Bacillus amyloliquefaciens derived from yeast for making hard liquor and having multiple tolerance and hexanoic acid production characteristics as well as separation method and application of bacillus amyloliquefaciens

Abstract

The invention discloses bacillus amyloliquefaciens which is derived from yeast for making hard liquor and has multiple tolerance and hexanoic acid production characteristics as well as a separation method and application of the bacillus amyloliquefaciens. The strain is separated from yeast for making hard liquor, can produce hexanoic acid, has the characteristics of high temperature resistance, acid resistance, ethanol resistance and the like, is identified as bacillus amyloliquefaciens DJ001, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and has the preservation number of CGMCC No.33299. The invention further discloses a method for preparing the caproic acid-resistant yeast for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor for making hard liquor. When the strain is added into yeast for making hard liquor, the hexanoic acid production capacity and saccharification capacity of the yeast for making hard liquor can be improved, and when the strain is added into fermented grains, the yield of white spirit and the content of ethyl hexanoate can be improved. The method has important values in the aspects of improving the yield and quality of the white spirit, saving the cost, improving the efficiency and the like, and can increase enterprise and social benefits.

Description

Technical Field

[0001] The invention relates to the field of caproic acid bacteria and liquor production, in particular to a bacillus amyloliquefaciens derived from Daqu and having multiple tolerances and caproic acid production characteristics, and an isolation method and application thereof, which can be used to increase caproic acid and ethyl caproate in liquor. Background Art

[0002] Chinese liquor is a type of alcoholic beverage with a long history. Luzhou-flavor liquor is the most produced and sold flavor type of Chinese liquor. Ethyl caproate is an important flavor component of Luzhou-flavor liquor. Caproic acid bacteria can produce caproic acid, which can be used as a precursor for the production of ethyl caproate. Therefore, the isolation, identification and application of caproic acid bacteria are of great significance in improving the quality of liquor.

[0003] In the winemaking environment, the contact area between the cellar mud and the mash is small; after the Daqu is crushed, it is evenly sprinkled into the mash, and the contact with the mash is more complete. Theoretically, the microorganisms in Daqu are more likely to play a role in the winemaking process than those in the cellar mud. However, in view of the growth characteristics of many caproic acid bacteria and some inherent understandings of people, in previous studies, the isolation of caproic acid bacteria mainly used cellar mud as raw material, and there were no reports on the isolation of caproic acid bacteria from Daqu. Therefore, screening new caproic acid bacteria from Daqu and applying them to the production of Luzhou-flavor liquor is of great significance to increase the content of ethyl caproate in liquor.

[0004] In addition, the process of making koji and brewing liquor is accompanied by extreme environmental conditions, such as high temperature, low pH, high ethanol, etc. These environmental conditions can affect the growth and function of caproic acid bacteria, thereby affecting their application effect. Therefore, it is of great significance to screen strains that are resistant to high temperature, acid, and ethanol. Summary of the invention

[0005] The present invention provides a Bacillus amyloliquefaciens derived from Daqu and having multiple tolerances and hexanoic acid production characteristics, and an isolation method and application thereof, so as to solve the problem of insufficient content of hexanoic acid, ethyl hexanoate and the like in the products of some Luzhou-flavor liquor enterprises.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: The strains of the present invention Bacillus amyloliquefaciens DJ001 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit date being January 3, 2025 and the deposit number being CGMCC No.33299.

[0007] Furthermore, the strain B. amyloliquefaciens DJ001 grows well at a pH of 1-11.

[0008] Furthermore, the strain B. amyloliquefaciensDJ001 grows well at 22-52℃.

[0009] Furthermore, the strain B. amyloliquefaciens DJ001 grows well in ethanol content of 0-12% vol.

[0010] A Bacillus amyloliquefaciens strain derived from Daqu with multiple tolerance and hexanoic acid production characteristics B. amyloliquefaciens And the separation method comprises the following steps: Step 1: Using Daqu as the screening source, isolate and purify bacterial strains by dilution plate method and plate streak method; Step 2: Detecting the hexanoic acid production ability of the bacteria obtained in step 1 by copper sulfate colorimetric method, and further screening candidate strains producing hexanoic acid according to the detection results; Step 3: Detecting the ability of the bacteria obtained in step 2 to produce caproic acid by gas chromatography, and screening out strains with high caproic acid production according to the detection results; 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.

[0011] An application of the Bacillus amyloliquefaciens in the production of Daqu.

[0012] An application of the above-mentioned Bacillus amyloliquefaciens in wine production.

[0013] A method for isolating caproic acid-producing strains using Daqu as a source and its application.

[0014] Compared with the prior art, the advantages of the present invention are: The present invention is the first to isolate Bacillus amyloliquefaciens with multiple tolerance and hexanoic acid production characteristics from Daqu B. amyloliquefaciens DJ001, adding the strain to Daqu can improve the Daqu's hexanoic acid production and saccharification capacity, and adding the strain to fermented grains can improve the yield and ethyl hexanoate content of liquor. The present invention has important value in improving the yield and quality of liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison chart of caproic acid production in Daqu culture fluid. Different lowercase letters indicate significant differences (P < 0.05); the values ​​in the figure were repeated more than 3 times.

[0016] Figure 2 This is the color reaction diagram of copper sulfate.

[0017] Figure 3This is a graph of caproic acid production of caproic acid bacteria. Different lowercase letters indicate significant differences (P < 0.05); the values ​​in the figure were repeated more than 3 times.

[0018] Figure 4 The comparison chart of caproic acid production of various caproic acid bacteria at different temperatures. Among them: different lowercase letters indicate significant differences (P < 0.05); the values ​​in the figure were repeated more than 3 times.

[0019] Figure 5 This is a comparison of caproic acid production of various caproic acid bacteria under different ethanol concentrations. Different lowercase letters indicate significant differences (P < 0.05); the values ​​in the figure were repeated more than 3 times.

[0020] Figure 6 The comparison chart of caproic acid production of various caproic acid bacteria at different pH values. Different lowercase letters indicate significant differences (P < 0.05); the values ​​in the figure were repeated more than 3 times.

[0021] Figure 7 This is the agarose gel electrophoresis of the 16S rDNA PCR product of strain DJ001, where: M, marker5000; -, negative control; A, DJ001.

[0022] Figure 8 This is the phylogenetic tree of strain DJ001 based on 16S rDNA sequence.

[0023] Fig. 9 This is a graph of caproic acid production of strain DJ001 cultured in simulated fermentation broth. Among them: "****", P<0.0001; the values ​​in the figure were repeated more than 3 times.

[0024] Fig.10 This is a temperature change curve during the cultivation of Daqu.

[0025] Fig.11 This is a graph showing the caproic acid content in the culture fluid of the koji powder with strain DJ001 added during the production of Daqu. Among them: "***", P<0.001; the values ​​in the figure were repeated more than 3 times.

[0026] Fig.12 This is the saccharification power diagram of Daqu. Among them: "*", P<0.05; the values ​​in the figure were repeated more than 3 times.

[0027] Fig.13 This is a graph of liquor production. Among them: "****", P<0.0001; "*", P<0.05; the values ​​in the graph were repeated more than 3 times.

[0028] Fig.14The graph shows the contents of caproic acid and ethyl caproate in liquor. “*”, P<0.05; ns, no significant difference; the values ​​in the graph were repeated more than 3 times.

[0029] Fig.15 This is the mass diagram of caproic acid and ethyl caproate in liquor. Among them: “****”, P<0.0001; ns, no significant difference; the values ​​in the figure were repeated more than 3 times. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0031] Preparation method of ethanol sodium acetate culture medium in the following examples Sodium ethanol acetate medium: 5 g anhydrous sodium acetate, 1 g Yeast Extract, 0.2 g magnesium sulfate heptahydrate, 0.4 g anhydrous potassium hydrogen phosphate, 0.5 g ammonium sulfate, 10 g calcium carbonate. After weighing and dissolving, the solution is fixed to 1 L, natural pH. Sterilize at 121℃ for 20 min, add 2% (volume fraction) anhydrous ethanol when used after cooling. Solid culture medium should add 2% agar powder before sterilization.

[0032] Extraction method of organic substances such as caproic acid and ethyl caproate in the following embodiments The obtained culture solution was filtered through a 0.22 μm microporous filter membrane, 500 μL of the filtrate was taken into a 5 mL volumetric flask, and anhydrous ether was added to make up to 5 mL, shaken thoroughly, and allowed to stand for 1 min. The upper extract was aspirated to obtain the test solution.

[0033] Gas chromatography detection method in the following examples The contents of hexanoic acid and ethyl hexanoate in the ether extract were detected by gas chromatography. The chromatographic conditions were as follows: column flow rate 2.15 mL / min; column temperature set at 50°C; detector temperature set at 240°C; temperature rise according to the program: the starting temperature was set at 60°C and maintained for 1 min, then increased to 220°C at a rate of 10°C / min and maintained for 2 min; injection volume set at 1 μL; split injection, split ratio 20:1. Embodiment 1

[0034] This embodiment discloses a method for isolating caproic acid-producing Bacillus amyloliquefaciens, comprising the following steps: Step 1: Select high-quality Daqu.

[0035] There are 5 kinds of Luzhou-flavor Daqu in the laboratory, which are numbered as DQ1, DQ2, DQ3, DQ4, and DQ5. The Daqu is fully ground into powder and evenly added to ethanol sodium acetate culture medium. After 7 days of cultivation, the caproic acid production is detected. The results are as follows: Figure 1 Among them, the caproic acid production of Daqu DQ2 and DQ3 was relatively high, which was 0.64 mg / mL and 0.52 mg / mL respectively. Therefore, the subsequent separation and screening of high caproic acid producing bacteria was mainly carried out from Daqu DQ2 and DQ3.

[0036] Step 2: Select strains in Daqu that have the ability to produce caproic acid based on the copper sulfate colorimetric method.

[0037] Take 200 uL of Daqu culture medium in a 2 mL centrifuge tube, dip a small amount of culture medium for streaking and coating to isolate and purify the strain; select a single colony and place it in ethanol sodium acetate medium for 7 days; take 2 mL of culture medium and place it in a 10 mL centrifuge tube, add 1 mL of 20% copper sulfate solution, and observe whether there is a blue-green precipitate. The results are as follows Figure 2 As shown, some strains produced blue-green precipitates, and the corresponding strains were preserved.

[0038] Step 3: Using gas chromatography to detect the hexanoic acid production capacity of the strain obtained in step 2, and screening strains with high hexanoic acid production according to the detection results.

[0039] Specifically, the strain isolated in step 2 was cultured in ethanol sodium acetate medium, and after culturing at 37°C for 7 days, the caproic acid production was detected by gas chromatography. Finally, three caproic acid bacteria with strong caproic acid production ability were obtained, among which DJ001 had the highest caproic acid production. Figure 3 As shown, the hexanoic acid production of DJ001 was 1.56 mg / mL.

[0040] Step 4: Test the tolerance of the candidate strain DJ001 obtained in step 3. The process is as follows: (4.1) Temperature tolerance test of strains Take 200 μL of each of the three bacterial suspensions of DJ001, 1.39-1, and 4.39-1 and transfer them to ethanol sodium acetate liquid culture medium for anaerobically culture at 37°C for 7 days to activate the strains. Inoculate DJ001, 1.39-1, and 4.39-1 bacterial suspensions into ethanol sodium acetate liquid culture medium at a 2% inoculation rate, with a liquid volume of 90%. The culture temperatures were set to 22°C, 27°C, 32°C, 37°C, 42°C, 47°C, and 52°C, respectively. Cultured anaerobically for 7 days. The caproic acid production of each group was tested, and the results are as follows: Figure 4 As the culture temperature increased, the caproic acid production of DJ001, 1.39-1, and 4.39-1 increased first and then decreased; DJ001 had the strongest tolerance to temperature, and when the temperature reached 52°C, DJ001 still had a high caproic acid production.

[0041] (4.2) Ethanol tolerance test of strains Take 200 μL of each of the three bacterial suspensions of DJ001, 1.39-1, and 4.39-1 and transfer them to ethanol sodium acetate liquid culture medium, and culture them anaerobically at 37℃ for 7 days to activate the strains. Inoculate DJ001, 1.39-1, and 4.39-1 bacterial suspensions into ethanol sodium acetate liquid culture medium with ethanol concentrations of 0%, 2%, 4%, 6%, 8%, 10%, and 12% vol at a 2% inoculation rate, and the liquid volume is 90%. Culture them anaerobically at 37℃ for 7 days. Detect the caproic acid production of each group, and the results are as follows: Figure 5 As shown in the figure, with the increase of ethanol concentration, the caproic acid production of DJ001, 1.39-1 and 4.39-1 first increased and then decreased; when the ethanol concentration was 12% vol, DJ001 still had a high caproic acid production.

[0042] (4.3) pH tolerance test of strains Take 200 μL of each of the three bacterial suspensions of DJ001, 1.39-1, and 4.39-1 and transfer them to ethanol sodium acetate liquid culture medium, and culture them anaerobically at 37℃ for 7 days to activate the strains. Inoculate DJ001, 1.39-1, and 4.39-1 bacterial suspensions into ethanol sodium acetate liquid culture medium with pH values ​​of 1, 3, 5, 7, 9, and 11 at a 2% inoculation rate, with a liquid volume of 90%. Culture them anaerobically at 37℃ for 7 days. Detect the caproic acid production of each group, and the results are as follows: Figure 6 As shown in the figure, the caproic acid production of DJ001, 1.39-1, and 4.39-1 increased first and then decreased with the increase of pH; DJ001 had the best adaptability to pH, and when the pH was 1-11, DJ001 had the highest caproic acid production.

[0043] Based on the above hexanoic acid production and tolerance analysis results, DJ001 was selected as the strain for subsequent research and application.

[0044] Step 5: Identification of strains (5.1) Extraction of genomic DNA of strains The genomic DNA of DJ001 was extracted using the SPARKeasy Bacterial Genomic DNA Rapid Extraction Kit.

[0045] (5.2) PCR amplification The genomic DNA was used as a template for PCR amplification and the universal primers 27F / 1492R were used for PCR amplification. The primer sequences are as follows: 27F: 5-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTG TTACGACTT-3'; PCR reaction program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min.

[0046] (5.3) Gel electrophoresis detection of amplified products After mixing 3 μL of amplified product with 3 μL of Loading Buffer, 3 μL of the mixed product was electrophoresed in 1% agarose gel. Figure 7 As shown, the amplified band is relatively single and is approximately 1500 bp in size.

[0047] (5.4) DNA sequencing and sequence alignment The purified PCR product was sent to Shanghai BGI Genomics Co., Ltd. for sequencing to obtain the 16S rDNA sequence of the DJ001 strain. The sequence information is as follows.

[0048] The above sequence was submitted to the NCBI database for Blast comparison, and the results 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 Therefore, DJ001 is B. amyloliquefaciens , name it B. amyloliquefaciens DJ001.

[0049] Bacillus amyloliquefaciens obtained in this example B. amyloliquefaciens DJ001 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 3, 2025, and the deposit number is CGMCC No.33299. Embodiment 2

[0050] This example discloses the Bacillus amyloliquefaciens obtained in Example 1. B. amyloliquefaciens The simulated fermentation of DJ001 in the laboratory is described as follows: Take 1 kg of mash that a wine company is preparing to make wine and set it aside.

[0051] Boil 1 kg of mash with 2 L of sterile water for 30 min, filter, and place the filtrate in a beaker, dilute to 5 L, divide into portions, and sterilize at 115 °C for 30 min as the simulated fermentation liquid.

[0052] CK group: 3 g koji powder, 5 g fermented grains, and 5 mL sterile water were added to a conical flask containing 270 mL simulated fermentation liquid; DJ001 group: 3 g koji powder, 5 g fermented grains, and 5 mL DJ001 bacterial liquid were added to a conical flask containing 270 mL simulated fermentation liquid. After fermentation for 7 days in both groups, the caproic acid production was measured. Fig. 9 As shown, the caproic acid production in the CK group was 0.038 mg / mL, and the caproic acid production in the DJ001 group was 0.73 mg / mL, and the caproic acid production increased by 1821.05%.

[0053] Therefore, adding B. amyloliquefaciens DJ001 has a good effect in producing caproic acid. Embodiment 3

[0054] This example discloses the Bacillus amyloliquefaciens obtained in Example 1. B. amyloliquefaciens The application of DJ001 in the production of large music is described as follows: (1) Strengthening the production method of Daqu The Bacillus amyloliquefaciens obtained in Example 1 B. amyloliquefaciensDJ001 bacterial liquid was evenly added to the koji making raw materials to make medium-high temperature Daqu. The amount of DJ001 bacterial liquid used for each Daqu was 50 L; the conventional Daqu without bacterial strain addition was the control (CK). There were 3840 Daqu blocks in each Daqu room.

[0055] (2) Temperature monitoring of Daqu After the Daqu is placed in the room, the temperature of the Daqu is recorded at 8:00 am every day until the Daqu is taken out of the room 24 days later (after the temperature stabilizes, the temperature of the room is no longer recorded). Fig.10 The temperature changes of the two-room Daqu were basically the same, indicating that the management during the cultivation of Daqu was stable.

[0056] (3) Selection of Daqu samples Five pieces of Daqu were selected from each room by a 5-point sampling method. The Daqu samples from the same room were ground, crushed, and mixed evenly before use in subsequent experiments.

[0057] (4) Detection of the Caproic Acid Production Capacity of Koji Powder Culture Medium The koji powder was added to the ethanol sodium acetate medium at an inoculum of 1.5%, cultured at 37℃ for 7 days, and the caproic acid production was detected. Fig.11 As shown, the caproic acid content of the fortified Daqu powder culture medium prepared by adding DJ001 bacterial solution increased by 600.00% compared with the control Daqu CK without adding bacterial strain.

[0058] (5) Daqu saccharification capacity test The results are as follows Fig.12 As shown in the figure, the saccharification capacity of the fortified Daqu made by adding DJ001 bacterial liquid is 31.11% higher than that of the control Daqu CK without bacterial strain addition. DJ001 is Bacillus amyloliquefaciens. Adding DJ001 during Daqu production may help convert starch into glucose, thereby improving the saccharification capacity of Daqu.

[0059] In summary, the Bacillus amyloliquefaciens described in Example 1 is added to the Daqu B. amyloliquefaciens DJ001 can significantly improve the ability of koji powder culture medium to produce caproic acid and significantly improve the saccharification capacity of Daqu. Embodiment 4

[0060] This example discloses the Bacillus amyloliquefaciens obtained in Example 1. B. amyloliquefaciens The application of DJ001 in liquor production is described as follows: In the production of Luzhou-flavor liquor, the strain is inoculated at a 1% rate. B. amyloliquefaciens DJ001 was evenly added to the mash, which was fermented in a pool for 70 days. After distillation, ordinary and high-quality liquors were obtained. The yield of each grade of liquor was obtained by weighing. Fig.13As shown in the figure, the production of ordinary and premium liquor in the DJ001 group increased by 17.76% and 32.69% respectively compared with the CK group. The overall liquor production of each group was calculated to be 23.04% higher in the DJ001 group than in the CK group.

[0061] The caproic acid and ethyl caproate content of each wine sample was detected by gas chromatography, and then the average concentration of caproic acid and ethyl caproate of each group of wine samples was calculated based on the individual yields. Fig.14 As shown, compared with the CK group, there was no significant difference in the caproic acid concentration in the DJ001 group, while the ethyl caproate concentration increased by 22.11%.

[0062] Combining the concentration and the yield of each wine sample, the mass of caproic acid and ethyl caproate in each group of wine samples can be calculated. Fig.15 As shown, compared with the CK group, the mass of caproic acid in the DJ001 group increased by 15.37% (not statistically significant), and the mass of ethyl caproate increased by 51.71%.

[0063] Therefore, adding functional bacteria B. amyloliquefaciens DJ001 has a significant effect on increasing liquor yield and ethyl hexanoate content. There was no significant difference in the hexanoic acid concentration of liquor between the DJ001 group and the CK group, which may be related to two factors: (1) the increase in liquor yield diluted the concentration of hexanoic acid; (2) hexanoic acid was converted into ethyl hexanoate.

[0064] In summary, the present invention provides a Bacillus amyloliquefaciens strain derived from Daqu and having multiple tolerance and hexanoic acid production characteristics and a method for isolating the strain. Adding the strain to Daqu can improve the hexanoic acid production capacity and saccharification capacity of Daqu, and adding the strain to fermented grains can improve the yield and ethyl hexanoate content of liquor. The present invention has important value in improving the yield and quality of liquor, saving costs, and improving efficiency, and can increase corporate and social benefits.

[0065] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. The embodiments described in the present invention are merely descriptions of the preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. The various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction, and such combinations should also be regarded as the contents disclosed by the present invention as long as they do not violate the concept of the present invention. In order to avoid unnecessary repetition, the present invention will not further explain various possible combinations.

[0066] The present invention is not limited to the specific details of the above-mentioned embodiments. Within the technical concept of the present invention and without departing from the design concept of the present invention, various modifications and improvements made to the technical solution of the present invention by technical personnel in this field should fall within the protection scope of the present invention. The technical contents for which protection is sought in the present invention have been fully recorded in the claims.

Claims

1. A Bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and hexanoic acid production characteristics, characterized in that: The Bacillus amyloliquefaciens is a strain Bacillus amyloliquefaciens DJ001 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit date being January 3, 2025, and the deposit number being CGMCC No.33299.

2. The Bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and hexanoic acid production characteristics according to claim 1, characterized in that: The strain was isolated from Daqu.

3. The Bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and hexanoic acid production characteristics according to claim 1, characterized in that: It grows well at a pH of 1-11.

4. The Bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and hexanoic acid production characteristics according to claim 1, characterized in that: It grows well at 22-52 ℃.

5. The Bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and hexanoic acid production characteristics according to claim 1, characterized in that: It grows well in ethanol content of 0-12% vol.

6. The Bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and hexanoic acid production characteristics according to claim 1, characterized in that: The hexanoic acid yield can reach 1.56 mg / mL.

7. A method for isolating caproic acid-producing strains from Daqu as described in any one of claims 1 to 6 and its use.

8. Use of the Bacillus amyloliquefaciens as claimed in any one of claims 1 to 6 in the production of Daqu.

9. Use of the Bacillus amyloliquefaciens according to any one of claims 1 to 6 in wine production.

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