A type of Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) exhibiting multiple tolerances and hexanoic acid production, along with its isolation method and applications.

Abstract

This invention discloses a *Bacillus amyloliquefaciens* strain derived from daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid production characteristics, along with its isolation method and applications. This strain, isolated from daqu, produces hexanoic acid and exhibits resistance to high temperatures, acids, and ethanol; it has been identified as *Bacillus amyloliquefaciens*. B. amyloliquefaciens DJ001, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33299, enhances the hexanoic acid production and saccharification power of daqu (a type of starter culture) when added to it, and increases the yield and ethyl hexanoate content of baijiu (Chinese liquor) when added to the mash. This invention has significant value in improving baijiu yield and quality, saving costs, and increasing efficiency, thus increasing benefits for enterprises and society.

Description

Technical Field

[0001] This invention relates to the field of hexanoic acid bacteria and baijiu production, specifically to a Bacillus amyloliquefaciens derived from daqu (a type of starter culture) that has multiple tolerances and hexanoic acid production characteristics, as well as its isolation method and application, which can be used to increase the hexanoic acid and ethyl hexanoate content in baijiu. Background Technology

[0002] Chinese Baijiu is a type of alcoholic beverage with a long history, and strong-aroma Baijiu is the most produced and sold type of Baijiu. Ethyl hexanoate is an important flavor component of strong-aroma Baijiu. Hexanoic acid bacteria can produce hexanoic acid, which can serve as a precursor for the formation of ethyl hexanoate. Therefore, the isolation, identification, and application of hexanoic acid bacteria are of great significance in improving the quality of Baijiu.

[0003] In the brewing environment, the contact area between cellar mud and fermented mash is small; however, after being crushed, the Daqu (a type of starter culture) is evenly sprinkled into the mash, allowing for more thorough contact. Theoretically, microorganisms in Daqu are more likely to play a role in the brewing process than those in cellar mud. However, given the growth characteristics of many caproic acid bacteria and some existing knowledge, previous studies primarily used cellar mud as raw material for the isolation of caproic acid bacteria, with no reports of isolating them from Daqu. Therefore, screening novel caproic acid bacteria from Daqu and applying them to the production of strong-aroma baijiu is of great significance for increasing the ethyl hexanoate content in baijiu.

[0004] Furthermore, the production of yeast starter and the brewing of baijiu (Chinese liquor) involve extreme environmental conditions, such as high temperature, low pH, and high ethanol levels. These conditions can affect the growth and function of caproic acid bacteria, thus impacting their application effectiveness. Therefore, screening for strains that simultaneously possess characteristics such as heat resistance, acid resistance, and ethanol resistance is of great significance. Summary of the Invention

[0005] This invention provides a Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing characteristics, along with its isolation method and applications, to address the problem of insufficient hexanoic acid and ethyl hexanoate content in the products of some strong-aroma baijiu enterprises.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The strains in this invention Bacillus amyloliquefaciens DJ001 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 3, 2025, with accession number CGMCC No. 33299.

[0008] Furthermore, the strain B. amyloliquefaciens DJ001 grows well under conditions with pH values ​​of 1-11.

[0009] Furthermore, the strain B. amyloliquefaciensDJ001 grows well in the range of 22-52℃.

[0010] Furthermore, the strain B. amyloliquefaciens DJ001 grows well under conditions with an ethanol content of 0-12% vol.

[0011] A type of Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing properties. B. amyloliquefaciens The separation method includes the following steps:

[0012] Step 1: Using Daqu (a type of starter culture) as the screening source, bacterial strains were isolated and purified by dilution plating and streak plating methods.

[0013] Step 2: The ability of the bacteria obtained in Step 1 to produce hexanoic acid was detected by the copper sulfate colorimetric method, and candidate strains for hexanoic acid production were further screened based on the detection results.

[0014] Step 3: Detect the ability of the bacteria obtained in Step 2 to produce hexanoic acid using gas chromatography, and screen out strains with high hexanoic acid production based on the test results;

[0015] Step 4: Test the tolerance of the strains obtained in Step 3, and select strains with multiple tolerances based on the test results. B. amyloliquefaciens DJ001.

[0016] An application of the above-mentioned Bacillus amyloliquefaciens in the production of Daqu (a type of starter culture).

[0017] An application of the above-mentioned Bacillus amyloliquefaciens in alcoholic beverage production.

[0018] A method for isolating hexanoic acid-producing strains from Daqu (a type of starter culture) and its application.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] This invention is the first to isolate Bacillus amyloliquefaciens with multiple tolerances and hexanoic acid-producing properties from Daqu (a type of starter culture). B. amyloliquefaciens DJ001 strain, when added to Daqu (a type of starter culture), can enhance the Daqu's hexanoic acid production capacity and saccharification power; when added to the mash, it can increase the yield and ethyl hexanoate content of Baijiu (Chinese liquor). This invention has significant value in improving the yield and quality of Baijiu. Attached Figure Description

[0021] Figure 1 This is a comparison chart of hexanoic acid production in Daqu culture medium. Note: Different lowercase letters indicate significant differences (P < 0.05); all values ​​in the chart are repeated more than three times.

[0022] Figure 2 This is a colorimetric diagram of copper sulfate reaction.

[0023] Figure 3 The graph shows the hexanoic acid production of *Hexanoic acid bacteria*. Note: Different lowercase letters indicate significant differences (P < 0.05); all values ​​in the graph are repeated more than three times.

[0024] Figure 4 This is a comparison of hexanoic acid production by various hexanoic acid bacteria at different temperatures. Note: Different lowercase letters indicate significant differences (P < 0.05); all values ​​in the figure are repeated more than three times.

[0025] Figure 5 This is a comparison of hexanoic acid production by various hexanoic acid bacteria at different ethanol concentrations. Note: Different lowercase letters indicate significant differences (P < 0.05); all values ​​in the figure are repeated more than three times.

[0026] Figure 6 This is a comparison of hexanoic acid production by various hexanoic acid bacteria at different pH values. Note: Different lowercase letters indicate significant differences (P < 0.05); all values ​​in the figure are repeated more than three times.

[0027] Figure 7 The image shows an agarose gel electrophoresis image of the 16S rDNA PCR product of strain DJ001, where: M, marker5000; -, negative control; A, DJ001.

[0028] Figure 8 Phylogenetic tree diagram of strain DJ001 based on 16S rDNA sequence.

[0029] Figure 9 This is a graph showing the hexanoic acid production of strain DJ001 in a simulated fermentation broth. Note: "****" indicates P < 0.0001; all values ​​in the graph are repeated at least three times.

[0030] Figure 10 This is a temperature change curve during the cultivation of Daqu (a type of starter culture).

[0031] Figure 11 The graph shows the hexanoic acid content in the koji powder culture medium after adding strain DJ001 during the koji production process. Note: "***" indicates P < 0.001; all values ​​in the graph are repeated at least three times.

[0032] Figure 12 This is a saccharification force graph of Daqu (a type of starter culture). * indicates P < 0.05; all values ​​in the graph are repeated more than three times.

[0033] Figure 13 The graph shows the production volume of baijiu (Chinese liquor). Where: “****”, P<0.0001; “*”, P<0.05; all values ​​in the graph are repeated more than 3 times.

[0034] Figure 14This is a graph showing the content of hexanoic acid and ethyl hexanoate in baijiu (Chinese liquor). * indicates P < 0.05; ns indicates no significant difference; all values ​​in the graph are repeated at least three times.

[0035] Figure 15 This is a graph showing the mass of hexanoic acid and ethyl hexanoate in baijiu (Chinese liquor). Where: "****" indicates P < 0.0001; ns indicates no significant difference; all values ​​in the graph are repeated more than three times. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] The following examples illustrate the preparation method of sodium ethoxyacetate culture medium.

[0038] Sodium ethanolacetate medium: Weigh and dissolve 5 g anhydrous sodium acetate, 1 g yeast extract, 0.2 g magnesium sulfate heptahydrate, 0.4 g anhydrous dipotassium hydrogen phosphate, 0.5 g ammonium sulfate, and 10 g calcium carbonate. Adjust the volume to 1 L, set to natural pH. Sterilize at 121°C for 20 min. After cooling, add 2% anhydrous ethanol (volume fraction) before use. For solid media, add 2% agar powder before sterilization.

[0039] The extraction methods of organic compounds such as hexanoic acid and ethyl hexanoate in the following examples

[0040] The obtained culture medium was filtered through a 0.22 μm microporous membrane. 500 μL of the filtrate was placed in a 5 mL volumetric flask, and anhydrous diethyl ether was added to bring the volume to 5 mL. The mixture was shaken thoroughly, allowed to stand for 1 min, and the upper extract was collected as the test solution.

[0041] Gas chromatography detection methods in the following examples

[0042] The contents of hexanoic acid and ethyl hexanoate in the diethyl ether extract were determined by gas chromatography. The chromatographic conditions were as follows: column flow rate 2.15 mL / min; column temperature 50℃; detector temperature 240℃; programmed temperature ramp: initial temperature 60℃ for 1 min, ramped to 220℃ at a rate of 10℃ / min and held for 2 min; injection volume 1 μL; split injection with a split ratio of 20:1. Example 1

[0043] This embodiment discloses a method for isolating hexanoic acid-producing Bacillus amyloliquefaciens, comprising the following steps:

[0044] Step 1: Select high-quality Daqu (a type of starter culture).

[0045] The laboratory had five types of strong-aroma koji, numbered DQ1, DQ2, DQ3, DQ4, and DQ5. The koji were thoroughly ground into powder and evenly added to sodium ethoxyacetate medium. After 7 days of cultivation, the hexanoic acid yield was measured. The results are as follows: Figure 1 As shown in the figure. Among them, Daqu DQ2 and DQ3 had higher hexanoic acid production, at 0.64 mg / mL and 0.52 mg / mL, respectively. Therefore, subsequent screening and isolation of high hexanoic acid-producing bacteria will mainly focus on Daqu DQ2 and DQ3.

[0046] Step 2: Select strains in Daqu (a type of Chinese liquor) that have the ability to produce hexanoic acid based on the copper sulfate colorimetric method.

[0047] Take 200 μL of Daqu culture medium into a 2 mL centrifuge tube, dip a small amount of culture medium into the tube, streak and spread it on a plate to isolate and purify the strain; select a single colony and incubate it in sodium ethanolacetate medium for 7 days; take 2 mL of culture medium into a 10 mL centrifuge tube, add 1 mL of 20% copper sulfate solution, and observe whether a blue-green precipitate is formed. Results are as follows: Figure 2 As shown, some strains produced blue-green precipitates, and the corresponding strains were preserved.

[0048] Step 3: Combine gas chromatography to detect the hexanoic acid production capacity of the strains obtained in Step 2, and screen out strains with high hexanoic acid production based on the detection results.

[0049] Specifically, the strains isolated in step 2 were cultured in sodium ethoxyacetate medium at 37°C for 7 days, and the hexanoic acid production was detected using gas chromatography. Three hexanoic acid-producing bacteria with strong hexanoic acid production capabilities were ultimately obtained, with DJ001 exhibiting the highest hexanoic acid production. Figure 3 As shown, DJ001 produced 1.56 mg / mL of hexanoic acid.

[0050] Step 4: Test the tolerance of the candidate strain DJ001 obtained in Step 3. The process is as follows:

[0051] (4.1) Temperature tolerance test of the strain

[0052] 200 μL of each of the preserved bacterial suspensions (DJ001, 1.39-1, and 4.39-1) were transferred to sodium glycoacetate liquid medium and anaerobically cultured at 37°C for 7 days to activate the strains. The bacterial suspensions of DJ001, 1.39-1, and 4.39-1 were inoculated into sodium glycoacetate liquid medium at a 2% inoculum volume (90% capacity), and cultured at temperatures of 22°C, 27°C, 32°C, 37°C, 42°C, 47°C, and 52°C, respectively, for 7 days of anaerobic culture. The hexanoic acid yield of each group was measured, and the results are as follows: Figure 4As shown, with increasing culture temperature, the hexanoic acid yield of DJ001, 1.39-1, and 4.39-1 all first increased and then decreased; DJ001 showed the strongest temperature tolerance, and even at 52℃, DJ001 still had a high hexanoic acid yield.

[0053] (4.2) Detection of ethanol tolerance in strains

[0054] 200 μL of each of the three bacterial suspensions (DJ001, 1.39-1, and 4.39-1) were transferred to sodium ethanolacetate liquid medium and anaerobically cultured at 37°C for 7 days to activate the strains. The bacterial suspensions of DJ001, 1.39-1, and 4.39-1 were then inoculated into sodium ethanolacetate liquid medium with ethanol concentrations of 0%, 2%, 4%, 6%, 8%, 10%, and 12% vol, respectively, at a 2% inoculation rate, with a liquid volume of 90%. Anaerobic culture was then performed at 37°C for 7 days. The hexanoic acid yield of each group was measured, and the results are as follows: Figure 5 As shown, with increasing ethanol concentration, the hexanoic acid yield of DJ001, 1.39-1, and 4.39-1 all first increased and then decreased; when the ethanol concentration was 12% vol, DJ001 still had a relatively high hexanoic acid yield.

[0055] (4.3) Detection of strain pH tolerance

[0056] 200 μL of each of the preserved bacterial suspensions of DJ001, 1.39-1, and 4.39-1 were transferred to sodium glycoacetate liquid medium and anaerobically cultured at 37°C for 7 days to activate the strains. The bacterial suspensions of DJ001, 1.39-1, and 4.39-1 were then inoculated into sodium glycoacetate liquid medium at pH 1, 3, 5, 7, 9, and 11, respectively, at a 2% inoculation rate, with a liquid volume of 90%. Anaerobic culture was then performed at 37°C for 7 days. The hexanoic acid yield of each group was measured, and the results are as follows: Figure 6 As shown, the hexanoic acid yields of DJ001, 1.39-1, and 4.39-1 all increased and then decreased with increasing pH; DJ001 showed the best pH adaptability, exhibiting the highest hexanoic acid yield across pH ranges of 1-11.

[0057] Based on the above analysis results of hexanoic acid yield and tolerance, DJ001 was selected as the strain for further research and application.

[0058] Step 5: Identification of the strain

[0059] (5.1) Extraction of genomic DNA from the strain

[0060] Genomic DNA was extracted from DJ001 using the SPARKeasy Bacterial Genomic DNA Rapid Extraction Kit.

[0061] (5.2) PCR amplification

[0062] Genomic DNA was used as a template for PCR amplification, and universal primers 27F / 1492R were employed. The primer sequences are as follows:

[0063] 27F: 5-AGAGTTTGATCCTGGCTCAG-3';

[0064] 1492R: 5'-GGTTACCTTG TTACGACTT-3';

[0065] PCR reaction program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 5 min.

[0066] (5.3) Gel electrophoresis detection of amplification products

[0067] After mixing 3 μL of amplification product with 3 μL of loading buffer, 3 μL of the mixture was then subjected to electrophoresis on a 1% agarose gel. The results are as follows: Figure 7 As shown, the amplified band is relatively simple, with a size of approximately 1500 bp.

[0068] (5.4) DNA sequencing and sequence alignment

[0069] The purified PCR product was sent to BGI Genomics Co., Ltd. in Shanghai for sequencing, and the 16S rDNA sequence of strain DJ001 was obtained. The sequence information is as follows.

[0070]

[0071] The above sequence was submitted to the NCBI database for BLAST comparison, and the result showed that it was *Bacillus amyloliquefaciens* (…). B. amyloliquefaciens A phylogenetic tree was constructed based on their sequences, and the results are as follows: Figure 8 As shown, DJ001 and B. amyloliquefaciens They cluster into one category. Therefore, DJ001 is... B. amyloliquefaciens Name it B. amyloliquefaciens DJ001.

[0072] The Bacillus amyloliquefaciens obtained in this embodiment B. amyloliquefaciens DJ001 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 3, 2025, with accession number CGMCC No. 33299. Example 2

[0073] This embodiment discloses the Bacillus amyloliquefaciens obtained in Example 1. B. amyloliquefaciens The simulated fermentation process conducted by DJ001 in the laboratory is described in detail below:

[0074] Take 1 kg of mash prepared for brewing by a winery for later use.

[0075] Boil 1 kg of fermented mash in 2 L of sterile water for 30 min, filter, put the filtrate into a beaker, make up to 5 L, dispense, sterilize at 115℃ for 30 min, and use as a simulated fermentation broth.

[0076] Group CK: 3 g of koji powder, 5 g of fermented mash, and 5 mL of sterile water were added to an Erlenmeyer flask containing 270 mL of simulated fermentation broth. Group DJ001: 3 g of koji powder, 5 g of fermented mash, and 5 mL of DJ001 bacterial culture were added to an Erlenmeyer flask containing 270 mL of simulated fermentation broth. Both groups fermented for 7 days, and the yield of hexanoic acid was measured. The results are as follows: Figure 9 As shown, the hexanoic acid yield in the CK group was 0.038 mg / mL, while the hexanoic acid yield in the DJ001 group was 0.73 mg / mL, representing an increase of 1821.05% in hexanoic acid yield.

[0077] Therefore, add B. amyloliquefaciens DJ001 has a good effect on producing hexanoic acid. Example 3

[0078] This embodiment discloses the Bacillus amyloliquefaciens obtained in Example 1. B. amyloliquefaciens The application of DJ001 in the production of large-scale music is explained in detail below:

[0079] (1) Strengthen the production method of Daqu (a type of Chinese liquor).

[0080] The Bacillus amyloliquefaciens obtained in Example 1 B. amyloliquefaciens DJ001 bacterial solution was evenly added to the raw materials for making medium- and high-temperature Daqu (a type of starter culture). The amount of DJ001 bacterial solution used in each Daqu cell was 50 L. Conventional Daqu without bacterial strain was used as the control (CK). There were a total of 3840 pieces of Daqu per cell.

[0081] (2) Temperature monitoring of Daqu

[0082] After the Daqu (a type of starter culture) is placed in the fermentation room, its temperature is recorded daily at 8:00 AM until it is removed from the room after 24 days (once the temperature stabilizes, the temperature in the fermentation room is no longer recorded). The Daqu temperature curve is shown below. Figure 10 As shown, the temperature changes in the two chambers of Daqu were basically the same, indicating that the management during the Daqu cultivation process was stable.

[0083] (3) Selection of Daqu samples

[0084] Five samples of Daqu were selected from each room using a five-point sampling method. The Daqu samples obtained from the same room were ground, pulverized, and mixed evenly before being used in subsequent experiments.

[0085] (4) Detection of the hexanoic acid production capacity of koji powder culture medium

[0086] The koji powder was added to sodium ethoxyacetate medium at an inoculum of 1.5% and cultured at 37°C for 7 days. The yield of hexanoic acid was then measured. The results are as follows: Figure 11 As shown, the hexanoic acid content in the enhanced Daqu powder culture medium prepared with DJ001 bacterial solution was increased by 600.00% compared with the control Daqu CK without bacterial strain.

[0087] (5) Saccharification power test of Daqu

[0088] The results are as follows Figure 12 As shown, the enhanced Daqu (a type of starter culture) prepared with added DJ001 bacterial solution exhibited a 31.11% higher saccharification power than the control Daqu (CK) without added strain. DJ001 is a Bacillus amyloliquefaciens; the addition of DJ001 during Daqu production may facilitate the conversion of starch into glucose, thereby improving the saccharification power of the Daqu.

[0089] In summary, the addition of Bacillus amyloliquefaciens as described in Example 1 to the Daqu (a type of starter culture) B. amyloliquefaciens DJ001 can significantly improve the hexanoic acid production capacity of koji culture medium and significantly enhance the saccharification power of koji. Example 4

[0090] This embodiment discloses the Bacillus amyloliquefaciens obtained in Example 1. B. amyloliquefaciens The application of DJ001 in baijiu production is explained in detail below:

[0091] In the production of strong-aroma baijiu, the strain is inoculated at a rate of 1%. B. amyloliquefaciens DJ001 was evenly added to the mash, and the mixture was fermented in a fermentation tank for 70 days. After distillation, ordinary and premium grades of baijiu were obtained. The yield of each grade of baijiu was calculated by weighing. The results are as follows: Figure 13 As shown, the production of ordinary and premium grade baijiu in the DJ001 group increased by 17.76% and 32.69% respectively compared to the CK group. Calculations show that the overall baijiu production of the DJ001 group increased by 23.04% compared to the CK group.

[0092] The hexanoic acid and ethyl hexanoate content of each wine sample was detected by gas chromatography, and then the average concentrations of hexanoic acid and ethyl hexanoate in the overall wine samples of each group were calculated based on the yield. The results are as follows: Figure 14 As shown, compared with the CK group, the concentration of hexanoic acid in the DJ001 group was not significantly different, but the concentration of ethyl hexanoate increased by 22.11%.

[0093] By combining the concentration and yield of each wine sample, the mass of hexanoic acid and ethyl hexanoate in the total wine samples of each group can be calculated. The results are as follows: Figure 15 As shown, compared with the CK group, the mass of hexanoic acid in the DJ001-added group increased by 15.37% (not statistically significant), and the mass of ethyl hexanoate increased by 51.71%.

[0094] Therefore, add functional bacteria B. amyloliquefaciens DJ001 has a significant effect on increasing the yield of baijiu and the content of ethyl hexanoate. The lack of significant difference in hexanoate concentration between the DJ001 group and the CK group may be related to two factors: (1) the concentration of hexanoate was diluted after the baijiu yield increased; (2) hexanoate was converted into ethyl hexanoate.

[0095] In summary, this invention provides a Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) that exhibits multiple tolerances and hexanoic acid production characteristics, along with its isolation method. Adding this strain to Daqu can enhance its hexanoic acid production and saccharification power; adding it to the mash can increase the yield and ethyl hexanoate content of Baijiu (Chinese liquor). This invention has significant value in improving Baijiu yield and quality, saving costs, and increasing efficiency, thus increasing benefits for enterprises and society.

[0096] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. These embodiments are merely descriptions of preferred embodiments and are not intended to limit the scope or concept of the invention. The specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. Such combinations, as long as they do not violate the spirit of the present invention, should also be considered as part of the disclosure of the present invention. To avoid unnecessary repetition, the present invention will not further describe the various possible combinations.

[0097] This invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this invention and without departing from the design idea of ​​this invention, all modifications and improvements made by those skilled in the art to the technical solutions of this invention should fall within the protection scope of this invention. The technical content for which protection is sought in this invention has been fully described in the claims.

Claims

1. A type of Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing properties, characterized in that, The Bacillus amyloliquefaciens strain mentioned is DJ001, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 3, 2025, with accession number CGMCCNo.33299.

2. The *Bacillus amyloliquefaciens* derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing characteristics according to claim 1, characterized in that... The strain was isolated from Daqu (a type of starter culture).

3. The *Bacillus amyloliquefaciens* derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing characteristics according to claim 1, characterized in that... It grows well under conditions with a pH of 1-11.

4. The *Bacillus amyloliquefaciens* derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing characteristics according to claim 1, characterized in that... It grows well under conditions of 22-52 ℃.

5. A type of Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing characteristics, as described in claim 1, is characterized in that... It grows well under conditions with an ethanol content of 0-12% vol.

6. A type of Bacillus amyloliquefaciens derived from Daqu (a type of starter culture) and possessing multiple tolerances and hexanoic acid-producing characteristics, as described in claim 1, is characterized in that... The yield of hexanoic acid can reach 1.56 mg / mL.

7. The application of Bacillus amyloliquefaciens as described in any one of claims 1-6 in the production of Daqu (a type of starter culture).

8. The application of Bacillus amyloliquefaciens as described in any one of claims 1-6 in alcoholic beverage production.

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