Bacillus amyloliquefaciens for producing heptanoic acid and nonanoic acid as well as isolated culture method and application of bacillus amyloliquefaciens

Abstract

The invention discloses bacillus amyloliquefaciens capable of producing heptanoic acid and nonanoic acid as well as an isolated culture method and application of the bacillus amyloliquefaciens, and belongs to the technical field of microorganisms. The bacillus amyloliquefaciens LZLJ2-11 is separated and screened from high-temperature yeast for making hard liquor, the preservation number is CCTCC NO: M 2025367, the bacillus amyloliquefaciens LZLJ2-11 can produce heptanoic acid and nonanoic acid at the same time through wheat, the performance is stable, the bacillus amyloliquefaciens LZLJ2-11 can adapt to the high-temperature environment, the high-acid environment and other environments in the liquor making process, flavor precursor substances are effectively provided when the bacillus amyloliquefaciens LZLJ2-11 is applied to liquor making, and the production efficiency is improved. The content of ethyl heptanoate and ethyl nonanoate in the fermented grains is increased, the microbial resource library of white spirit brewing is enriched, functional division of microbial communities in the white spirit brewing process is facilitated, and microbial resources are provided for research of the white spirit brewing complex microbial inoculants.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus amyloliquefaciens capable of producing heptanoic acid and nonanoic acid, and its isolation, culture method and application. Background Art

[0002] Heptanoic acid and nonanoic acid belong to medium and long-chain fatty acids. Heptanoic acid has a weak oily and cheesy smell, and nonanoic acid presents a similar waxy or oily smell. However, the flavor contribution of heptanoic acid and nonanoic acid to baijiu is weak. They mainly serve as precursor substances to react with ethanol under the action of esterase to generate corresponding esters, namely ethyl heptanoate and ethyl nonanoate. Ethyl heptanoate has an aroma similar to that of apples or pears, and ethyl nonanoate has a fruity and rose aroma, which can increase the layering and coordination of the baijiu body flavor. However, in existing research, there are few strains capable of producing both heptanoic acid and nonanoic acid, and there is no Bacillus amyloliquefaciens. Summary of the Invention

[0003] In view of the deficiencies in existing research, the present invention screened a Bacillus amyloliquefaciens capable of producing both heptanoic acid and nonanoic acid from high-temperature daqu for baijiu brewing. It can adapt to the baijiu brewing environment and increase the contents of ethyl heptanoate and ethyl nonanoate.

[0004] In the first aspect, the present invention provides a strain of Bacillus amyloliquefaciens named Bacillus amyloliquefaciens LZLJ2-11, which was deposited at the China Center for Type Culture Collection (CCTCC) on March 4, 2025, and its deposit number is CCTCC NO: M 2025367.

[0005] Among them, the biological characteristics of the above-mentioned Bacillus amyloliquefaciens LZLJ2-11 are as follows: on DSMZ 1 agar medium, the colony characteristics are irregularly round, with serrated edges, a raised and wrinkled middle, milky white, and opaque.

[0006] Among them, the 16S rDNA sequence of the above-mentioned Bacillus amyloliquefaciens LZLJ2-11 is shown as SEQ ID NO:1.

[0007] The 16S rDNA sequence of Bacillus amyloliquefaciens LZLJ2-11 of SEQ ID NO:1 is as follows:

[0008] CGGTGGGGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTG

[0009] AGTGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATA

[0010] ACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAA

[0011] AAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGG

[0012] TAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG

[0013] GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATG

[0014] GACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGC

[0015] TCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAA

[0016] CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCG

[0017] TTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAA

[0018] GCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAG

[0019] GAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTG

[0020] GCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAA

[0021] CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGT

[0022] TTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGC

[0023] AAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT

[0024] AATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGA

[0025] TAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGT

[0026] CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCAT

[0027] TCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0028] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAA

[0029] GGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCA

[0030] GTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCG

[0031] GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACAC

[0032] CCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGTGACAGAGG。

[0033] Second aspect, the present invention also provides a method for isolating and culturing the above-mentioned Bacillus amyloliquefaciens LZLJ2-11, which comprises the following steps: A. Mix high-temperature Daqu and water, and make a bacterial suspension by shaking in a shaker. Take the supernatant of the bacterial suspension and mix it with water, and make dilutions of 10 -2 、10 -3 、10 -4 、10 -5 and 10 -6 by gradient dilution; coat the dilutions of each dilution degree on DSMZ 1 solid medium for culture; then pick out the vigorously growing single colonies and perform multiple streakings until all single colonies are purified, and inoculate the purified strains into TSB liquid medium for culture to isolate and purify a number of strains;

[0034] B. After activating the several strains obtained in step A with TSB liquid medium respectively, obtain bacterial solutions; inoculate the bacterial solutions into TSB liquid medium to obtain seed solutions; inoculate the seed solutions into wheat solid medium, simulate the temperature rising program of high-temperature Daqu, and perform gradient temperature culture, the culture temperature is 37±1℃ - 48±1℃ - 52±1℃ - 55±1℃ - 59±1℃ - 63±1℃ - 63±1℃, each gradient is cultured for 24 - 36h, detect the volatile substances of the obtained wheat fermentation samples, and screen to obtain Bacillus amyloliquefaciens LZLJ2-11 that simultaneously produces heptanoic acid and nonanoic acid.

[0035] Among them, in the above-mentioned isolation and culture method, in step A, when mixing high-temperature Daqu and water, the mass ratio of high-temperature Daqu to water is 1 - 2:9 - 8.

[0036] Among them, in the above-mentioned isolation and culture method, in step A, when preparing the bacterial suspension, the conditions of shaking in the shaker are: room temperature of the shaker, rotating at 150 - 180 r / min for 30 - 60 min.

[0037] Among them, in the above-mentioned isolation and culture method, in step A, when mixing the supernatant of the bacterial suspension and water, the volume ratio of the supernatant of the bacterial suspension to water is 1±0.01:9±0.1.

[0038] Among them, in the above-mentioned isolation and culture method, in step A, the culture conditions of DSMZ 1 solid medium are: culture at 45℃ for 1 - 3 d.

[0039] Among them, in the above-mentioned isolation and culture method, in step A, the culture conditions of TSB liquid medium are: culture temperature 37 - 45℃, shaker rotation speed 120 - 150 rpm, culture for 1 - 2 d.

[0040] Among them, in the above-mentioned isolation and culture method, in step A, 15 strains are isolated and purified.

[0041] Among them, in the above separation and culture method, in step B, the specific operation of activation is as follows: inoculate several strains obtained in step A into TSB liquid medium at an inoculation amount of 1% - 10%, and culture them on a shaker at 37 - 45°C and 120 - 150 r / min for 1 - 2 days.

[0042] Among them, in the above separation and culture method, in step B, inoculate the bacterial liquid into TSB liquid medium at an inoculation amount of 1% - 10%.

[0043] Among them, in the above separation and culture method, in step B, the conditions for culturing the seed liquid are: culture on a shaker at 37 - 45°C and 120 - 150 r / min for 1 - 2 days.

[0044] Among them, in the above separation and culture method, in step B, inoculate the seed liquid into wheat solid medium at an inoculation amount of 2% - 10%.

[0045] Among them, in the above separation and culture method, the formula of DSMZ 1 solid medium is: peptone 5.0 g, meat extract powder 3.0 g, agar 15.0 g, add water to 1000 mL, adjust the pH to 7.0 ± 0.2, and autoclave at 121°C for 15 minutes.

[0046] Among them, in the above separation and culture method, the formula of TSB liquid medium is: tryptone 17.0 g, sodium chloride 5.0 g, soybean papain hydrolyzate 3.0 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g, add water to 1000 mL, adjust the pH to 7.2 ± 0.2, and autoclave at 121°C for 15 minutes.

[0047] Among them, in the above separation and culture method, the formula of wheat solid medium is: take crushed wheat, add water and mix evenly according to the ratio of material to water of 1 g: 0.25 - 0.3 mL, moisten for 1 - 2 h, sterilize at 121°C for 30 min, and add sterile water accounting for 20% - 60% of the wheat quality after cooling, and stir evenly.

[0048] In the third aspect, the present invention also provides the application of the above Bacillus amyloliquefaciens LZLJ2 - 11 in the simultaneous production of heptanoic acid and nonanoic acid.

[0049] Fourth aspect, the present invention also provides a method for simultaneously producing heptanoic acid and nonanoic acid by using the above-mentioned Bacillus amyloliquefaciens LZLJ2-11, which comprises the following steps: activating Bacillus amyloliquefaciens LZLJ2-11 in a TSB liquid medium to obtain a bacterial solution; inoculating the bacterial solution into the TSB liquid medium and culturing to obtain a seed solution; inoculating the seed solution into a wheat solid medium, simulating the temperature rising procedure of high-temperature Daqu, and performing gradient temperature culture, the culture temperature is 37±1°C - 48±1°C - 52±1°C - 55±1°C - 59±1°C - 63±1°C - 63±1°C, and each gradient is cultured for 24 - 36 h to obtain a wheat fermentation sample containing both heptanoic acid and nonanoic acid.

[0050] Among them, in the above method for producing heptanoic acid and nonanoic acid, the specific operation of activation is: inoculating Bacillus amyloliquefaciens LZLJ2-11 into the TSB liquid medium at an inoculation amount of 1% - 10%, and culturing in a shaker at 37°C - 45°C and 120 - 150 r / min for 1 - 2 d.

[0051] Among them, in the above method for producing heptanoic acid and nonanoic acid, the bacterial solution is inoculated into the TSB liquid medium at an inoculation amount of 1% - 10%.

[0052] Among them, in the above method for producing heptanoic acid and nonanoic acid, the conditions for culturing to obtain a seed solution are: culturing in a shaker at 37°C - 45°C and 120 - 150 r / min for 1 - 2 d.

[0053] Among them, in the above method for producing heptanoic acid and nonanoic acid, the seed solution is inoculated into the wheat solid medium at an inoculation amount of 2% - 10%.

[0054] Among them, in the above method for producing heptanoic acid and nonanoic acid, the formula of the TSB liquid medium is: 17.0 g of tryptone, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolyzate, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, adding water to 1000 mL, adjusting the pH to 7.2±0.2, and autoclaving at 121°C for 15 minutes.

[0055] Among them, in the above method for producing heptanoic acid and nonanoic acid, the formula of the wheat solid medium is: taking the crushed wheat, adding water and mixing evenly according to the material-water ratio of 1 g: 0.25 - 0.3 mL, moistening for 1 - 2 h, sterilizing at 121°C for 30 min, and adding sterile water accounting for 20% - 60% of the wheat mass after cooling, and stirring evenly.

[0056] Among them, in the above method for producing heptanoic acid and nonanoic acid, the content of heptanoic acid in the obtained wheat fermentation sample containing both heptanoic acid and nonanoic acid is 0.159 μg / g, and the content of nonanoic acid is 0.120 μg / g.

[0057] Fifth aspect, the present invention also provides the application of the above-mentioned Bacillus amyloliquefaciens LZLJ2-11 in Baijiu brewing.

[0058] Sixth aspect, the present invention also provides a method for brewing Chinese liquor by using the above-mentioned Bacillus amyloliquefaciens LZLJ2-11, which comprises the following steps: After adding water to moisten the crushed sorghum and spreading it out to cool, inoculate Daqu starter powder and the seed liquid of Bacillus amyloliquefaciens LZLJ2-11, and carry out stacking fermentation. The fermentation temperature is 28±1℃-35±1℃-45±1℃, and each gradient is cultured for 24-36 h. After the stacking fermentation is completed, seal and ferment at 30℃-35℃ for 20-30 d to obtain fermented grains; The ratio of the seed liquid of Bacillus amyloliquefaciens LZLJ2-11 to the dry weight of sorghum is 2-5 mL: 100 g.

[0059] Among them, in the method for brewing Chinese liquor, the addition amount of Daqu starter powder is 13%-16% of the dry weight of sorghum.

[0060] Among them, in the method for brewing Chinese liquor, the concentration of the seed liquid of Bacillus amyloliquefaciens LZLJ2-11 is 10 9 ~10 10 CFU / mL.

[0061] Among them, in the method for brewing Chinese liquor, the content of ethyl heptanoate in the obtained fermented grains is 22.068 μg / kg, and the content of ethyl nonanoate is 55.723 μg / kg.

[0062] Among them, in the method for brewing Chinese liquor, the seed liquid of Bacillus amyloliquefaciens LZLJ2-11 is prepared by the following method: Inoculate Bacillus amyloliquefaciens LZLJ2-11 into TSB liquid medium at an inoculation amount of 1%-10%, and culture it in a shaker at 37℃-45℃ and 120-150 r / min for 1-2 d to obtain a bacterial liquid; Inoculate the bacterial liquid into TSB liquid medium at an inoculation amount of 1%-10%, and culture it in a shaker at 37℃-45℃ and 120-150 r / min for 1-2 d to obtain a seed liquid.

[0063] In the present invention, when using Bacillus amyloliquefaciens LZLJ2-11 to brew Chinese liquor, adding water to moisten the crushed sorghum and spreading it out to cool adopt conventional operations in the art.

[0064] Advantages of the present invention:

[0065] The Bacillus amyloliquefaciens LZLJ2-11 provided by the present invention is derived from high-temperature Daqu, adapts to high-temperature fermentation during the preparation of high-temperature Daqu, and can utilize wheat to produce heptanoic acid and nonanoic acid. When it is applied to the brewing of Chinese liquor, it effectively provides flavor precursor substances, increases the content of ethyl heptanoate and ethyl nonanoate in the fermented grains, and provides microbial resources for the research of compound microbial agents for Chinese liquor brewing.

[0066] The preservation number of the Bacillus amyloliquefaciens of the present invention is CCTCC NO: M 2025367, the preservation time is March 4, 2025, the preservation center is the China Center for Type Culture Collection (CCTCC), the address is Wuhan University, Wuhan, China, the postal code is 430072, and the taxonomic name is Bacillus amyloliquefaciens LZLJ2-11. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the colony morphology diagram of Bacillus amyloliquefaciens LZLJ2-11 of the present invention.

[0068] Figure 2 This is the GC-MS diagram of volatile substances in the solid-state fermentation sample of wheat by Bacillus amyloliquefaciens LZLJ2-11.

[0069] Figure 3 This is the GC-MS diagram of volatile substances in fermented grains with the addition of Bacillus amyloliquefaciens LZLJ2-11. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0070] The present invention will be further described in detail below through examples, but the protection scope of the present invention is not limited to the scope of the described examples.

[0071] In the examples, the medium formulations involved are as follows:

[0072] DSMZ 1 solid medium: 5.0 g of peptone, 3.0 g of meat extract powder, 15.0 g of agar, add water to 1000 mL, adjust the pH to 7.0, and sterilize at 121 °C for 15 minutes under high-pressure steam.

[0073] TSB liquid medium: 17.0 g of tryptone, 5.0 g of sodium chloride, 3.0 g of soy peptone hydrolysate, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, add water to 1000 mL, adjust the pH to 7.2, and sterilize at 121 °C for 15 minutes under high-pressure steam.

[0074] Wheat solid medium: Take the pulverized wheat, add water and mix evenly according to the ratio of material to water of 1:0.25 - 0.3, moisten for 1 h, dispense into 100 mL Erlenmeyer flasks, about 40 g of material in each flask, sterilize at 121 °C for 30 min, and add 16 mL of sterile water to each flask after cooling, and stir evenly.

[0075] In the examples, the specific method for determining volatile products by headspace solid-phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS) is as follows:

[0076] Take the sample and add it to a headspace vial. Add 2 g of NaCl, 5 mL of pure water, and 5 μL of internal standard (2-octanol at 0.264 mg / mL). After equilibrating the prepared sample at 55 °C for 5 min, extract it with a 50 / 30 μm DVB / CAR / PDMS extraction head at 55 °C for 45 min. After extraction, desorb it at 250 °C in the GC injection port for 5 min.

[0077] In the examples, the GC-MS detection chromatographic conditions are as follows:

[0078] Gas chromatography conditions: HP-INNOWAX chromatographic column (60 m × 0.25 mm × 0.25 μm); temperature programming: initial temperature 40 °C, hold for 1 min, increase to 75 °C at 2.5 °C / min, increase to 162 °C at 3 °C / min, increase to 230 °C at 6 °C / min, hold for 5 min, carrier gas is high-purity helium (1.0 mL / min); injection port temperature 250 °C, splitless.

[0079] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40 - 450 m / z.

[0080] Using 2-octanol at 0.264 mg / mL as the internal standard, the compound retrieval results are matched with the NIST standard spectral library, and compounds with a similarity of more than 80% are confirmed as the target compounds.

[0081] Example 1: Isolation and purification of strains

[0082] Weigh 10 g of high-temperature Daqu and add it to a 250 mL conical flask containing 90 mL of sterile water with glass beads (90 mL of sterile water, physiological saline can also be used). Shake it on a shaker at room temperature with a rotation speed of 150 r / min for 30 min to prepare a sample bacterial suspension for standby. Pipette 1 mL of the supernatant of the bacterial suspension and 9 mL of sterile water (or physiological saline) into a 15 mL centrifuge tube to prepare a dilution of 10 -2 , and perform gradient dilution in this way to obtain dilutions of 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 respectively; Take 100 μL of each dilution and spread it on DSMZ 1 solid medium, and incubate it in an incubator at 45 °C for 1 - 3 d; Then pick out the vigorously growing single colonies and perform multiple streakings until all single colonies are purified. The purified strains are inoculated into TSB liquid medium, cultured at 45 °C with a shaker rotation speed of 120 rpm. After culturing for 1 day, store the strains at -80 °C with 30% glycerol for further analysis. A total of 15 strains are isolated and purified.

[0083] Example 2: Fermentation experiment on wheat solid medium

[0084] Take the glycerol preservation tubes of 15 strains and inoculate them into 1 mL of TSB liquid medium at an inoculation amount of 10%. Incubate them on a shaker at 37 °C and 120 r / min for 1 day to activate the strains; transfer the bacterial liquid to 5 mL of TSB liquid medium at an inoculation amount of 10% and incubate it on a shaker at 37 °C and 120 r / min for 1 day to prepare the seed liquid; inoculate the seed liquid into the wheat solid medium at an inoculation amount of 5%, simulate the temperature-rising program of high-temperature Daqu, and perform gradient temperature cultivation. The cultivation temperatures are 37 °C - 48 °C - 52 °C - 55 °C - 59 °C - 63 °C - 63 °C, and each gradient is cultivated for 1 day. Set up a blank control group experiment under the same cultivation conditions. After the experiment, use the HS-SPME / GC-MS method to detect the volatile substances in the samples. Take the culture solution without added bacteria fermentation as the blank control group and calculate the content.

[0085] One strain that can produce heptanoic acid and nonanoic acid simultaneously was screened out from 15 strains, and the contents were 0.159 μg / g and 0.120 μg / g respectively. Its GCMS detection spectrum is as Figure 2 shown.

[0086] Example 3: Molecular identification of the target strain

[0087] After the target strain was amplified and cultured, take the fresh bacterial liquid in the logarithmic growth phase, centrifuge to collect the bacterial cells, and use the Ezup column bacterial genomic DNA extraction kit of Shanghai Sangon Biotech Co., Ltd. to extract genomic DNA. Use the bacterial universal primers 27F / 1492R to amplify the full-length sequence of its 16S rDNA, as follows.

[0088] 27F (SEQ ID NO:2: 5′-AGAGTTTGATCCTGGCTCAG-3′).

[0089] 1492R (SEQ ID NO:3: 5′-GGTTACCTTGTTACGACTT-3′).

[0090] Table 1 Reaction system

[0091]

[0092]

[0093] Table 2 Reaction program

[0094]

[0095] According to the above process, perform 30 cycles of "denaturation - annealing - extension" PCR reactions. The PCR products are separated and examined by 1.0% agarose gel electrophoresis at a voltage of about 11 V / cm for 20 min.

[0096] The purification of the PCR products was carried out according to the instructions of the small - volume gel recovery PCR product purification kit from Sangon Biotech (Shanghai) Co., Ltd., and the sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0097] The gene sequence of the 16S rDNA fragment obtained by sequencing was aligned by BLAST of NCBI, combined with the colony morphology. As Figure 1 shown, on DSMZ 1 agar medium, the colony characteristics were irregularly round, with serrated edges, a raised and wrinkled middle, milky white, opaque. The species information of the strain was determined, and it was identified as Bacillus amyloliquefaciens and named Bacillus amyloliquefaciens LZLJ2 - 11.

[0098] Example 4: Application in Baijiu brewing

[0099] Take crushed sorghum and add hot water at 50% (v / m) with a water temperature of 95°C. After moistening the grains for 2 h, steam them for 30 min; after steaming, supplement 16% of water (95°C); spread the sorghum to cool to 28 - 30°C and inoculate with Daqu koji powder. The inoculation amount of Daqu is 16% of the dry weight of sorghum. In the experimental group, add the seed liquid of Bacillus amyloliquefaciens LZLJ2 - 11, and the preparation method of the seed liquid is the same as that in Example 2. The blank control group adds TSB liquid medium, and the added liquid volume is 2% (v / m) of the dry weight of sorghum. After mixing well, put them into a sealed bag and place it in a constant - temperature incubator with the opening exposed. The fermentation temperature is 28°C - 35°C - 45°C, and each gradient is cultured for 1 d. After stacking fermentation, put them into a sealed tank and ferment at 30°C for 30 d. Then take the fermented grains and detect them by HS - SPME / GC - MS method.

[0100] The experimental results are as Figure 3 shown. The detected contents of ethyl heptanoate and ethyl nonanoate are shown in Table 3. The contents of ethyl heptanoate and ethyl nonanoate in the fermented grains added with Bacillus amyloliquefaciens LZLJ2 - 11 increased. Ethyl heptanoate was not detected in the control group, and the content in the experimental group was 22.068 μg / kg. The content of ethyl nonanoate increased from 17.685 μg / kg in the control group to 55.723 μg / kg.

[0101] Table 3 Contents of ethyl heptanoate and ethyl nonanoate in fermented grains (μg / kg)

[0102]

[0103]

[0104] It can be seen that the Bacillus amyloliquefaciens LZLJ2-11 provided by the present invention can utilize wheat to simultaneously produce heptanoic acid and nonanoic acid, with the contents being 0.159 μg / g and 0.120 μg / g respectively. It can adapt to environments such as high temperature and high acid during the white liquor brewing process. When applied to white liquor brewing, the ethyl heptanoate in the fermented grains has never been detected and increased to 22.068 μg / kg, and the ethyl nonanoate has increased from 17.685 μg / kg to 55.723 μg / kg, effectively providing flavor precursor substances and providing microbial resources for the research of compound microbial agents for white liquor brewing.

Claims

1. Bacillus amyloliquefaciens, characterized in that: The strain of Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens LZLJ2-11, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025367.

2. The method for isolating and culturing Bacillus amyloliquefaciens according to claim 1, characterized in that: It includes the following steps: A. Mix high-temperature Daqu and water, and make a bacterial suspension by shaking on a shaker. Take the supernatant of the bacterial suspension and mix it with water, and prepare dilutions of 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 by gradient dilution; Spread the dilutions of each dilution factor on DSMZ1 solid medium for cultivation; Then pick the vigorously growing single colonies and streak them multiple times until all single colonies are purified. The purified strains are inoculated into TSB liquid medium for cultivation, and several strains are obtained by isolation and purification; B. After activating several strains obtained in step A in TSB liquid medium respectively, bacterial solutions are obtained; the bacterial solutions are inoculated into TSB liquid medium to obtain seed solutions; the seed solutions are inoculated into wheat solid medium, and the temperature-rising program of high-temperature Daqu is simulated for gradient temperature cultivation. The cultivation temperatures are 37±1°C - 48±1°C - 52±1°C - 55±1°C - 59±1°C - 63±1°C - 63±1°C, and each gradient is cultivated for 24 - 36 h. The volatile substances of the obtained wheat fermentation samples are detected, and Bacillus amyloliquefaciens LZLJ2-11 that can produce both heptanoic acid and nonanoic acid is screened out.

3. The isolation and culture method of Bacillus amyloliquefaciens according to claim 2, wherein: At least meet one of the following: In step A, when the high-temperature Daqu and water are mixed, the mass ratio of high-temperature Daqu to water is 1 - 2:9 - 8; In step A, when preparing the bacterial suspension, the conditions of the shaker oscillation are: at room temperature of the shaker, oscillating at a speed of 150 - 180 r / min for 30 - 60 min; In step A, when the supernatant of the bacterial suspension and water are mixed, the volume ratio of the supernatant of the bacterial suspension to water is 1±0.01:9±0.1; In step A, the cultivation conditions of DSMZ 1 solid medium are: cultivating at 45°C for 1 - 3 d; In step A, the cultivation conditions of TSB liquid medium are: cultivation temperature 37 - 45°C, shaker speed 120 - 150 rpm, cultivating for 1 - 2 d; In step A, 15 strains are obtained by separation and purification; In step B, the specific operation of activation is: inoculating several strains obtained in step A into TSB liquid medium at an inoculation amount of 1% - 10% respectively, and culturing in a shaker at 37 - 45°C and 120 - 150 r / min for 1 - 2 d; In step B, the bacterial solution is inoculated into TSB liquid medium at an inoculation amount of 1% - 10%; In step B, the conditions for obtaining the seed solution are: culturing in a shaker at 37 - 45°C and 120 - 150 r / min for 1 - 2 d; In step B, the seed solution is inoculated into wheat solid medium at an inoculation amount of 2% - 10%; The formula of DSMZ 1 solid medium is: peptone 5.0 g, meat extract powder 3.0 g, agar 15.0 g, adding water to 1000 mL, adjusting the pH to 7.0±0.2, and autoclaving at 121°C for 15 minutes; The formula of TSB liquid medium is: tryptone 17.0 g, sodium chloride 5.0 g, soy peptone 3.0 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g, adding water to 1000 mL, adjusting the pH to 7.2±0.2, and autoclaving at 121°C for 15 minutes; The formula of wheat solid medium is: take the crushed wheat, add water and mix evenly according to the material-water ratio of 1 g:0.25 - 0.3 mL, moisten for 1 - 2 h, sterilize at 121°C for 30 min, and add sterile water accounting for 20% - 60% of the wheat mass after cooling, and stir evenly.

4. Use of Bacillus amyloliquefaciens described in claim 1 in the simultaneous production of heptanoic acid and nonanoic acid.

5. A method for simultaneously producing heptanoic acid and nonanoic acid using Bacillus amyloliquefaciens as claimed in claim 1, characterized in that: It includes the following steps: After activating Bacillus amyloliquefaciens LZLJ2-11 in TSB liquid medium, a bacterial solution is obtained; the bacterial solution is inoculated into TSB liquid medium and cultured to obtain a seed solution; The seed solution is inoculated into wheat solid medium, and the temperature increase program of high-temperature Daqu is simulated for gradient temperature culture. The culture temperatures are 37±1°C - 48±1°C - 52±1°C - 55±1°C - 59±1°C - 63±1°C - 63±1°C, and each gradient is cultured for 24 - 36 h to obtain a wheat fermentation sample containing both heptanoic acid and nonanoic acid.

6. The method according to claim 5, wherein: At least one of the following is satisfied: The specific operation of activation is: inoculating Bacillus amyloliquefaciens LZLJ2-11 into TSB liquid medium at an inoculation amount of 1% - 10%, and culturing on a shaker at 37°C - 45°C and 120 - 150 r / min for 1 - 2 d; The bacterial solution is inoculated into TSB liquid medium at an inoculation amount of 1% - 10%; The conditions for culturing to obtain a seed solution are: culturing on a shaker at 37°C - 45°C and 120 - 150 r / min for 1 - 2 d; The seed solution is inoculated into wheat solid medium at an inoculation amount of 2% - 10%; The formula of TSB liquid medium is: 17.0 g of tryptone, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolyzate, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, adding water to 1000 mL, adjusting the pH to 7.2±0.2, and autoclaving at 121°C for 15 minutes; The formula of wheat solid medium is: taking crushed wheat, adding water and mixing evenly according to the material-water ratio of 1 g: 0.25 - 0.3 mL, moistening for 1 - 2 h, sterilizing at 121°C for 30 min, and adding sterile water accounting for 20% - 60% of the wheat mass after cooling and stirring evenly; The content of heptanoic acid in the obtained wheat fermentation sample containing both heptanoic acid and nonanoic acid is 0.159 μg / g, and the content of nonanoic acid is 0.120 μg / g.

7. Use of Bacillus amyloliquefaciens described in claim 1 in Baijiu brewing.

8. A method for brewing Chinese liquor using Bacillus amyloliquefaciens described in claim 1, characterized in that: It includes the following steps: After adding water to moisten and spreading out crushed sorghum, inoculating Daqu koji powder and the seed solution of Bacillus amyloliquefaciens LZLJ2-11, and carrying out stacking fermentation. The fermentation temperatures are 28±1°C - 35±1°C - 45±1°C, and each gradient is cultured for 24 - 36 h. After the stacking fermentation is completed, sealed fermentation is carried out at 30°C - 35°C for 20 - 30 d to obtain fermented grains; the ratio of the seed solution of Bacillus amyloliquefaciens LZLJ2-11 to the dry weight of sorghum is 2 - 5 mL: 100 g.

9. The method according to claim 8, characterized in that: At least one of the following is satisfied: The addition amount of Daqu koji powder is 13% - 16% of the dry weight of sorghum; The concentration of the Bacillus amyloliquefaciens LZLJ2-11 seed liquid is 10 9 ~10 10 CFU / mL; The content of ethyl heptanoate in the obtained fermented grains is 22.068 μg / kg, and the content of ethyl nonanoate is 55.723 μg / kg.

10. The method according to claim 8, wherein: The Bacillus amyloliquefaciens LZLJ2-11 seed liquid is prepared by the following method: inoculate Bacillus amyloliquefaciens LZLJ2-11 into TSB liquid medium at an inoculation amount of 1% - 10%, and culture it on a shaker at 37°C - 45°C and 120 - 150 r / min for 1 - 2 days to obtain a bacterial liquid; inoculate the bacterial liquid into TSB liquid medium at an inoculation amount of 1% - 10%, and culture it on a shaker at 37°C - 45°C and 120 - 150 r / min for 1 - 2 days to obtain the seed liquid.

Images