Bacillus amyloliquefaciens capable of producing dimethyl trisulfide as well as isolated culture method and application of bacillus amyloliquefaciens

Abstract

The invention discloses bacillus amyloliquefaciens capable of producing dimethyl trisulfide as well as an isolated culture method and application of the bacillus amyloliquefaciens, and belongs to the technical field of microorganisms. The bacillus amyloliquefaciens LZLJ2-10 is separated and screened from high-temperature yeast for making hard liquor, the preservation number of the bacillus amyloliquefaciens LZLJ2-10 is CCTCC NO: M 2025366, the bacillus amyloliquefaciens LZLJ2-10 can produce dimethyl trisulfide from wheat, the performance is stable, the bacillus amyloliquefaciens LZLJ2-10 can adapt to high-temperature, high-acid and other environments in the liquor brewing process, when the bacillus amyloliquefaciens LZLJ2-10 is applied to liquor brewing, the content of dimethyl trisulfide in fermented grains is increased to 0.105 microgram / g from undetected, and the content of dimethyl trisulfide in the fermented grains is increased to 0.105 microgram / g from The method enriches a Baijiu brewing microbial resource library, is helpful for researching functional division of microbial communities in the Baijiu brewing process, and provides a theoretical basis for modernized transformation of a traditional process.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a dimethyl trisulfide-producing Bacillus amyloliquefaciens and an isolation and cultivation method and application thereof. Background Art

[0002] Dimethyl trisulfide has a strong characteristic sulfide odor (such as cooked cabbage, onion, or garlic) and can be perceived at extremely low concentrations (threshold of about 0.1-1 μg / L). When present in trace amounts, dimethyl trisulfide can add a complex aged aroma, cellar bottom aroma, or sauce aroma to liquor, especially in strong-flavor and sauce-flavor liquors; but when the content is too high, it will produce a corrupted smell, a pungent odor, destroy the harmony of the liquor body, and reduce the sensory quality. For example, Chen Lu et al. (Chen Lu, Shi Dongmei, He Hongkui et al. Changes in some volatile active components during the distillation of mash [J]. Food Science, 2020, (24): 137-143.) studied the changes in volatile active components during the distillation process and found that the mass concentration of dimethyl trisulfide in the original liquor reached 47.43-580.12 μg / L. Wang Lingling (Wang Lingling. Research on trace components of sauce-flavor liquor and aroma substances of Daqu [J]. Jiangnan University [2025-02-24].) conducted a comparative study on the aroma components of sauce-flavor and other flavor-flavored liquors, and analyzed the aroma activity value (OAV) of the compounds. The results showed that the OAV value of dimethyl trisulfide in the four flavor-flavored Daqu was greater than 1, indicating that it is a relatively important flavor component.

[0003] Therefore, identifying strains capable of producing dimethyl trisulfide during liquor brewing provides a scientific basis for regulating microbial community composition and optimizing process parameters, thereby achieving targeted regulation of dimethyl trisulfide production, avoiding odors (such as rancidity and sulfur odors) caused by excessive production, and ensuring the stability of the liquor flavor. However, existing research has shown that few strains are capable of producing dimethyl trisulfide, and none have been found to be Bacillus amyloliquefaciens. Summary of the Invention

[0004] In response to the shortcomings of existing research, the present invention obtains a dimethyl trisulfide-producing Bacillus amyloliquefaciens strain from high-temperature liquor koji, which helps to study the functional division of microbial communities in the liquor brewing process and provides a theoretical basis for the modernization of traditional processes.

[0005] In a first aspect, the present invention provides a strain of Bacillus amyloliquefaciens, which is named Bacillus amyloliquefaciens LZLJ2-10 and has been deposited in the China Center for Type Culture Collection (CCTCC) on March 4, 2025, with a deposit number of CCTCC NO: M 2025366.

[0006] The biological characteristics of the Bacillus amyloliquefaciens LZLJ2-10 are as follows: on LB solid culture medium, the colonies are round, have neat edges, are off-white, and are opaque.

[0007] Among them, the flavor metabolism characteristics of the above-mentioned Bacillus amyloliquefaciens LZLJ2-10 are: dimethyl trisulfide can be produced using wheat solid culture medium, with a content of 0.144μg / g; in addition, it can metabolize and produce a variety of other flavor substances, including 8 alcohols, 7 acids, 6 aldehydes, 3 ketones, 3 phenols, 5 nitrogen-containing compounds and 2 esters; specifically, the alcohols include n-hexanol, 1-octen-3-ol, 2-ethylhexanol, n-octanol, 1-nonanol, benzyl alcohol, phenylethyl alcohol, styraxol, and the acids include isobutyric acid, isovaleric acid, 3,3-dimethyl Acrylic acid, 4-methylpentanoic acid, hexanoic acid, (R)-(-)-4-methylhexanoic acid, nonanoic acid; aldehydes such as n-hexanal, (E)-2-heptenal, nonanal, trans-2-octenal, benzaldehyde, phenylacetaldehyde; ketones such as 3-octen-2-one, acetophenone, 3-hydroxy-2-butanone; phenols such as 4-ethylresorcinol, phenol, guaiacol; nitrogen-containing compounds such as 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, indole; esters such as gamma-nonalactone and ethyl hexanoate.

[0008] The 16S rDNA sequence of the Bacillus amyloliquefaciens LZLJ2-10 is shown in SEQ ID NO: 1.

[0009] The 16S rDNA sequence of SEQ ID NO: 1 Bacillus amyloliquefaciens LZLJ2-10 is as follows:

[0010] GCGCCGCTAGCAGTAGAGAGTGATCGTGCGAGCGAGAAGACGGAGAGCTGGCTCC

[0011] CTGATGTTAGCGGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGAT

[0012] AACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAGACATA

[0013] AAAGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAG

[0014] GTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACT

[0015] GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAAT

[0016] GGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAG

[0017] CTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTA

[0018] ACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC

[0019] GTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAA

[0020] AGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGA

[0021] GGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGT

[0022] GGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGA

[0023] ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGG

[0024] TTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCG

[0025] CAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTT

[0026] TAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAG

[0027] ATAGGACGTCCCTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTG

[0028] TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCA

[0029] TTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC

[0030] GTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAA

[0031] AGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGC

[0032] AGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGC

[0033] GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTACAC

[0034] ATACGCAGATAGTCTGCTGGGCGTCACTAAGTCGAACAAGTACCCATAGAGTTAAACCC.

[0035] In a second aspect, the present invention also provides a method for isolating and culturing the above-mentioned Bacillus amyloliquefaciens LZLJ2-10, which comprises the following steps: mixing high-temperature Daqu with water, shaking on a shaker to prepare a bacterial suspension, taking the supernatant of the bacterial suspension and mixing it with water, and performing gradient dilution to prepare 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6dilution; each dilution was spread on LB solid medium for culture; then, a single colony with vigorous growth was picked and streaked multiple times until all single colonies were purified. The purified strain was inoculated into TSB liquid medium for culture, and the volatile substances in the obtained bacterial liquid were detected to screen and obtain Bacillus amyloliquefaciens LZLJ2-10.

[0036] Wherein, in the above separation and culture method, when the high-temperature Daqu and water are mixed, the mass ratio of the high-temperature Daqu to water is 1-2:9-8.

[0037] Wherein, in the above separation and culture method, the shaking conditions are: shaking at room temperature, a rotation speed of 150 to 180 r / min, and shaking for 30 to 60 minutes.

[0038] Wherein, in the above separation and culture method, when the bacterial suspension supernatant and water are mixed, the volume ratio of the bacterial suspension supernatant and water is 1±0.01:9±0.1.

[0039] Wherein, in the above separation and culture method, the culture conditions of LB solid culture medium are: culture at 50°C for 1 to 3 days.

[0040] Among them, in the above-mentioned separation and culture method, the culture conditions of the TSB liquid culture medium are: culture temperature 45-50°C, shaker speed 120-150rpm, and culture for 2-3 days.

[0041] Among them, in the above-mentioned isolation and culture method, the formula of LB solid culture medium is: yeast extract 5.0g, tryptone 10.0g, sodium chloride 10.0g, agar 15.0g, add water to 1000mL, adjust the pH to 7.0±0.2, and sterilize by high-pressure steam at 121°C for 15 minutes.

[0042] Among them, in the above-mentioned isolation and culture method, the formula of TSB liquid culture medium is: 17.0g of trypticase, 5.0g of sodium chloride, 3.0g of soybean papain hydrolyzate, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, add water to 1000mL, adjust the pH to 7.2±0.2, and sterilize by high-pressure steam at 121°C for 15 minutes.

[0043] In a third aspect, the present invention also provides the use of the above-mentioned Bacillus amyloliquefaciens LZLJ2-10 in producing dimethyl trisulfide.

[0044] In a fourth aspect, the present invention also provides a method for producing dimethyl trisulfide using the above-mentioned Bacillus amyloliquefaciens LZLJ2-10, which comprises the following steps: activating the Bacillus amyloliquefaciens LZLJ2-10 with TSB liquid culture medium to obtain a bacterial liquid; inoculating the bacterial liquid into TSB liquid culture medium and culturing to obtain a seed liquid; inoculating the seed liquid into a wheat solid culture medium, and performing a gradient temperature culture simulating a high-temperature Daqu heating program, the culture temperature being 37±1°C-48±1°C-52±1°C-55±1°C-59±1°C-63±1°C-63±1°C, each gradient culture being 24 to 36 hours, to obtain a wheat fermentation sample containing dimethyl trisulfide.

[0045] In the above-mentioned method for producing dimethyl trisulfide, the formula of TSB liquid culture medium is as follows: 17.0 g of trypticase, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolysate, 2.5 g of dipotassium hydrogen phosphate, and 2.5 g of glucose. Water is added to 1000 mL, the pH is adjusted to 7.2±0.2, and high-pressure steam sterilization is performed at 121°C for 15 minutes.

[0046] Among them, in the above-mentioned method for producing dimethyl trisulfide, the specific operation of activation is: inoculating Bacillus amyloliquefaciens LZLJ2-10 into TSB liquid culture medium at an inoculum rate of 1% to 10%, and culturing at 37° C. to 50° C. and 120 to 150 r / min on a shaking table for 1 to 2 days.

[0047] Wherein, in the above method for producing dimethyl trisulfide, the bacterial liquid is inoculated into TSB liquid culture medium at an inoculation rate of 1% to 10%.

[0048] Wherein, in the above-mentioned method for producing dimethyl trisulfide, the conditions for culturing to obtain the seed solution are: culturing at 37°C to 50°C and shaking at 120 to 150 r / min for 1 to 2 days.

[0049] Among them, in the above-mentioned method for producing dimethyl trisulfide, the formula of the wheat solid culture medium is: take the crushed wheat, add water at a material-water ratio of 1g: 0.25-0.3mL and mix evenly, moisten for 1-2h, sterilize at 121°C for 30min, cool, add sterile water at 20%-60% of the mass of the wheat, and stir evenly.

[0050] Wherein, in the above method for producing dimethyl trisulfide, the seed liquid is inoculated into the wheat solid culture medium at an inoculation rate of 2% to 10%.

[0051] Wherein, in the above-mentioned method for producing dimethyl trisulfide, the content of dimethyl trisulfide in the obtained wheat fermentation sample containing dimethyl trisulfide is 0.144 μg / g.

[0052] In a fifth aspect, the present invention also provides the use of the above-mentioned Bacillus amyloliquefaciens LZLJ2-10 in liquor brewing.

[0053] In a sixth aspect, the present invention also provides a method for brewing liquor using the above-mentioned Bacillus amyloliquefaciens LZLJ2-10, which comprises the following steps: adding water to the crushed sorghum to moisten the grain and spreading it to dry, inoculating large qu powder and Bacillus amyloliquefaciens LZLJ2-10 seed liquid, and performing stacking fermentation. The fermentation temperature is 28±1°C-35±1°C-45±1°C, and each gradient culture is 24 to 36 hours. After the stacking fermentation is completed, the mixture is sealed and fermented at 30°C to 35°C for 20 to 30 days to obtain the mash; the ratio of the Bacillus amyloliquefaciens LZLJ2-10 seed liquid to the dry weight of the sorghum is 2 to 5 mL: 100 g.

[0054] Among them, in the above-mentioned method for brewing white wine, the addition amount of Daququ powder is 13% to 16% of the dry weight of sorghum.

[0055] Among them, in the above-mentioned method for brewing liquor, the concentration of Bacillus amyloliquefaciens LZLJ2-10 seed liquid is 10 9 ~10 10 CFU / mL.

[0056] Among them, in the above-mentioned method for brewing white wine, the content of dimethyl trisulfide in the obtained mash is 0.105 μg / g.

[0057] Among them, in the above-mentioned method for brewing white wine, the Bacillus amyloliquefaciens LZLJ2-10 seed liquid is prepared by the following method: Bacillus amyloliquefaciens LZLJ2-10 is inoculated into TSB liquid culture medium at an inoculum rate of 1% to 10%, and cultured on a shaking table at 37°C to 50°C and 120 to 150 r / min for 1 to 2 days to obtain a bacterial liquid; the bacterial liquid is inoculated into TSB liquid culture medium at an inoculum rate of 1% to 10%, and cultured on a shaking table at 37°C to 50°C and 120 to 150 r / min for 1 to 2 days to obtain a seed liquid.

[0058] In the present invention, when using Bacillus amyloliquefaciens LZLJ2-10 to brew liquor, adding water to the crushed sorghum and spreading it to dry are carried out according to conventional operations in the art.

[0059] Beneficial effects of the present invention:

[0060] The Bacillus amyloliquefaciens LZLJ2-10 provided by the present invention is derived from high-temperature Daqu, can utilize wheat to produce dimethyl trisulfide, has stable performance, and is adaptable to high-temperature, high-acid environments and the like during the liquor brewing process. When used in liquor brewing, the dimethyl trisulfide content in the mash increases from an undetectable level to 0.105 μg / g, thereby enriching the liquor brewing microbial resource library, providing a basis for subsequent functional strain development, and promoting the development of the liquor industry.

[0061] The Bacillus amyloliquefaciens of the present invention has a deposit number of CCTCC NO: M 2025366, a deposit date of March 4, 2025, a deposit center of the China Center for Type Culture Collection (CCTCC), an address of Wuhan University, Wuhan, China, a postal code of 430072, and a classification name of Bacillus amyloliquefaciens LZLJ2-10. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a colony morphology diagram of Bacillus amyloliquefaciens LZLJ2-10 of the present invention.

[0063] Figure 2 This is the GC-MS graph of volatile substances in wheat solid-state fermentation samples produced by Bacillus amyloliquefaciens LZLJ2-10.

[0064] Figure 3 This is the GC-MS graph of volatile substances in the mash with the addition of Bacillus amyloliquefaciens LZLJ2-10. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below through examples, but the protection scope of the present invention is not limited to the scope of the examples.

[0066] In the embodiment, the culture medium formula involved is:

[0067] LB solid medium: 5.0 g yeast extract, 10.0 g tryptone, 10.0 g sodium chloride, 15.0 g agar, add water to 1000 mL, adjust the pH to 7.0, and sterilize by high-pressure steam at 121°C for 15 min.

[0068] TSB liquid medium: 17.0 g of trypticase, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolysate, 2.5 g of dipotassium hydrogen phosphate, and 2.5 g of glucose. Add water to 1000 mL, adjust the pH to 7.2, and sterilize by high-pressure steam at 121°C for 15 min.

[0069] Wheat solid culture medium: Take the crushed wheat, add water at a material-water ratio of 1:0.25-0.3 and mix evenly. Moisten for 1 hour and divide into 100mL Erlenmeyer flasks, with about 40g in each bottle. Sterilize at 121℃ for 30min. After cooling, add 16mL of sterile water to each bottle and stir evenly.

[0070] In the examples, the specific method for determining volatile products using headspace solid phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS) is as follows:

[0071] The sample was placed in a headspace vial, and 2 g of NaCl, 5 mL of pure water, and 5 μL of internal standard (0.264 mg / mL 2-octanol) were added. The prepared sample was kept equilibrated at 55°C for 5 minutes, and then extracted with a 50 / 30 μm DVB / CAR / PDMS extraction head at 55°C for 45 minutes. After the extraction, it was desorbed at the GC injection port at 250°C for 5 minutes.

[0072] In the embodiment, GC-MS detection chromatographic conditions:

[0073] Gas chromatography conditions: HP-INNOWAX column (60 m × 0.25 mm × 0.25 μm); temperature program: starting 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); inlet temperature 250°C, no split.

[0074] 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.

[0075] Using 0.264 mg / mL 2-octanol as the internal standard, the compound search results were matched with the NIST standard spectral library, and the target compound was confirmed with a similarity of more than 80%.

[0076] Example 1: Isolation and purification of strains

[0077] Weigh 10g of high-temperature Daqu and add 90mL of sterile water with glass balls (90mL of sterile water, normal saline can be used) into a 250mL conical flask. Oscillate on a shaker at room temperature and 150r / min for 30min to prepare a sample bacterial suspension for later use. Pipette 1mL of the bacterial suspension supernatant and 9mL of sterile water (or normal saline) into a 15mL centrifuge tube to prepare 10 -2 The dilution was diluted in this way to prepare 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6The researchers then plated 100 μL of each dilution onto LB solid medium and incubated in an incubator at 50°C for 1–3 days. Actively growing colonies were then selected and streaked multiple times until all colonies were purified. The purified strains were then inoculated into TSB liquid medium and incubated at 50°C on a shaker at 120 rpm for 2–3 days. After 2–3 days, 0.2 mL of the culture medium was aspirated and analyzed for volatile metabolites using HS-SPME / GC-MS. A strain capable of producing dimethyl trisulfide was identified. The strain was then stored in 30% glycerol at -80°C for further analysis.

[0078] Example 2: Molecular identification of target strains

[0079] After amplifying the target strain, fresh bacterial culture medium from the logarithmic growth phase was collected by centrifugation. Genomic DNA was extracted using the Ezup Column-Based Bacterial Genomic DNA Extraction Kit from Shanghai Sangon Biotechnology Co. The full-length 16S rDNA sequence was amplified using the universal bacterial primers 27F / 1492R as follows.

[0080] 27F (SEQ ID NO:2: 5'-AGAGTTTGATCCTGGCTCAG-3').

[0081] 1492R (SEQ ID NO: 3: 5′-GGTTACCTTGTTACGACTT-3′).

[0082] Table 1 Reaction system

[0083] Reagents Volume (μL) Template (genomic DNA 20-50 ng / μl) 0.5 <![CDATA[10×Buffer(with Mg 2+ )]]> 2.5 dNTPs (2.5 mM each) 1 Taq DNA polymerase 0.2 27F(10uM) 0.5 1492R(10uM) 0.5 <![CDATA[Add double-distilled H2O to]]> 25

[0084] Table 2 Reaction procedure

[0085]

[0086]

[0087] According to the above process, 30 cycles of "denaturation-annealing-extension" PCR reaction were performed, and the PCR products were separated and tested by 1.0% agarose gel electrophoresis at a voltage of about 11 V / cm and an electrophoresis time of 20 min.

[0088] The PCR products were purified according to the instructions of the mini-gel recovery PCR product purification kit of Shanghai Sangon Biotechnology Co., Ltd., and the sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0089] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST, combined with the colony morphology, such as Figure 1As shown, the colony characteristics on LB solid culture medium were round, with neat edges, grayish white, and opaque. The strain species information was determined and it was identified as Bacillus amyloliquefaciens and named Bacillus amyloliquefaciens LZLJ2-10.

[0090] Example 3: Fermentation experiment of wheat solid medium with Bacillus amyloliquefaciens LZLJ2-10

[0091] A 10% inoculum of strain LZLJ2-10 from a glycerol stock tube was inoculated into 1 mL of TSB liquid culture medium and cultured at 37°C, 120 rpm, and shaken for 1 day to activate the strain. A 10% inoculum of the bacterial suspension was transferred to 5 mL of TSB liquid culture medium and cultured at 37°C, 120 rpm, and shaken for 1 day to prepare a seed solution. A 5% inoculum of the seed solution was inoculated into wheat solid culture medium and cultured at a gradient temperature of 37°C, 48°C, 52°C, 55°C, 59°C, 63°C, and 63°C, simulating the high-temperature Daqu process. Each gradient temperature was incubated for 1 day. A blank control group was also established under the same culture conditions. After the experiment, volatile metabolites in the samples were analyzed using HS-SPME / GC-MS. The culture medium without the added bacteria was used as a blank control, and the content of each volatile compound was calculated.

[0092] Through the above method, the experimental results are as follows Figure 2 The analysis results of flavor metabolites are shown in Table 3. The content of dimethyl trisulfide is 0.144 μg / g. In addition, Bacillus amyloliquefaciens LZLJ2-10 can metabolize many other flavor substances, including alcohols such as n-hexanol, 1-octen-3-ol, 2-ethylhexanol, n-octanol, 1-nonanol, benzyl alcohol, phenylethyl alcohol and styraxol, and acids such as isobutyric acid, isovaleric acid, 3,3-dimethylacrylic acid, 4-methylvaleric acid, hexanoic acid, (R)-(-)-4-methylhexane Acid and nonanoic acid, aldehydes such as hexanal, (E)-2-heptenal, nonanal, trans-2-octenal, benzaldehyde and phenylacetaldehyde, ketones such as 3-octen-2-one, acetophenone and 3-hydroxy-2-butanone, phenols such as 4-ethylresorcinol, phenol and guaiacol, nitrogen-containing compounds such as 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2-ethyl-3,5-dimethylpyrazine and indole, esters such as gamma-nonalactone and ethyl hexanoate.

[0093] Table 3 Volatile substance content of solid-state fermentation samples of strain LZLJ2-10 (μg / g)

[0094]

[0095]

[0096]

[0097] Example 4: Application of Bacillus amyloliquefaciens LZLJ2-10 in liquor brewing

[0098] Take crushed sorghum and add 50% (v / m) hot water at 95°C. After moistening the grain for 2 hours, steam it for 30 minutes; after steaming, add 16% water (95°C); spread the sorghum to 28-30°C and inoculate Daqu powder. The Daqu inoculum amount is 16% of the dry weight of the sorghum. The experimental group is added with Bacillus amyloliquefaciens LZLJ2-10 seed liquid. The seed liquid preparation method is the same as that in Example 3. The blank control group is added with TSB liquid culture medium. The liquid volume is 2% (v / m) of the dry weight of the sorghum. After fully mixing, the bag is placed in a sealed bag, and the bag is opened and placed in a constant temperature incubator. The fermentation temperature is 28°C-35°C-45°C. Each gradient culture is 1 day. After the stacking fermentation is completed, the bag is placed in a sealed tank. After fermentation at 30°C for 30 days, the mash is taken and detected by HS-SPME / GC-MS method.

[0099] The experimental results are as follows Figure 3 As shown in Table 4, the content of dimethyl trisulfide in the mash with the addition of Bacillus amyloliquefaciens LZLJ2-10 increased, while no dimethyl trisulfide was detected in the control group. The content in the mash of the experimental group was 0.105 μg / g.

[0100] Table 4 Dimethyl trisulfide content in fermented grains (μg / g)

[0101] Compound Experimental group control group dimethyl trisulfide 0.105 Not detected

[0102] It can be seen that the Bacillus amyloliquefaciens LZLJ2-10 provided by the present invention can adapt to high temperature, high acid and other environments during the liquor brewing process. When used in liquor brewing, the dimethyl trisulfide content in the mash is increased from undetectable to 0.105 μg / g, which helps to achieve targeted regulation of the dimethyl trisulfide content in liquor.

Claims

1. Bacillus amyloliquefaciens, characterized in that: The strain of Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens LZLJ2-10, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025366.

2. The method for isolating and culturing Bacillus amyloliquefaciens according to claim 1, characterized in that: The following steps are involved: The high temperature Daqu was mixed with water, and the suspension was prepared by shaking on a shaker. The supernatant of the suspension was mixed with water, and the mixture was diluted to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 dilution; each dilution was spread on LB solid culture medium for culture; then single colonies with vigorous growth were picked and streaked for multiple times until all single colonies were purified, the purified strains were inoculated into TSB liquid culture medium for culture, and the volatile substances of the obtained bacterial liquid were detected, so that Bacillus amyloliquefaciens LZLJ2-10 could be screened and obtained.

3. The method for separating and culturing Bacillus amyloliquefaciens according to claim 2, wherein: At least one of the following is met: When high-temperature Daqu and water are mixed, the mass ratio of high-temperature Daqu to water is 1-2:9-8; The conditions for shaking on a shaking table are: shaking at room temperature, a speed of 150-180 r / min, and shaking for 30-60 min; When the bacterial suspension supernatant and water are mixed, the volume ratio of the bacterial suspension supernatant to water is 1±0.01:9±0.1; The culture conditions of LB solid medium are: 50°C for 1 to 3 days; The culture conditions of TSB liquid medium are: culture temperature 45-50°C, shaker speed 120-150 rpm, culture for 2-3 days; The formula of LB solid medium is: 5.0 g yeast extract, 10.0 g tryptone, 10.0 g sodium chloride, 15.0 g agar, add water to 1000 mL, adjust the pH to 7.0 ± 0.2, and sterilize by high pressure steam at 121 °C for 15 min; The formula of TSB liquid culture medium is: 17.0 g trypticase, 5.0 g sodium chloride, 3.0 g soybean papain hydrolysate, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, add water to 1000 mL, adjust the pH to 7.2±0.2, and sterilize by high-pressure steam at 121°C for 15 minutes.

4. Use of the Bacillus amyloliquefaciens according to claim 1 in producing dimethyl trisulfide.

5. The method for producing dimethyl trisulfide using the Bacillus amyloliquefaciens according to claim 1, characterized in that: The following steps are involved: After Bacillus amyloliquefaciens LZLJ2-10 was activated in TSB liquid culture medium, a bacterial liquid was obtained; the bacterial liquid was inoculated into TSB liquid culture medium to obtain seed liquid; the seed liquid was inoculated into wheat solid culture medium, and gradient temperature culture was carried out to simulate the high-temperature Daqu heating program, the culture temperature was 37±1℃-48±1℃-52±1℃-55±1℃-59±1℃-63±1℃-63±1℃, each gradient culture was 24 to 36 hours, and a wheat fermentation sample containing dimethyl trisulfide was obtained.

6. The method according to claim 5, characterized in that: At least one of the following is met: The formula of TSB liquid medium is as follows: 17.0 g of trypticase, 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, add water to 1000 mL, adjust the pH to 7.2 ± 0.2, and sterilize by high pressure steam at 121 °C for 15 min; The specific operation of activation is: inoculating Bacillus amyloliquefaciens LZLJ2-10 into TSB liquid culture medium at an inoculum amount of 1% to 10%, and culturing at 37° C. to 50° C. and 120 to 150 r / min on a shaking table for 1 to 2 days; Inoculate the bacterial solution into TSB liquid culture medium at an inoculum rate of 1% to 10%; The conditions for culturing the seed solution are: 37°C to 50°C, 120 to 150 r / min shaking culture for 1 to 2 days; The formula of wheat solid culture medium is as follows: take the crushed wheat, add water according to the material-water ratio of 1g: 0.25-0.3mL and mix evenly, moisten for 1-2h, sterilize at 121℃ for 30min, add sterile water of 20%-60% of the wheat mass after cooling, and stir evenly; Inoculate the seed liquid into the wheat solid culture medium at an inoculum rate of 2% to 10%; The content of dimethyl trisulfide in the obtained wheat fermentation sample containing dimethyl trisulfide was 0.144 μg / g.

7. Use of the Bacillus amyloliquefaciens according to claim 1 in liquor brewing.

8. A method for brewing liquor using the Bacillus amyloliquefaciens according to claim 1, characterized in that: The following steps are involved: After the crushed sorghum is moistened with water and spread out to dry, it is inoculated with Daqu powder and Bacillus amyloliquefaciens LZLJ2-10 seed liquid for stacking fermentation. The fermentation temperature is 28±1℃-35±1℃-45±1℃, and each gradient culture is 24-36h. After the stacking fermentation is completed, it is sealed and fermented at 30℃-35℃ for 20-30d to obtain the mash. The ratio of the Bacillus amyloliquefaciens LZLJ2-10 seed liquid to the dry weight of sorghum is 2-5mL:100g.

9. The method according to claim 8, characterized in that: At least one of the following is met: The amount of Daququ powder added is 13% to 16% of the dry weight of sorghum; The concentration of Bacillus amyloliquefaciens LZLJ2-10 seed solution was 10 9 ~10 10 CFU / mL; The content of dimethyl trisulfide in the obtained mash is 0.105 μg / g.

10. The method according to claim 8, characterized in that: The Bacillus amyloliquefaciens LZLJ2-10 seed liquid is prepared by the following method: inoculating the Bacillus amyloliquefaciens LZLJ2-10 into a TSB liquid culture medium at an inoculation rate of 1% to 10%, and culturing the culture on a shaking platform at 37° C. to 50° C. and 120 to 150 r / min for 1 to 2 days to obtain a bacterial liquid; inoculating the bacterial liquid into a TSB liquid culture medium at an inoculation rate of 1% to 10%, and culturing the culture on a shaking platform at 37° C. to 50° C. and 120 to 150 r / min for 1 to 2 days to obtain a seed liquid.

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