Caproic acid bacillus ZCY20-5 and its application

Abstract

The present invention discloses a caproicibacterium ZCY20-5 and its application, and relates to the field of microbial resources and microbial technology. The key points of its technical scheme are: the bacteria are preserved in the Guangdong Microbial Culture Collection Center, and the preservation time is March 27, 2023, and the preservation number is GDMCC 63296; the nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.1. Caproicibacterium sp. ZCY20-5 screened by the present invention can produce high caproic acid under optimized culture medium, and the caproic acid yield can reach 12.57g / L; optimized culture medium formula: glucose 10g / L, yeast powder 2%, sodium butyrate 0.7%, sodium chloride 5g / L, L-cysteine ​​hydrochloride 0.5g / L.

Description

Technical Field

[0001] The invention relates to the field of microbial resources and microbial technology, and more specifically, to hexanoic acid bacillus ZCY20-5 and application thereof. Background Art

[0002] Cellar mud is the basis of solid-state brewing of liquor. It is rich in a variety of functional microorganisms for brewing. The community composition and diversity of microorganisms in the cellar mud reflect the quality of the cellar mud and largely determine the quality of Luzhou-flavor liquor. Caproic acid bacteria are a type of functional microorganisms in the cellar mud of Luzhou-flavor liquor. The caproic acid produced by their metabolism is one of the organic acid components in liquor and is also the precursor of the main aromatic caproic acid ethyl ester of Luzhou-flavor liquor. In addition to caproic acid, caproic acid bacteria can also metabolize butyric acid, octanoic acid and other trace components. Therefore, the content of caproic acid bacteria in the cellar mud and the acid production performance have an important influence on the formation of liquor flavor and the improvement of liquor quality.

[0003] Caproic acid bacteria are the key functional bacteria in the Luzhou-flavored pit mud, and their content in the pit mud determines the quality of the pit mud. At present, the caproic acid bacteria that have been screened out from the pit mud mainly include Clostridium, Bacillus, Ruminnococcaceae, etc. Studies have found that caproic acid bacteria have different degrees of dependence on different carbon sources. Xue Zhengkai et al. studied strain K-2 and proved that it can use a variety of carbon sources such as glucose, mannitol, inulin, arabinose, and sucrose to produce caproic acid. Nitrogen is also an indispensable element for organisms. It can form substances such as amino acids, proteins, and nucleic acids necessary for microorganisms. Nitrogen can increase the caproic acid production of caproic acid-producing bacteria. Yao Wanchun et al. studied the effects of five nitrogen sources on the growth and metabolism of caproic acid bacteria and found that caproic acid bacteria can use organic nitrogen sources and (NH4)2SO4, and have poor utilization of nitrates. At present, after long-term use, the cellar mud will degenerate into hardened and white crystals mainly composed of ferrous lactate, which will lead to a decrease in the production of caproic acid by caproic acid bacteria, and even cause the death of caproic acid-producing bacteria, and reduce the content of aromatic components such as ethyl caproate. In order to stabilize and improve the quality of liquor, the cellar mud must be maintained. The application of high-concentration caproic acid bacteria fermentation liquid to the maintenance of the cellar mud and the rapid aging of artificial cellar mud have become the focus. Summary of the invention

[0004] The invention aims to provide a caproic acid bacillus ZCY20-5 and an application thereof, which can effectively solve the problems of low yield and poor acid production performance commonly existing in existing caproic acid-producing bacteria, and can also maintain Luzhou-flavor liquor cellars and improve the quality of Luzhou-flavor liquor.

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: a caproic acid bacillus ZCY20-5, the strain preservation number of which is GDMCC 63296.

[0006] Furthermore, the nucleotide sequence of 16S rDNA of the strain is as follows:

[0007]

[0008] The present invention also provides a method for screening Hexanoic acid bacillus ZCY20-5, which specifically comprises the following steps:

[0009] S1: dilute the cellar mud enrichment solution and spread it on the solid medium for enrichment and separation of YE Clostridium to obtain a single colony of the strain, and inoculate it into a fermentation liquid medium after purification for multiple times to obtain a strain seed culture solution;

[0010] S2: Inoculate the seed culture medium at a ratio of 5%-10% into the liquid fermentation medium, ferment and culture at 30-37°C for 10-12 days, perform preliminary screening by copper sulfate colorimetric method, select strains with obvious color reaction, and then screen Hexanoic acid Bacillus ZCY20-5 by detecting the hexanoic acid content in the fermentation liquid.

[0011] The present invention also provides an application of Hexanoic acid Bacillus ZCY20-5 in preparing a bacterial agent for improving hexanoic acid production. The bacterial agent is prepared by inoculating Hexanoic acid Bacillus ZCY20-5 into a fermentation medium.

[0012] Furthermore, the fermentation medium includes 1% carbon source, 1% nitrogen source, 5 g / L sodium chloride and 0.5 g / L L-cysteine ​​hydrochloride.

[0013] Furthermore, the fermentation medium is RCM liquid fermentation medium, and the fermentation substrate includes 3 g / L sodium acetate, 3 g / L sodium lactate and 3 g / L sodium butyrate.

[0014] Furthermore, the fermentation medium includes 10 g / L of glucose, 1%-4% of yeast powder, 0.1%-0.9% of sodium butyrate, 5 g / L of sodium chloride, and 0.5 g / L of L-cysteine ​​hydrochloride.

[0015] The present invention also provides an application of Hexanoic acid bacillus ZCY20-5 in the preparation of microbial preparations.

[0016] Furthermore, the microbial preparation is a microbial preparation for winemaking.

[0017] In summary, the present invention has the following beneficial effects:

[0018] 1. The Caproicibacterium sp. ZCY20-5 strain screened by the present invention produces 3.84 g / L caproic acid when using RCM liquid medium as fermentation medium. This indicates that the ZCY20-5 strain has a high capacity to produce caproic acid. It is the strain with the highest capacity to produce caproic acid reported so far, and has important application prospects;

[0019] 2. The present invention simplifies and optimizes the formula of the fermentation medium of caproic acid bacteria ZCY20-5, and increases the caproic acid yield of strain ZCY20-5 to 12.57 g / L, which is 3.43 times higher than the caproic acid yield before optimization. While increasing the caproic acid yield, the cost is also reduced. The high-concentration caproic acid bacteria fermentation liquid is used to maintain the Luzhou-flavor liquor cellar, thereby improving the quality of the Luzhou-flavor liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a peak diagram of a standard sample under the gas chromatography conditions in Example 1 of the present invention;

[0021] Figure 2 This is a colony diagram of the high-caproic acid-producing strain ZCY20-5 in Example 1 of the present invention cultured anaerobically on RCM solid medium (without calcium carbonate);

[0022] Figure 3 This is a microscopic morphology of the high-caproic acid-producing strain ZCY20-5 in Example 1 of the present invention;

[0023] Figure 4 This is the electrophoresis diagram of the 16S rDNA PCR product of the high-caproic acid-producing strain ZCY20-5 in Example 1 of the present invention; wherein, band 1 is a DNA marker, and band 2 is a 16S rDNA PCR product;

[0024] Figure 5 is a 16SrDNA phylogenetic tree diagram of strain ZCY20-5 in Example 1 of the present invention;

[0025] Figure 6 The effects of different carbon sources on the growth and pH of strain ZCY20-5 in Example 2 of the present invention;

[0026] Figure 7 This is the effect of different nitrogen sources on the growth and pH of strain ZCY20-5 in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The following is combined with Figure 1-7 The present invention is described in further detail.

[0028] Example 1: Method for screening Hexanoic acid bacillus ZCY20-5.

[0029] The caproic acid strain ZCY20-5 with high caproic acid production in the present embodiment was isolated from a high-quality old cellar in a Luzhou-flavor liquor cellar in Sichuan. The YE enrichment separation medium used was composed of: 0.5 g / L glucose, 1.5 g / L tryptone, 1.0 g / L yeast extract powder, 2.5 g / L starch, 3 g / L sodium acetate, 3 ml / L sodium lactate, 0.5 g / L sodium sulfate, 0.4 g / L magnesium chloride hexahydrate, 0.5 g / L MES, 0.5 g / L potassium dihydrogen phosphate, 0.15 g / L calcium chloride dihydrate, pH 6.8, and a solid medium containing 20 g / L agar.

[0030] The components of RCM liquid medium are: peptone 10g / L, beef extract 10g / L, yeast extract powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate 3g / L, L-cysteine ​​hydrochloride 0.5g / L, pH 7.0;

[0031] Gas chromatography conditions: DB-WAX (60m×250μm×0.25μm) column;

[0032] The carrier gas was high-purity N2, with an initial temperature of 80°C (1 min), which was increased to 200°C at 20°C / min, and then increased to 250°C at 50°C / min (1 min);

[0033] SIM (selected ion scan mode), scanning ions are 60, 87, 88, 116;

[0034] Split ratio 20:1, carrier gas flow rate 1 mL / min;

[0035] Under this condition, the peak of the standard sample is as follows Figure 1 As shown, the peak time of hexanoic acid is 29.094 min.

[0036] Sample preparation: Pick a purified single colony and inoculate it into YE liquid medium. After culturing at 37℃ for 5 days, obtain the seed liquid, inoculate it into 100mL fermentation liquid medium at a 5% inoculation amount, ferment at 37℃ for 10 days, take an appropriate amount of supernatant and filter it through a 0.2μm microporous membrane. Take 1mL and put it into a sample injection bottle for gas chromatography qualitative and quantitative analysis using the internal standard method.

[0037] The specific steps for screening Hexanobacterium ZCY20-5 are:

[0038] S1: Enrichment of pit mud: Weigh 10 g of pit mud and add it into an anaerobic bottle containing 10 mL of sterile water, treat it in a constant temperature water bath at 80°C for 10 min, inoculate it into the sterilized YE enrichment and separation medium at a 5% inoculum after cooling, and enrich and culture it in an anaerobic box at 35°C for 5-7 days to obtain an enrichment solution;

[0039] S2: Isolation and purification of strains: The dilution coating method was used to separate the caproic acid bacteria in the enrichment solution. The enrichment solution was diluted to 10 -1 -10 -6 , absorb 10 -3 -10 -6 0.1 mL of bacterial solution was spread on YE enrichment and separation solid medium (deoxygenated in an anaerobic incubator for 24 h) and cultured in an anaerobic incubator at 35°C for 3-5 days. Colonies with different morphological characteristics were streaked and purified again for at least three times to obtain single colonies;

[0040] S3: Screening of bacteria with high caproic acid production: single colonies grown in S2 were selected, inoculated into test tube fermentation liquid culture medium under sterile conditions using an inoculation loop, and then cultured in an anaerobic incubator at 35°C for 3-5 days to obtain a primary seed solution. 5% of the primary seed solution of the obtained bacteria was inoculated into 100 mL of sterilized fermentation liquid culture medium and cultured at 35°C for ten days. Initial screening was performed using the copper sulfate colorimetric method, and strains with obvious color development reactions were selected. The caproic acid content in the fermentation broth was detected by gas chromatography, and finally a strain with high caproic acid production was screened out and named ZCY20-5.

[0041] This example identifies the screened ZCY20-5:

[0042] 1. Morphological characteristics

[0043] The morphological observation of ZCY20-5 showed that the strain was thick and long rod-shaped, with terminal spores, and was a Gram-positive bacterium. After 72 hours of culture in RCM solid medium (without calcium carbonate), it formed regular circular colonies. The colonies were large and opaque, with light yellow edges and a milky white protrusion in the middle. Figure 2 shown.

[0044] 2. Physiological and biochemical identification

[0045] ZCY20-5 was identified physiologically and biochemically with reference to the ninth edition of Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems (edited by Dong Xiuzhu, Science Press). The results are shown in Table 1.

[0046] Table 1 Physiological and biochemical identification results of ZCY20-5

[0047]

[0048] Note: + indicates a positive reaction; - indicates a negative reaction.

[0049] 3. 16S rDNA sequence determination and analysis

[0050] (1) DNA extraction

[0051] The extraction steps are:

[0052] 1) Take 1.5 mL of enrichment solution and centrifuge at 13000 rpm for 3 min, discard the supernatant, and repeat several times until there are enough bacteria;

[0053] 2) Add 450 μL of DNA extraction buffer and lysozyme to a final concentration of 20 mg / mL, and treat in a 37°C water bath for 1 h;

[0054] 3) Add proteinase K and 50 μL SDS solution and treat in a 70°C water bath for 1 h;

[0055] 4) Add an equal volume of phenol chloroform isoamyl alcohol (25:24:1), mix well, let stand for 5 minutes, and centrifuge at 13000 rpm for 15 minutes;

[0056] 5) Take the supernatant and add 0.6 volumes of isopropanol or 2 volumes of anhydrous ethanol, and centrifuge at 13000 rpm for 10 min;

[0057] 6) Remove the supernatant, add 100 μL 70% ethanol to wash, centrifuge at 13000 rpm for 1 min, repeat twice, and blow dry the ethanol in a sterile operating table;

[0058] 7) Add 100 μL of sterile water to dissolve the DNA and store it in a -20°C refrigerator.

[0059] (2) PCR amplification and phylogenetic tree construction

[0060] After bacterial DNA was extracted, PCR amplification was performed using the bacterial universal primer 27F-1492R. The reaction system was as follows: dNTPs: 1 μL, final concentration 200 μmol / L; Taq DNA polymerase 1 μL, final concentration 0.1 U / μL; template DNA 3 μL; 5× Buffer (containing Mg 2 + ) 10 μL, final concentration is 1x; forward and reverse primers 1 μL, final concentration 1.0 nmol / L, ultrapure water is added to 50 μL.

[0061] Reaction conditions: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1.5 min. After 30 cycles, final extension was performed at 72°C for 10 min.

[0062] The amplified products were detected using 1% agarose gel, and the PCR products of the correct size were sent to Chengdu Qingke Biotechnology Co., Ltd. for sequencing. The returned sequences were spliced ​​end to end and uploaded to the NCBI database for comparison. The sequences with the highest similarity were downloaded, and the phylogenetic tree was constructed using MEGA 6.0 software. The phylogenetic tree based on 16S rDNA is shown in Figure 1. Figure 5shown.

[0063] The PCR amplification results are shown in the figure. The size of the PCR product is 1512bp. After sequencing and identification, the 16SrDNA sequence of strain ZCY20-5 is shown in SEQ ID NO.1.

[0064] The strain ZCY20-5 was identified as Hexanoic acid bacterium through morphological, physiological and biochemical characteristics and 16S rDNA sequencing. The suitable growth temperature of the bacterium is 32-45℃, the optimum growth temperature is 37℃, the pH tolerance range is 4-8, the optimum pH is 5.5, and the ethanol concentration is 5%.

[0065] The bacteria were cultured at 37°C for 3 days in YE enrichment and separation solid medium and cultured for several generations. There was no significant change in its culture characteristics, morphological characteristics, physiological and biochemical characteristics, and other biological characteristics, showing good biological trait stability. The strain was identified as caproic acid-producing bacteria (Caproicibacterium amylolyticum) by 16S rDNA and named Caproicibacterium sp. ZCY20-5. It was deposited in the Guangdong Provincial Microbiological Culture Collection Management Center (Address: Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong, China) on March 27, 2023, with the deposit number GDMCC 63296.

[0066] Example 2: Application of Hexanoic Acid Bacillus ZCY20-5 in high production of caproic acid.

[0067] 1. Fermentation and production of caproic acid by ZCY20-5

[0068] The ZCY20-5 strain obtained in Example 1 was inoculated aseptically into a test tube containing a fermentation liquid medium using an inoculation loop, and cultured in an anaerobic incubator at 37°C for 3-5 days to obtain a seed solution; 5% of the seed solution was inoculated into 100 mL of sterilized fermentation liquid medium and cultured statically at 37°C. A disposable syringe was used to draw an appropriate amount of fermentation liquid through a 0.2 μm microporous filter membrane at 10 days of fermentation. 1 mL was taken and placed in a sample injection bottle for qualitative and quantitative analysis by gas chromatography using the internal standard method. The hexanoic acid production of ZCY20-5 was determined when RCM was used as the fermentation medium. Three replicates were made for each sample, and the test results are shown in Table 2.

[0069] Table 2 Acid production of ZCY20-5 in RCM fermentation medium for 10 days

[0070]

[0071] 2. Exploration of Carbon Sources for Caproic Acid Production by ZCY20-5 Fermentation

[0072] Carbon source selection fermentation medium: carbon source 1%, yeast powder 3g / L, sodium chloride 5g / L, L-cysteine ​​hydrochloride 0.5g / L.

[0073] According to the biolog plate, the carbon source of ZCY20-5 was verified. The seed solution obtained in Example 1 was inoculated into 100 mL of carbon source selection fermentation medium at a rate of 5%. After sealed culture at 37°C for 10 days, the OD value and pH change were compared to select the carbon source that promoted the growth and acid production of the strain for subsequent acid production verification. Figure 6 As shown, the changes in OD value and pH of ZCY20-5 after ten days of fermentation using different carbon sources show that glucose, lactose and D-maltose have a significant promoting effect on the growth and acid production of the strain.

[0074] The seed solution obtained in Example 1 was inoculated into 100 mL according to an inoculum of 5% with glucose (10 g / L), lactose (10 g / L), and D-maltose (10 g / L) as carbon sources, and the remaining components were 3 g / L yeast powder, 5 g / L sodium chloride, and 0.5 g / L L-cysteine ​​hydrochloride. The mixture was sealed and cultured at 37°C for 10 days. After aseptic operation, an appropriate amount of fermentation liquid was taken and filtered through a 0.2 μm filter membrane, and then the caproic acid production was detected by gas chromatography. Three replicates were made for each sample, and the test results are shown in Table 3. As can be seen from Table 3, when the carbon source is glucose, the caproic acid production is the highest, reaching 1.62 g / L, which is the best carbon source for producing caproic acid; secondly, the strain also showed good caproic acid production ability using maltose and lactose, and the caproic acid production was stable.

[0075] Table 3 Acid production of ZCY20-5 after 10 days of fermentation using different carbon sources

[0076]

[0077] 3. Exploration of nitrogen sources for caproic acid production by ZCY20-5 fermentation

[0078] Nitrogen source selection fermentation medium: glucose 10g / L, nitrogen source 1%, sodium chloride 5g / L, L-cysteine ​​hydrochloride 0.5g / L.

[0079] The common nitrogen sources in the laboratory were selected to culture the strain ZCY20-5. The seed solution obtained in Example 1 was inoculated into 100 mL of nitrogen source selection medium at a 5% inoculation rate. After sealed culture at 37°C for 10 days, the OD value and pH changes were compared to select nitrogen sources that promote strain growth and acid production for subsequent acid production verification. Figure 7 The results showed that beef extract and yeast powder can promote the growth and acid production of Hexanoic acid Bacillus ZCY20-5.

[0080] The seed liquid obtained in Example 1 was inoculated into 100 mL of fermentation medium with yeast powder (10 g / L) and beef extract (10 g / L) as nitrogen sources at an inoculum of 5%, and sealed and cultured at 37°C for 10 days. After aseptic operation, an appropriate amount of fermentation liquid was taken and filtered through a 0.2 μm filter membrane, and then the caproic acid production was detected by gas chromatography. Three replicates were made for each sample, and the test results are shown in Table 4. As can be seen from Table 4, when the nitrogen source is yeast powder, the caproic acid production is the highest, reaching 4.66 g / L, which is the best nitrogen source for producing caproic acid.

[0081] Table 4 Acid production of ZCY20-5 after 10 days of fermentation using different nitrogen sources

[0082]

[0083]

[0084] Effects of sodium acetate, sodium lactate, and sodium butyrate addition on caproic acid production by ZCY20-5

[0085] The seed solution obtained in Example 1 was inoculated into 100 mL of RCM liquid fermentation medium with sodium acetate (3 g / L), sodium lactate (3 g / L) and sodium butyrate (3 g / L) as fermentation substrates according to an inoculum size of 5%. After sealed culture at 37°C for 10 days, an appropriate amount of fermentation liquid was taken aseptically and filtered through a 0.2 μm filter membrane for gas chromatography to detect the caproic acid production. Three replicates were made for each sample, and the test results are shown in Table 5. As can be seen from Table 5, the addition of sodium butyrate has a good promoting effect on the production of caproic acid by ZCY20-5 fermentation, and the caproic acid production reaches 3.76 g / L.

[0086] Table 5 Production of caproic acid using sodium acetate and sodium butyrate in ZCY20-5

[0087]

[0088] Example 3: Optimization of caproic acid production by Hexanoic Acid Bacillus.

[0089] From Example 2, it can be seen that glucose is the best carbon source, yeast powder is the best nitrogen source, sodium butyrate is the best fermentation substrate, and yeast powder and sodium butyrate in the fermentation medium have a greater impact on the production of caproic acid by Hexanobacter ZCY20-5. Therefore, yeast powder and sodium butyrate are selected as variables to optimize the fermentation medium.

[0090] (1) Preparation of seed solution of Hexanoic acid bacterium ZCY20-5

[0091] The single colony obtained in S2 of the specific step of Hexanoic acid bacillus ZCY20-5 in Example 1 was selected, and an appropriate amount was picked up using an inoculation loop under sterile operation conditions and inoculated into a test tube fermentation liquid culture medium, and then cultured in an anaerobic incubator at 37° C. for 3-5 days to obtain a test tube seed solution.

[0092] (2) Optimization of yeast powder addition amount in fermentation medium

[0093] The seed liquid obtained in Example 1 was inoculated into 100 mL of fermentation liquid medium including 10 g / L glucose, 1%-4% yeast powder, 5 g / L sodium chloride, and 0.5 g / L L-cysteine ​​hydrochloride according to an inoculum amount of 5%. After sealed culture at 37°C for 10 days, an appropriate amount of fermentation liquid was taken aseptically and filtered through a 0.2 μm filter membrane for gas chromatography to detect its caproic acid production. Three replicates were made for each sample, and the test results are shown in Table 6. As can be seen from Table 6, as the amount of yeast powder added increases, the caproic acid production increases accordingly. When the amount of yeast powder added reaches 2%, the caproic acid production reaches the highest, which is 5.31 g / L.

[0094] Table 6 Effect of different yeast powder contents on caproic acid production in ZCY20-5

[0095]

[0096] (3) Optimization of sodium butyrate addition in fermentation medium

[0097] The seed solution obtained in Example 1 was inoculated into 100mL fermentation liquid medium including 10g / L glucose, 2% yeast powder, 0.1%-0.9% sodium butyrate, 5g / L sodium chloride, and 0.5g / L L-cysteine ​​hydrochloride according to an inoculum of 5%. After sealed culture at 37°C for 10 days, a suitable amount of fermentation liquid was taken aseptically and passed through a 0.2μm filter membrane for gas chromatography to detect the caproic acid production. Three replicates were made for each sample, and the test results are shown in Table 7. As can be seen from Table 7, as the amount of sodium butyrate added increased, the caproic acid production increased therewith, and when the amount of sodium butyrate added reached 0.7%, the caproic acid production reached the highest, which was 12.57g / L.

[0098] Table 7 Effect of different sodium butyrate contents on caproic acid production in ZCY20-5

[0099]

[0100] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A caproic acid bacillus ZCY20-5, characterized in that: The deposit number of the strain is GDMCC 63296.

2. The use of a caproic acid bacillus ZCY20-5 according to any one of claim 1 in the preparation of a bacterial agent for increasing caproic acid production, characterized in that: The bacterial agent is obtained by inoculating Hexanoic acid bacillus ZCY20-5 into a fermentation medium.

3. The use according to claim 2, characterized in that: The fermentation medium comprises 1% carbon source, 1% nitrogen source, 5g / L sodium chloride and 0.5g / L L-cysteine ​​hydrochloride.

4. The use according to claim 2, characterized in that: The fermentation medium is RCM liquid fermentation medium, and the fermentation substrate includes 3g / L sodium acetate, 3g / L sodium lactate and 3g / L sodium butyrate.

5. The use according to claim 2, characterized in that: The fermentation medium includes 10 g / L of glucose, 1%-4% of yeast powder, 0.1%-0.9% of sodium butyrate, 5 g / L of sodium chloride, and 0.5 g / L of L-cysteine ​​hydrochloride.

6. Use of the Hexanoic acid bacillus ZCY20-5 according to claim 1 in the preparation of microbial preparations.

7. The use according to claim 6, characterized in that: The microbial preparation is a microbial preparation for winemaking.

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