Bacillus sorola for high-yield production of 4-ethyl guaiacol as well as isolated culture method and application of bacillus sorola for high-yield production of 4-ethyl guaiacol

Abstract

The invention discloses bacillus sorola for high-yield production of 4-ethyl guaiacol as well as an isolated culture method and application of the bacillus sorola, and belongs to the technical field of microorganisms. According to the invention, a strain of Bacillus SooraDesert LZLJ2-9 is separated and screened from high-temperature yeast for making hard liquor, the preservation number is CCTCC NO: M 2025365, the Bacillus SooraDesert LZLJ2-9 has the advantages of easy culture, high temperature resistance, adaptability to a white liquor brewing environment and the like, 4-ethyl guaiacol can be produced at high yield by using a wheat solid culture medium, the fermentation temperature is gradually increased from 37 DEG C to 63 DEG C, the Bacillus SooraDesert LZLJ2-9 can adapt to the temperature change of a high-temperature yeast making process, and the yield of 4-ethyl guaiacol is increased. In the Baijiu brewing process, the content of the 4-ethyl guaiacol can be increased to 3.956 micrograms / g and is far higher than the existing level, a high-quality strain resource is provided for biosynthesis of the 4-ethyl guaiacol in the Baijiu, and the method has important significance on Baijiu brewing through a high-temperature process.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Bacillus sonorensis with high yield of 4-ethylguaiacol, and its isolation, culture method and application. Background Art

[0002] 4-ethylguaiacol is usually a colorless to light yellow liquid with smoky, woody and spicy odors, and is commonly used in foods and flavors. It is one of the important flavor compounds in Chinese liquor, especially in Maotai-flavor Chinese liquor (such as Moutai) and some Luzhou-flavor Chinese liquors, and it has a significant impact on the flavor, aroma and quality of the liquor body.

[0003] Bacillus sonorensis has characteristics such as high temperature resistance and enzyme production, and has potential application value in Chinese liquor brewing. According to the research on high-temperature Daqu in Maotai Town by Wang Yurong et al. (Wang Yurong, Hou Qiangchuan, Tian Longxin, et al. Analysis of the differences in microbial diversity of high-temperature Daqu from two enterprises in Maotai Town based on pure culture and ultra-high-depth metagenomic sequencing technology [J]. Food Science: 0-0 [2025-03-03]), among the 212 strains of Bacillus isolated, Bacillus sonorensis had the highest proportion (77 strains, about 36.3%), which was significantly higher than Bacillus licheniformis and Bacillus amyloliquefaciens, indicating its importance in Chinese liquor production.

[0004] Currently, there are few studies on the flavor products of Bacillus sonorensis. CN116694514A discloses a method for improving the high temperature resistance and flavor production ability of Bacillus sonorensis. It co-cultures Bacillus sonorensis CCTCC M2023288 with Bacillus lithotrophicus LBM32026. The highest content of 4-ethylguaiacol in pure culture of Bacillus sonorensis is 0.246 μg / kg, and the highest content of 4-ethylguaiacol in co-culture is 5.47 μg / kg. The content of 4-ethylguaiacol in both pure culture and co-culture is relatively low. Summary of the Invention

[0005] Aiming at the existing research gap, the present invention screens a strain of Bacillus sonorensis from high-temperature Daqu of Chinese liquor, which is easy to culture, high temperature resistant, adaptable to the Chinese liquor brewing environment and can produce high yield of 4-ethylguaiacol, and is of great significance for the research and innovation of high-temperature process Chinese liquor.

[0006] In the first aspect, the present invention provides a strain of Bacillus sonorensis named Bacillus sonorensis LZLJ2-9, which was deposited at the China Center for Type Culture Collection (CCTCC) on March 4, 2025, and its deposit number is CCTCC NO: M 2025365.

[0007] Among them, the biological characteristics of the above-mentioned Bacillus sonorensis LZLJ2-9 are as follows: it grows on ISP2 solid medium, and the colony characteristics are round, with neat edges, slightly yellow, and opaque.

[0008] Among them, the flavor metabolic characteristics of the above-mentioned Bacillus sonorensis LZLJ2-9 are as follows: it can produce a high yield of 4-ethylguaiacol on wheat solid medium, with a content of 4.120 μg / g; in addition, it can metabolize and produce various other flavor substances, including 3 alcohols, 1 ester, 5 acids, 3 aldehydes, 3 ketones, 6 phenols, 7 pyrazines, 2 anilines, and 1 benzoxazole compound; specifically, they include benzyl alcohol, phenethyl alcohol, and farnesol among the alcohols; ethyl hexanoate among the esters; isobutyric acid, isovaleric acid, 2-methyl-4-pentenoic acid, 4-methylpentanoic acid, and phenylacetic acid among the acids; benzaldehyde, vanillin, and α-ethylidene-benzeneacetaldehyde among the aldehydes; 3-hydroxy-2-butanone, acetophenone, and geranyl acetone among the ketones; phenol, 4-vinylguaiacol, 2,6-dimethyl-1,4-benzenediol, 4-ethylresorcinol, 2,4-di-tert-butylphenol, and guaiacol among the phenols; 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, and 2,6-diethylpyrazine among the pyrazines; 2-methyl-4-methoxyaniline and 4-methylacetanilide among the anilines; and 2,5,6-trimethylbenzoxazole among the benzoxazoles.

[0009] Among them, the 16S rDNA sequence of the above-mentioned Bacillus sonorensis LZLJ2-9 is as shown in SEQ ID NO:1.

[0010] The 16S rDNA sequence of Bacillus sonorensis LZLJ2-9 of SEQ ID NO:1 is as follows:

[0011] GGGTATGGCTACCAGTATAGATTGATCGTGCGAGCGAGCCGTCGGAACGCTGGATC

[0012] CCTTAGGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGA

[0013] TAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATA

[0014] AAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGA

[0015] GGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACAC

[0016] TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAA

[0017] TGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAA

[0018] ACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAACAGGGCGGTGCCTTGACGGTACCT

[0019] AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAG

[0020] CGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGA

[0021] AAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAG

[0022] AGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAG

[0023] TGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCG

[0024] AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGG

[0025] GTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTC

[0026] GCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT

[0027] TTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACACCCCTAGA

[0028] GATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGT

[0029] GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGC

[0030] ATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGA

[0031] CGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACA

[0032] AAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGCTCTCAGTTCGGATC

[0033] GCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCC

[0034] GCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAA

[0035] CACCACGTATAGTTCGGTGGAGTGTAACTCTTTGGAGCCAGCGTACCGCATAGAGTAAA

[0036] CC。

[0037] In a second aspect, the present invention also provides a method for isolating and culturing the above-mentioned Bacillus sonorensis LZLJ2-9, which comprises the following steps:

[0038] 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 respectively by gradient dilution; Spread the dilutions of each dilution factor on ISP2 solid medium for cultivation; Then pick the vigorously growing single colonies and perform multiple streakings until all single colonies are purified. The purified strains are inoculated into TSB liquid medium for cultivation to obtain several strains of bacteria, and then through identification, several strains belonging to Bacillus sonorensis are obtained;

[0039] B. After activating the strains belonging to Bacillus sonorensis obtained in step A respectively through TSB liquid medium, obtain bacterial solutions; Inoculate the bacterial solutions into the fermentation medium and cultivate to obtain fermentation broth; Detect the volatile substances in the fermentation broth and screen to obtain Bacillus sonorensis LZLJ2-9 with high yield of 4-ethylguaiacol.

[0040] Among them, in the above separation and cultivation 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.

[0041] Among them, in the above separation and cultivation method, in step A, the conditions of shaking on the shaker are: at room temperature of the shaker, shaking at a speed of 150-180 r / min for 30-60 min.

[0042] Among them, in the above separation and cultivation 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.

[0043] Among them, in the above separation and cultivation method, in step A, the cultivation conditions of ISP2 solid medium are: cultivate at 55°C for 1-6 d.

[0044] Among them, in the above separation and cultivation method, in step A, the cultivation conditions of TSB liquid medium are: cultivation temperature 50-55°C, shaker speed 120-150 rpm, cultivate for 1-2 d.

[0045] Among them, in the above separation and cultivation method, in step A, 38 strains of bacteria are obtained through cultivation in TSB liquid medium, and then through identification, 10 strains belonging to Bacillus sonorensis are obtained.

[0046] Among them, in the above separation and cultivation method, in step B, the specific operation of activation is: inoculate the strains belonging to Bacillus sonorensis obtained in step A into TSB liquid medium at an inoculation amount of 1%-10%, and cultivate on a shaker at 37-55°C and 120-150 r / min for 1-2 d.

[0047] Among them, in the above separation and culture method, in step B, the bacterial liquid is inoculated into the fermentation medium at an inoculation amount of 5% - 10%.

[0048] Among them, in the above separation and culture method, in step B, the conditions for obtaining the fermentation broth are: shaking culture at 50 - 55°C and 120 - 150 r / min for 1 - 3 days.

[0049] Among them, in the above separation and culture method, the formula of the ISP2 solid medium is: yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, agar 15.0 g, add water to 1000 mL, adjust the pH to 7.0 ± 0.2, and sterilize by high-pressure steam at 121°C for 15 minutes.

[0050] Among them, in the above separation and culture method, the formula of the 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 sterilize by high-pressure steam at 121°C for 15 minutes.

[0051] Among them, in the above separation and culture method, the formula of the fermentation 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, ferulic acid 2 mg, add water to 1000 mL, adjust the pH to 7.0 ± 0.2, and sterilize by high-pressure steam at 121°C for 15 minutes.

[0052] In the third aspect, the present invention also provides the application of the above Bacillus sonorensis LZLJ2 - 9 in the production of 4 - ethylguaiacol.

[0053] In the fourth aspect, the present invention also provides a method for producing 4 - ethylguaiacol using the above Bacillus sonorensis LZLJ2 - 9, which includes the following steps: activating Bacillus sonorensis LZLJ2 - 9 through the TSB liquid medium to obtain a bacterial liquid; inoculating the bacterial liquid into the TSB liquid medium to obtain a seed liquid; inoculating the seed liquid into the wheat solid medium, simulating the temperature rising program 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 4 - ethylguaiacol.

[0054] Among them, in the above method for producing 4 - ethylguaiacol, 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 at 37°C - 55°C and 120 - 150 r / min on a shaker for 1 - 2 days.

[0055] Among them, in the above method for producing 4-ethylguaiacol, the bacterial liquid is inoculated into the TSB liquid medium at an inoculation amount of 1% - 10%.

[0056] Among them, in the above method for producing 4-ethylguaiacol, the conditions for culturing the seed liquid are: shaking culture at 37°C - 55°C, 120 - 150 r / min for 1 - 2 d.

[0057] Among them, in the above method for producing 4-ethylguaiacol, the seed liquid is inoculated into the wheat solid medium at an inoculation amount of 2% - 10%.

[0058] Among them, in the above method for producing 4-ethylguaiacol, the formula of the TSB liquid medium is: 17.0 g of tryptone, 5.0 g of sodium chloride, 3.0 g of soy peptone, 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 at 121°C under high-pressure steam for 15 minutes.

[0059] Among them, in the above method for producing 4-ethylguaiacol, the formula of the 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.

[0060] Among them, in the above method for producing 4-ethylguaiacol, the content of 4-ethylguaiacol in the obtained wheat fermentation sample containing 4-ethylguaiacol is 4.120 μg / g.

[0061] In the fifth aspect, the present invention also provides the application of the above-mentioned Bacillus sonorensis LZLJ2-9 in liquor brewing.

[0062] In the sixth aspect, the present invention also provides a method for liquor brewing using the above-mentioned Bacillus sonorensis LZLJ2-9, which includes the following steps: After adding water to moisten and spreading out the crushed sorghum, inoculate the Daqu starter powder and the Bacillus sonorensis LZLJ2-9 seed liquid, and carry out stacking fermentation. The fermentation temperature is 28 ± 1°C - 35 ± 1°C - 45 ± 1°C, and each gradient is cultured for 24 - 36 h. After the stacking fermentation is completed, seal and ferment at 30 - 35°C for 20 - 30 d to obtain the fermented grains; the ratio of the Bacillus sonorensis LZLJ2-9 seed liquid to the dry weight of sorghum is 2 - 5 mL: 100 g.

[0063] Among them, in the above method for liquor brewing, the addition amount of the Daqu starter powder is 13% - 16% of the dry weight of sorghum.

[0064] Among them, in the above method for brewing Chinese liquor, the concentration of the Bacillus sonorensis LZLJ2-9 seed liquid is 10 9 ~10 10 CFU / mL.

[0065] Among them, in the above method for brewing Chinese liquor, the content of 4-ethylguaiacol in the resulting fermented grains is 3.956 μg / g.

[0066] Among them, in the above method for brewing Chinese liquor, the Bacillus sonorensis LZLJ2-9 seed liquid is prepared by the following method: inoculating Bacillus sonorensis LZLJ2-9 into a TSB liquid medium at an inoculation amount of 1% to 10%, culturing on a shaker at 37°C to 55°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 medium at an inoculation amount of 1% to 10%, culturing on a shaker at 37°C to 55°C and 120 to 150 r / min for 1 to 2 days to obtain a seed liquid.

[0067] In the present invention, when using Bacillus sonorensis LZLJ2-9 for brewing Chinese liquor, the crushing of sorghum, adding water to moisten the grains and spreading them out for cooling adopt conventional operations in the art.

[0068] Advantages of the present invention:

[0069] In the present invention, a Bacillus sonorensis capable of highly producing 4-ethylguaiacol using a wheat solid medium is obtained from a high-temperature Chinese liquor starter. The fermentation temperature gradually increases from 37°C to 63°C, which can adapt to the temperature change of the high-temperature starter-making process, and the content of 4-ethylguaiacol is much higher than that of Bacillus sonorensis reported in the existing literature, providing high-quality strain resources for the biosynthesis of 4-ethylguaiacol in the Chinese liquor industry.

[0070] The preservation number of the Bacillus sonorensis of the present invention is CCTCC NO: M 2025365, 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 sonorensis LZLJ2-9. Description of the Drawings

[0071] Figure 1 It is a colony morphology diagram of Bacillus sonorensis LZLJ2-9 of the present invention.

[0072] Figure 2 It is a GC-MS diagram of volatile substances of the wheat solid fermentation sample of Bacillus sonorensis LZLJ2-9.

[0073] Figure 3It is the GC-MS diagram of the volatile substances in the fermented grains added with Bacillus sonorensis LZLJ2-9. Detailed implementation mode

[0074] 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.

[0075] In the examples, the medium formulations involved are as follows:

[0076] ISP2 solid medium: yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, agar 15.0 g, add water to 1000 mL, adjust the pH to 7.0, and autoclave at 121 °C for 15 minutes.

[0077] TSB liquid medium: 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, and autoclave at 121 °C for 15 minutes.

[0078] Fermentation medium: 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, ferulic acid 2 mg, add water to 1000 mL, adjust the pH to 7.0, and autoclave at 121 °C for 15 minutes.

[0079] Wheat solid medium: Take the crushed wheat, mix it evenly with water according to the ratio of material to water of 1:0.25 - 0.3, moisten it for 1 h, divide it into 100 mL Erlenmeyer flasks, each flask contains about 40 g of the material, sterilize at 121 °C for 30 min, and after cooling, add 16 mL of sterile water to each flask and stir evenly.

[0080] 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:

[0081] 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 (0.264 mg / mL 2-octanol). 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 the extraction is completed, desorb it at 250 °C in the GC injection port for 5 min.

[0082] In the examples, the GC-MS detection chromatographic conditions are as follows:

[0083] Gas chromatography conditions: HP-INNOWAX chromatographic column (60m×0.25mm×0.25μm); 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 injection.

[0084] 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.

[0085] Using 0.264 mg / mL 2-octanol as the internal standard, the compound retrieval results were matched with the NIST standard spectral library, and compounds with a similarity of over 80% were confirmed as the target compounds.

[0086] Example 1: Screening of Bacillus sonorensis

[0087] (1) Isolation and purification of strains

[0088] Weigh 10 g of high-temperature Daqu, add it to a 250 mL conical flask containing sterile water with glass beads (90 mL of sterile water, physiological saline can be used), shake at room temperature on a shaker at 150 r / min for 30 min to prepare a sample bacterial suspension for later use. 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 -2 dilution, 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 an ISP2 solid medium, incubate in an incubator at 55°C for 1 - 6 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 a TSB liquid medium, cultured at a temperature of 55°C and a shaker speed of 120 rpm. After 1 - 2 days of culture, pipette the bacterial liquid and store it at -80°C with 30% glycerol. A total of 38 strains were screened out.

[0089] (2) Identification of strains

[0090] After expanding the culture of the 38 strains, take the fresh bacterial liquid in the logarithmic growth phase, centrifuge to collect the bacterial cells, and extract the genomic DNA using the Ezup column bacterial genomic DNA extraction kit from Sangon Biotech (Shanghai) Co., Ltd. Amplify the full-length 16S rDNA sequence using the bacterial universal primers 27F / 1492R as follows.

[0091] 27F (SEQ ID NO: 2: 5'-AGAGTTTGATCCTGGCTCAG-3').

[0092] 1492R (SEQ ID NO: 3: 5′-GGTTACCTTGTTACGACTT-3′).

[0093] Table 1 Reaction system

[0094] Reagent Volume (μL) Template (Genomic DNA 20 - 50 ng / μl) 0.5 <![CDATA[10×Buffer(with Mg 2+ )]]> 2.5 dNTP (each 2.5 mM) 1 Taq DNA Polymerase 0.2 27F (10 uM) 0.5 1492R (10 uM) 0.5 <![CDATA[Add double-distilled H2O to]]> 25

[0095] Table 2 Reaction procedure

[0096]

[0097] According to the above process, 30 cycles of "denaturation-annealing-extension" PCR reaction were carried out, and the PCR products were separated and tested by 1.0% agarose gel electrophoresis with a voltage of about 11 V / cm and an electrophoresis time of 20 min.

[0098] The purification of PCR products was carried out 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.

[0099] The gene sequences of the 16S rDNA fragments were sequenced and compared with NCBI's BLAST to determine the species information of the strains. Ten of the strains were identified as Bacillus sonorensis. Among them, the colony morphology of strain No. 2 was as follows Figure 1 As shown, the colonies grown on ISP2 solid medium are round, have neat edges, are slightly yellowish, and are opaque.

[0100] Example 2: Liquid fermentation experiment

[0101] Glycerol preservation tubes of 10 Sonora Desert Bacillus strains were inoculated with 10% inoculum into 1 mL TSB liquid culture medium and cultured in a shaking incubator at 37°C and 120 r / min for 1 day to activate the strains; the bacterial solution was transferred to 10 mL fermentation culture medium at 10% inoculum and cultured in a shaking incubator at 55°C and 120 r / min for 3 days; the culture medium without bacteria was used as the blank control group, and 0.2 mL of the bacterial solution was aspirated after the culture was completed, and the volatile metabolites in the sample were detected by HS-SPME / GC-MS method.

[0102] The experimental results are shown in Table 3. Among the 10 Sonora Desert Bacillus strains, 3 strains were able to produce 4-ethylguaiacol. Strains 2, 4, and 5 were selected for further analysis.

[0103] Table 3 4-Ethylguaiacol content in liquid fermentation samples of 10 strains (μg / mL)

[0104] Compound 1 2 3 4 5 4 - Ethylguaiacol / 0.382 / 0.145 0.207 Compound 6 7 8 9 10 4 - Ethylguaiacol / / / / /

[0105] Example 3: Wheat solid medium fermentation experiment

[0106] Take the glycerol preservation tubes of 3 strains numbered 2, 4, and 5, inoculate them into 1 mL of TSB liquid medium at an inoculation amount of 10%, and culture 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 culture 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 culture. The culture temperatures are 37 °C - 48 °C - 52 °C - 55 °C - 59 °C - 63 °C - 63 °C, and each gradient is cultured for 1 day, and a blank control group experiment is set under the same culture conditions. After the experiment, HS-SPME / GC-MS method is used to detect the volatile substances in the samples. The culture solution without added bacteria is used as the blank control group, and the content is calculated.

[0107] The experimental results are shown in Table 4. The highest content of 4-ethylguaiacol produced by the 3 strains using the wheat solid medium is strain No. 2, with a content of 4.120 μg / g. This strain is named Bacillus sonorensis LZLJ2-9 and was deposited in the China Center for Type Culture Collection (CCTCC) on March 4, 2025, with the deposit number: CCTCC NO: M2025365.

[0108] Table 4 Content of 4-ethylguaiacol in the solid fermentation samples of 3 strains (μg / g)

[0109] Volatile Substance 2 4 5 4 - Ethylguaiacol 4.120 0.887 1.174

[0110] The GC-MS diagram of the volatile substances detected in the wheat solid fermentation of Bacillus sonorensis LZLJ2-9 is as Figure 2As shown, the analysis results of its flavor metabolites are shown in Table 5. In addition to fermenting to produce high yields of 4-ethylguaiacol, this strain can metabolize a variety of other flavor substances, including benzyl alcohol, phenethyl alcohol, and farnesol among alcohols; ethyl hexanoate among esters; isobutyric acid, isovaleric acid, 2-methyl-4-pentenoic acid, 4-methylpentanoic acid, and phenylacetic acid among acids; benzaldehyde, vanillin, and α-ethylidene-benzeneacetaldehyde among aldehydes; 3-hydroxy-2-butanone, acetophenone, and geranyl acetone among ketones; phenol, 4-vinylguaiacol, 2,6-dimethyl-1,4-benzenediol, 4-ethylresorcinol, 2,4-di-tert-butylphenol, and guaiacol among phenols; 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, and 2,6-diethylpyrazine among pyrazines; 2-methyl-4-methoxyaniline and 4-methylacetanilide among anilines; and 2,5,6-trimethylbenzoxazole among benzoxazoles.

[0111] Table 5 Content of volatile substances in the solid-state fermentation sample of Bacillus sonorensis LZLJ2-9 (μg / g)

[0112]

[0113]

[0114] Example 4: Application of Bacillus sonorensis LZLJ2-9 in Baijiu brewing

[0115] Take crushed sorghum and add 50% (v / m) of hot water at 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 LZLJ2-9. The preparation method of the seed liquid is the same as in Example 3. In the blank control group, add TSB liquid medium, and the added liquid volume is 2% (v / m) of the dry weight of sorghum. After mixing well, put it into a sealed bag and place it in an incubator with an open mouth. The fermentation temperature is 28°C - 35°C - 45°C, and each gradient is cultured for 1 d. After stacking fermentation, put it 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.

[0116] The experimental results are as Figure 3 shown. Among them, the detected content of 4-ethylguaiacol is shown in Table 6. The content of 4-ethylguaiacol in the fermented grains added with Bacillus sonorensis LZLJ2-9 increased significantly, from the original 0.620 μg / g to 3.956 μg / g.

[0117] Table 6 Content of 4-ethylguaiacol in fermented grains (μg / g)

[0118] Compound Experimental Group Control Group 4 - Ethylguaiacol 3.956 0.620

[0119] It can be seen that Bacillus sonorensis LZLJ2-9 provided by the present invention can adapt to environments such as high temperature and high acid during the brewing process of Chinese liquor. When applied to the brewing of Chinese liquor, the content of 4-ethylguaiacol in fermented grains increases from 0.620 μg / g to 3.956 μg / g, significantly improving the content of 4-ethylguaiacol and providing high-quality strain resources for the biosynthesis of 4-ethylguaiacol.

Claims

1. Sonora Desert Bacillus, characterized in that: The strain of Sonora Desert Bacillus is named Sonora Desert Bacillus (Bacillus sonorensis) LZLJ2-9, which is deposited in China Center for Type Culture Collection, and its deposit number is CCTCC NO: M 2025365.

2. The method for isolating and culturing Sonora Desert Bacillus according to claim 1, characterized in that: The following steps are involved: A. Mix high temperature Daqu and water, shake on a shaker to make a bacterial suspension, take the bacterial suspension supernatant and mix with water, and dilute by gradient to make 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 dilutions of each dilution degree; spreading the dilutions of each dilution degree on ISP2 solid culture medium for culture; then picking up single colonies with vigorous growth, streaking for multiple times until all single colonies are purified, inoculating the purified strains in TSB liquid culture medium for culture, obtaining several strains, and then identifying and obtaining several strains of Sonora Desert Bacillus; B. The strains of Sonora Desert Bacillus obtained in step A are activated in TSB liquid culture medium to obtain bacterial liquid; the bacterial liquid is inoculated into a fermentation medium and cultured to obtain a fermentation liquid; the volatile substances in the fermentation liquid are detected, and the Sonora Desert Bacillus LZLJ2-9 with high yield of 4-ethylguaiacol is screened.

3. The method for separating and culturing Bacillus amyloliquefaciens according to claim 2, wherein: At least one of the following is met: In step A, when the high-temperature Daqu and water are mixed, the mass ratio of the high-temperature Daqu to water is 1-2:9-8; In step A, the shaking conditions are as follows: shaking at room temperature and a rotation speed of 150 to 180 r / min for 30 to 60 min; In step A, 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; In step A, the culture conditions of ISP2 solid medium are: 55°C for 1 to 6 days; In step A, the culture conditions of TSB liquid culture medium are: culture temperature 50-55° C., shaker speed 120-150 rpm, culture 1-2 days; In step A, 38 strains were obtained by culturing in TSB liquid medium, and 10 strains belonging to Sonora Desert Bacillus were obtained through identification; In step B, the specific operation of activation is: inoculating the strain of Sonora Desert Bacillus obtained in step A into TSB liquid culture medium at an inoculation rate of 1% to 10%, and culturing at 37 to 55° C. and 120 to 150 r / min on a shaking table for 1 to 2 days; In step B, the bacterial solution is inoculated into the fermentation medium at an inoculum rate of 5% to 10%; In step B, the conditions for culturing the fermentation liquid are: 50-55° C., 120-150 r / min shaking culture for 1-3 days; The formula of ISP2 solid medium is: 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 15.0 g agar, add water to 1000 mL, adjust pH to 7.0 ± 0.2, and sterilize by high pressure steam at 121 °C for 15 min; 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 formula of the fermentation medium is: 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, 2 mg of ferulic acid, add water to 1000 mL, adjust the pH to 7.0±0.2, and sterilize by high-pressure steam at 121°C for 15 minutes.

4. Use of the Sonora Desert Bacillus according to claim 1 in producing 4-ethylguaiacol.

5. A method for producing 4-ethylguaiacol using the Sonora Desert Bacillus according to claim 1, characterized in that: The following steps are involved: The Sonora Desert Bacillus LZLJ2-9 was activated with TSB liquid culture medium to obtain a bacterial liquid; the bacterial liquid was inoculated into TSB liquid culture medium to obtain a seed liquid; the seed liquid was inoculated into a wheat solid culture medium, and a gradient temperature culture was carried out to simulate a high-temperature Daqu heating program, with the culture temperature being 37±1℃-48±1℃-52±1℃-55±1℃-59±1℃-63±1℃-63±1℃, and each gradient culture was carried out for 24 to 36 hours to obtain a wheat fermentation sample containing 4-ethylguaiacol.

6. The method according to claim 5, characterized in that: At least one of the following is met: The specific operation of activation is: inoculating Bacillus amyloliquefaciens LZLJ2-11 into TSB liquid culture medium at an inoculum amount of 1% to 10%, and culturing at 37° C. to 55° 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 55°C, 120 to 150 r / min shaking culture for 1 to 2 days; Inoculate the seed liquid into the wheat solid culture medium at an inoculum rate of 2% to 10%; 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 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; The content of 4-ethylguaiacol in the obtained wheat fermentation sample containing 4-ethylguaiacol was 4.120 μg / g.

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

8. A method for brewing liquor using the Sonora Desert Bacillus according to claim 1, characterized in that: The following steps are involved: After the crushed sorghum is moistened with water and spread out to air, it is inoculated with Daqu powder and Sonora Desert Bacillus LZLJ2-9 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 Sonora Desert Bacillus LZLJ2-9 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 the Sonora Desert Bacillus LZLJ2-9 seed solution was 10 9 ~10 10 CFU / mL; The content of 4-ethylguaiacol in the obtained mash is 3.956 μg / g.

10. The method according to claim 8, characterized in that: The Sonora Desert Bacillus LZLJ2-9 seed liquid is prepared by the following method: inoculating the Sonora Desert Bacillus LZLJ2-9 into a TSB liquid culture medium at an inoculation rate of 1% to 10%, and culturing the culture on a shaking table at 37°C to 55°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 table at 37°C to 55°C and 120 to 150 r / min for 1 to 2 days to obtain a seed liquid.

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