. The strain can produce a variety of white wine flavor substances such as ethyl formate, 2,6-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-pentyl butyrate, ethyl glycolate, etc.

[0005] The present invention provides a liquor thiamine bacillus ( Aneurinibacillus baijiui ), whose deposit number is GDMCC NO.65698.

[0006] Specifically, it adopts NA medium for culture, shaking culture, culture temperature is 25-35°C, pH is 6-8, and the concentration of sodium chloride in the culture 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 culture 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: 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 make up to volume.

[0016] Example 1: Isolation of Thiamine Bacillus in Liquor 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, take 200μL and spread it on NA medium and culture it aerobically at 30℃. After the coating, pick single colonies of different morphologies on the plate every 2-3 days for separation and purification. A total of about 323 colonies were initially screened. This strain was obtained after aerobic culture at 30℃ for 48h. It was purified three times by three-zone line to obtain purified cultured bacteria. Extract the purified cultured bacterial DNA, amplify the 16S rRNA sequence by PCR technology, and determine that the strain belongs to the genus Thiamine Bacillus by sequence comparison. Pure culture is stored on plates / test tubes and frozen in glycerol.

[0017] Example 2: Identification of Thiamine Bacillus in Liquor The pure culture was identified at the morphological, physiological and biochemical, cytochemical and molecular biological levels.

[0018] 1. Morphological characteristics The pure culture was inoculated into NA medium and cultured for 48 hours before observing the morphological characteristics of the colonies. The colonies were found to be smooth, round, and milky white in color. Microscopic observation showed that the strain was rod-shaped and Gram-positive.

[0019] 2. Physiological and biochemical characteristics The optimum growth temperature of Bacillus thiamine in white wine is 30℃, 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-uronidase, β-glucosidase, N-acetyl-glucosaminidase and α-mannosidase.

[0020] 3. Cytochemical characteristics The cellular chemical components such as fatty acids, quinone compounds and polar lipids of Bacillus thiaminesecreting were detected by GC gas chromatography, HPLC liquid chromatography and TLC thin layer chromatography.

[0021] (1) The main fatty acid component of white wine thiamine Bacillus is 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 cellular fatty acid composition is shown in Table 1.

[0022] Table 1 Main fatty acid components of Bacillus thiamine in liquor

[0023] (2) The types and quantities of quinone compounds in microorganisms are important indicators of the diversity of microbial population composition. After testing, the quinone compound of Bacillus thiamine in white wine was MK-7.

[0024] (3) There are 5 polar lipids in the white wine thiamine Bacillus (such as Figure 1 As shown in the figure), they are diphosphatidylglycerol, Dphosphatidylglycerol (DPG); phosphatidylethanolamine, phosphatidylethanolamine (PE); phosphatidylglycerol, phosphatidylglycerol (PG); unidentified phospholipids (PL1-3), phosphatidylcholine (PC).

[0025] 4. Molecular biology level (1) Extraction of genomic DNA from Bacillus thiamine The pure culture was inoculated into NA medium and cultured for 48 hours, then the bacteria were picked and the whole genome DNA of the strain was extracted using a bacterial genome extraction kit.

[0026] (2) 16S rRNA sequence alignment and phylogenetic tree construction of Bacillus thiamine The 16S rRNA sequence of the strain was amplified and sequenced using universal primers 27F (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.3: 5'-TACGGCTACCTTGTTACGACTT-3'). Aneurinibacillus baijiui

[0027] The 16S rRNA sequences were compared on NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The comparison results showed that the white wine thiamine Bacillus of the present invention was Aneurinibacillus migulanus DSM2895 T and Aneurinibacillus aneurinilyticus ATCC 12856 T The closest relationship is 97.6%. Based on sequence similarity, the model species with the closest relationship are selected to construct the phylogenetic tree. Figure 2 , indicating that the strain is most closely related to the genus Thiamine Bacillus and forms an independent branch, belonging to the genus Thiamine Bacillus.

[0028] (3) Whole genome sequencing of Bacillus thiamine The whole genome of Bacillus thiamine was sequenced to obtain the whole genome sequence. The whole genome sequence was compared with the most similar strain Aneurinibacillus migulanus DSM 2895 T and Aneurinibacillus aneurinolytic ATCC 12856 T The whole genome sequence of the present invention was compared with that of the present invention (http: / / ggdc.dsmz.de / home.php). Aneurinibacillus migulanus DSM2895 T and Aneurinibacillus aneurinilyticus ATCC 12856 T The genome homology (DDH) of the two bacteria were 19.7% and 19.5%, respectively, both less than the threshold of 70%. Therefore, the recognized standard for identification of new bacteria preliminarily determined it to be a new species of the genus Thiaminobacillus.

[0029] Example 3: Analysis of flavor substances produced by Thiamine Bacillus in liquor Inoculate the liquor thiamine Bacillus into NA liquid medium and shake culture for 48 hours. Take 10 mL of culture solution into a 50 mL centrifuge tube, transfer 5.0 mL of supernatant to a gas chromatography-mass spectrometer after centrifugation, add 2 g of sodium chloride and 20.0 μL of 99.07 mg / L internal standard 2-octanol, and tighten the sample bottle. After aging at 250 °C for 30 min, insert the extraction head into the sample bottle, balance at 40 °C for 30 min, extract for 40 min, and desorb the extraction head at 250 °C for 10 min at the GC injection port.

[0030] HS-SPME / GC-MS operation program: GC conditions: injection port temperature was 250°C, chromatographic column specifications were DB-WAX (60 m×250 μm, 0.25μm, Thermo, USA), helium was used as carrier gas, and the flow rate was 1.0 mL / min for splitless injection; the temperature program was: 40°C for 3 min, increased to 150°C at 4°C / min, maintained for 3 min, and increased to 240°C at 9°C / min, maintained for 9 min. Mass spectrometry conditions: electron bombardment ion source; electron bombardment energy 70 eV; transfer line temperature 230°C; quadrupole temperature 150°C; ion source temperature 230°C; mass scan range m / z 35-600.

[0031] After GC-MS flavor analysis, it was found that Bacillus thiamine 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.

[0032] Table 2 Statistics of flavor substances produced by Thiamine Bacillus in liquor .

Claims

1. A liquor thiamine bacillus ( Aneurinibacillus baijiui ), characterized in that, Its deposit number is GDMCC NO.65698.

2. The method for culturing liquor thiamine bacillus according to claim 1, characterized in that: The culture medium is NA, the culture temperature is 25-35°C, the pH is 6-8, and the concentration of sodium chloride in the culture medium is 0.3-0.8%.

3. The method for culturing liquor thiamine bacillus according to claim 2, characterized in that: The culture temperature was 30°C, the pH was 7, and the sodium chloride concentration in the culture medium was 0.5%.

4. The culture of the white wine thiamin Bacillus obtained by the culturing method according to claims 2 and 3.

5. Use of the liquor thiamine bacillus as claimed in claim 1 in fermentation winemaking.

6. The use according to claim 5, characterized in that The invention is used for producing liquor flavor substances, wherein 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.

7. An additive for fermentation and winemaking, which is prepared from the white wine thiamine Bacillus as claimed in claim 1 and is used to produce flavor substances in fermentation and winemaking.

8. The additive according to claim 7, characterized in that It is prepared by the culture according to claim 4.

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