Screening, mutagenesis and application of high-yield tetramethylpyrazine strain

Abstract

The invention provides screening, mutagenesis and application of a high-yield tetramethylpyrazine strain. The invention relates to a high-yield tetramethylpyrazine strain, which is named as salt-tolerant bacillus (Bacillus halotolans) YB13, the strain is preserved in the China General Microbiological Culture Collection Center on January 12, 2024, and the preservation number is CGMCC No.28161. The tetramethylpyrazine strain has the advantages that the tetramethylpyrazine yield is high, and the tetramethylpyrazine yield is high. According to screening, mutagenesis and application of the high-yield tetramethylpyrazine strain, the high-yield tetramethylpyrazine strain is screened in a liquid culture medium and a solid culture medium through fermentation respectively, the amount of tetramethylpyrazine produced by the salt-tolerant bacillus YB13 in the solid culture medium is remarkably higher than that of other strains, and the salt-tolerant bacillus YB13 is the high-yield tetramethylpyrazine strain. When the strain is applied to white spirit brewing, detection shows that the quality of raw wine added with the salt-tolerant bacillus is higher than that of a control group, and the content of TTMP in the brewing process is also remarkably increased.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, in particular to the screening, mutagenesis and application of a high-yield tetramethylpyrazine strain. Background Art

[0002] Tetramethylpyrazine (TTMP) is an important heterocyclic flavor compound widely found in fermented foods and beverages, such as baijiu (white wine), soy sauce, vinegar, and bread. Its unique nutty and toasty aroma makes it an indispensable flavor component in many foods. In recent years, as consumers' expectations for food flavor and quality have increased, research on TTMP has become a hot topic in fields such as food science, microbiology, and bioengineering. TTMP not only contributes significantly to flavor but has also been found to possess diverse biological activities, such as antioxidant, anti-inflammatory, and improved blood circulation, thus showing promising applications in medicine and cosmetics. Its synthesis primarily relies on microbial metabolic activity, with its synthetic pathway involving multiple biochemical reactions, including amino acid and sugar metabolism. The TTMP content is closely related to the aroma and taste of wine. Screening for high-TTMP-producing strains can effectively increase the TTMP content, thereby improving food flavor quality.

[0003] The yield of TTMP produced by traditional microbial fermentation is relatively low, but plasma mutagenesis technology has become an important means to improve the performance of TTMP production strains due to its advantages such as high efficiency, safety and ease of operation. Plasma mutagenesis uses active particles (free radicals, ultraviolet rays, charged particles, etc.) generated by low-temperature plasma (such as helium, argon or air plasma) to act on microbial cells, inducing DNA damage and mutations, thereby obtaining high-yield mutant strains. Targeted enhanced fermentation using high-yield TTMP strains has become a new strategy for improving the flavor quality of liquor. The application of high-yield TTMP strains in liquor brewing not only directly contributes to the aroma, but also synergizes with other flavor substances in the liquor (such as esters and phenols) to form a more complex flavor profile. In sensory evaluation, liquor brewed using high-yield strains has significantly improved "mellowness" and "lasting aftertaste" without affecting the balance of the main flavor.

[0004] Baijiu, a traditional Chinese solid-state fermented distilled liquor, develops its unique flavor through a close relationship with its microbial community. In recent years, the potential for enhanced application of microbial strains that produce high amounts of TTMP in baijiu brewing has been gaining increasing attention. Microbial tolerance to high-salt, high-ethanol, and high-fat brewing environments promotes the production of pyrazines such as tetramethylpyrazine (TTMP), imparting a toasted aroma to the liquor.

[0005] Therefore, the present invention provides the screening, mutagenesis and application of salt-tolerant strains with high tetramethylpyrazine production to solve the above technical problems. Summary of the Invention

[0006] The present invention provides screening, mutagenesis and application of a high-yield tetramethylpyrazine strain, solving the problems in the background technology.

[0007] In order to solve the above technical problems, the present invention provides a high-yield tetramethylpyrazine strain, which is named Bacillus halotolerans YB13. The strain is deposited in the China General Microbiological Culture Collection Center with a deposit date of January 12, 2024 and a deposit number of CGMCC No. 28161.

[0008] A solid-state fermentation medium for white wine lees of halotolerant Bacillus YB13 comprises the following components: bran and soybean meal powder.

[0009] Preferably, the bran and soybean meal powder are evenly mixed in a ratio of 8:2, and then an appropriate amount of water is added to adjust the humidity to 30% for culturing, and each 50 g is placed in a 250 mL conical flask.

[0010] A strain of halotolerant Bacillus subtilis YB13 screened from solid-state fermentation medium of white wine lees was optimized by plasma mutagenesis.

[0011] A method for screening a high-yield tetramethylpyrazine strain specifically comprises the following steps:

[0012] (1) The high-temperature koji in the koji room was randomly collected at 5 sampling points, crushed and sieved for use;

[0013] (2) Place the crushed koji powder in LB liquid medium and culture it at 37°C for enrichment;

[0014] (3) The enriched sample was diluted and spread on LB solid medium. After culturing in a 37°C incubator overnight for 24 hours, a single colony was picked and placed on LB solid slant medium. After culturing at 37°C for 24 hours, the single colony was stored in a -4°C refrigerator for later use.

[0015] (4) The single colony strains isolated at room temperature (37°C) were inoculated in batches into LB liquid seed culture medium for enrichment culture at 37°C for 24 h, and then spread on LB solid culture medium for high-temperature culture. The growth of the plate colonies was observed, and the strains with better growth were selected.

[0016] (5) The strains with better growth obtained in step (4) were inoculated into VP liquid culture medium respectively, and cultured on a shaker at 180 rpm for 48 h to obtain culture solution;

[0017] (6) Add O'Meara reagent (40% NaOH containing 0.3% creatine) to the culture medium, shake evenly, and incubate in a 30°C incubator for 15 minutes. After the culture medium is incubated, place it in a spectrophotometer at a wavelength of 560 nm to measure the absorbance.

[0018] (7) The strain with the higher absorbance value was inoculated into liquid fermentation medium (sucrose 100 g / L, peptone 30 g / L, yeast powder 10 g / L, diammonium phosphate 30 g / L, pH 7.5, sterilized at 121°C for 20 min) and cultured at 37°C, 150 rpm for 48 h;

[0019] (8) The culture solution was centrifuged at 8000 rpm for 5 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The filtered sample was placed in a sample bottle and the TTMP content in the sample was determined by HPLC. The strain with the highest TTMP production was selected for solid-state fermentation rescreening.

[0020] (9) A single colony of a functional bacterial strain producing high TTMP yield in liquid fermentation was inoculated into a 250 mL shake flask containing 50 mL of liquid fermentation medium and cultured at 37°C for 24 h. The liquid fermentation culture was used as a seed solution and inoculated at an 8% (v / w) inoculum into a 250 mL shake flask containing 50 g of bran solid culture medium and cultured at 37°C for 5 d.

[0021] (10) The strain with the highest TTMP production content during solid-state fermentation was inoculated into LB medium and cultured overnight at 37°C and 200 rpm for activation;

[0022] (11) Take 1 mL of bacterial suspension in an EP tube, centrifuge at 12,000 rpm for 2 min, discard the supernatant, add 500 μL of sterile water to the bacterial suspension and shake evenly to prepare a new suspension;

[0023] (12) 20 μL of the suspension was applied to a quenched metal sheet for ARTP mutagenesis. The irradiation time was 0-120 s, with a monitoring point every 20 s (i.e., the mutagenesis time was: 0, 20, 40, 60, 80, 100, 120 s);

[0024] (13) After the mutagenesis, use sterile tweezers to move the metal piece to a tube containing 1 mL of 25% glycerol, shake it evenly to make a new bacterial suspension, and dilute the bacterial suspension by 10 -6 Spread onto LB medium and culture at 37°C for 24 h;

[0025] (14) Single colonies were randomly selected from the plates, and the TTMP content was detected after fermentation culture. The strain with the highest yield was identified by 16S rDNA strain identification and was identified as halotolerant Bacillus YB13 by molecular identification.

[0026] Preferably, the sieve aperture in step (1) is 100 mesh, and the enrichment culture time in step (2) is 1 hour.

[0027] Preferably, the screened strain is cultured at room temperature of 37°C for 24 hours and at high temperature of 50°C for 48 hours.

[0028] Preferably, the TTMP production of the screened strain under solid-state fermentation conditions is between 0.021 and 1.366 mg / g, that is, the TTMP production per gram of dry weight of the solid culture medium is between 0.021 and 1.366 mg.

[0029] Preferably, after plasma mutagenesis, the maximum TTMP yield of the strain under solid-state fermentation conditions is 2.859 mg / g, that is, the yield of TTMP per gram of dry weight of solid culture medium is 2.859 mg.

[0030] The invention discloses an application of a high-yield tetramethylpyrazine strain. The bacterial seed liquid is prepared from salt-tolerant Bacillus YB13, which is evenly mixed with other bacterial seed liquids. A bacterial bran koji is prepared using a disc, and the bacterial bran koji is mixed with yeast bran koji and white koji in a certain proportion to form a fusion bran koji, which is finally applied to the brewing of fusion-fragrant liquor.

[0031] Compared with related technologies, the screening, mutagenesis and application of high-yield tetramethylpyrazine strains provided by the present invention have the following beneficial effects:

[0032] The present invention provides screening, mutagenesis and application of a high-yield tetramethylpyrazine strain. The present invention respectively screens the high-yield tetramethylpyrazine strain by fermentation in liquid and solid culture media. The amount of tetramethylpyrazine produced by halogen-tolerant Bacillus YB13 in the solid culture medium is significantly higher than that of other strains, and the strain is a high-yield tetramethylpyrazine strain. The strain is first prepared into a bacterial seed liquid using a fermenter, and then uniformly mixed with other prepared bacterial seed liquids. A pure bacterial bran koji is then prepared using a disc, and the mixture is mixed with yeast bran koji and white koji in a certain proportion to form a blended bran koji. The blended bran koji is applied to liquor brewing. Testing shows that the quality of the original liquor added with the halogen-tolerant Bacillus is higher than that of the control group, and the TTMP content is increased, thereby improving the quality of the liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the phylogenetic tree of strain YB13;

[0034] Figure 2 is the yield of TTMP during the grain mash stacking and brewing process of the experimental group;

[0035] Figure 3 is the yield of TTMP during the mash stacking and brewing process in the control group;

[0036] Figure 4 The sensory evaluation results of the original liquor of the experimental group are shown in Figure 2.

[0037] Figure 5 The sensory evaluation results of the control group's original liquor are shown in Figure 2.

[0038] Preservation Instructions

[0039] Bacillus halotolerans YB13, the strain is deposited in the China General Microbiological Culture Collection Center, the preservation date is January 12, 2024, and the preservation number is CGMCC No. 28161. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Please refer to Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 ,in, Figure 1 is the phylogenetic tree of strain YB13; Figure 2 、 3 To strengthen the effect of mash stacking and TTMP production during brewing; Figure 4 、 5 This is a sensory evaluation chart of the fortified base liquor.

[0042] The present invention provides a high-yield tetramethylpyrazine strain, which is named Bacillus halophilus (Bacillus halophilus) YB13. The strain is deposited in the China General Microbiological Culture Collection Center with a deposit date of January 12, 2024 and a deposit number of CGMCC No. 28161.

[0043] A solid-state fermentation medium for white wine grains of halotolerant Bacillus YB13 comprises the following components: bran, soybean meal powder and water.

[0044] The bran and soybean meal powder are evenly mixed in a ratio of 8:2, and then an appropriate amount of water is added to adjust the humidity to 30% for culturing. Each 50 g of the mixture is placed in a 250 mL conical flask.

[0045] A strain of halotolerant Bacillus subtilis YB13 screened from solid-state fermentation medium of white wine lees was optimized by plasma mutagenesis.

[0046] A method for screening a high-yield tetramethylpyrazine strain specifically comprises the following steps:

[0047] (1) The high-temperature koji in the koji room was randomly collected at 5 sampling points, crushed and sieved for use;

[0048] (2) Place the crushed koji powder in LB liquid medium and culture it at 37°C for enrichment;

[0049] (3) The enriched sample was diluted and spread on LB solid medium. After culturing in a 37°C incubator overnight for 24 hours, a single colony was picked and placed on LB solid slant medium. After culturing at 37°C for 24 hours, the single colony was stored in a -4°C refrigerator for later use.

[0050] (4) The single colony strains isolated at room temperature (37°C) were inoculated in batches into LB liquid seed culture medium for enrichment culture at 37°C for 24 h, and then spread on LB solid culture medium for high-temperature culture. The growth of the plate colonies was observed, and the strains with better growth were selected.

[0051] (5) The strains with better growth obtained in step (4) were inoculated into VP liquid culture medium respectively, and cultured on a shaker at 180 rpm for 48 h to obtain culture solution;

[0052] (6) Add O'Meara reagent (40% NaOH containing 0.3% creatine) to the culture medium, shake evenly, and incubate in a 30°C incubator for 15 minutes. After the culture medium is incubated, place it in a spectrophotometer at a wavelength of 560 nm to measure the absorbance.

[0053] (7) The strain with the higher absorbance value was inoculated into liquid fermentation medium (sucrose 100 g / L, peptone 30 g / L, yeast powder 10 g / L, diammonium phosphate 30 g / L, pH 7.5, sterilized at 121°C for 20 min) and cultured at 37°C, 150 rpm for 48 h;

[0054] (8) The culture solution was centrifuged at 8000 rpm for 5 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The filtered sample was placed in a sample bottle and the TTMP content in the sample was determined by HPLC. The strain with the highest TTMP production was selected for solid-state fermentation rescreening.

[0055] (9) The strain with the highest TTMP production content during solid-state fermentation was inoculated into LB medium and cultured overnight at 37°C and 200 rpm for activation;

[0056] (10) Take 1 mL of bacterial suspension in an EP tube, centrifuge at 12,000 rpm for 2 min, discard the supernatant, add 500 μL of sterile water to the bacterial suspension, and shake evenly to prepare a new suspension;

[0057] (11) Pipette 20 μL of the suspension onto a quenched metal sheet for ARTP mutagenesis. The irradiation time was 0-120 s, with a monitoring point every 20 s (i.e., the mutagenesis time was: 0, 20, 40, 60, 80, 100, 120 s).

[0058] (12) After the mutagenesis, use sterile tweezers to move the metal piece to a tube containing 1 mL of 25% glycerol, shake it evenly to make a new bacterial suspension, and dilute the bacterial suspension by 10 -6 Spread onto LB medium and culture at 37°C for 24 h;

[0059] (13) Single colonies were randomly selected from the plates, and the TTMP content was detected after fermentation culture. The strain with the highest yield was identified by 16S rDNA strain identification and was identified as halotolerant Bacillus YB13 by molecular identification.

[0060] Preferably, the sieve aperture in step (1) is 100 mesh, and the enrichment culture time in step (2) is 1 hour.

[0061] Preferably, the screened strain is cultured at room temperature of 37°C for 24 hours and at high temperature of 50°C for 48 hours.

[0062] Preferably, the TTMP production of the screened strain under solid-state fermentation conditions is between 0.021 and 1.366 mg / g, that is, the TTMP production per gram of dry weight of the solid culture medium is between 0.021 and 1.366 mg.

[0063] Preferably, after plasma mutagenesis, the maximum TTMP yield of the strain under solid-state fermentation conditions is 2.859 mg / g, that is, the yield of TTMP per gram of dry weight of solid culture medium is 2.859 mg.

[0064] The invention discloses an application of a high-yield tetramethylpyrazine strain, and applies the salt-tolerant Bacillus YB13 to liquor brewing.

[0065] Example 1

[0066] The high-temperature koji in the koji room was randomly harvested at 5 sampling points, crushed and sieved for use. The crushed koji powder was placed in LB liquid culture medium and enriched at 37°C. The sample after enrichment culture was diluted and spread on LB solid culture medium. After culturing in a 37°C incubator overnight for 24 hours, 115 single colonies were picked on LB solid slant culture medium, and after culturing at 37°C for 24 hours, they were stored in a -4°C refrigerator for standby use. The single colony strains isolated under normal temperature of 37°C were inoculated in batches into LB liquid seed culture medium for enrichment culture at 37°C for 24 hours, spread on LB solid culture medium for high-temperature culture, and the growth of the plate colonies was observed, and the strains with better growth were selected. The strains with better growth obtained in step (4) were respectively inoculated into VP liquid culture medium and cultured on a shaker at 180rpm for 48 hours to obtain culture solution. The culture broth was added with O'Meara reagent (40% NaOH containing 0.3% creatine), shaken evenly, and incubated in a 30°C incubator for 15 minutes. The absorbance of the culture broth was measured at 560 nm using a spectrophotometer. Thirty-seven strains with high absorbance were inoculated into liquid fermentation medium (100 g / L sucrose, 30 g / L peptone, 10 g / L yeast extract, 30 g / L diammonium phosphate, pH 7.5, sterilized at 121°C for 20 minutes) and incubated at 37°C, 150 rpm, for 48 hours. Thirty-six potential high-TTMP-producing strains were obtained.

[0067] Thirty-seven strains showed red color in the VP test. These 37 strains could produce acetoin through liquid fermentation, so these strains were further screened for target strains in subsequent fermentation tests. The results are shown in Table 1.

[0068] Table 1 OD values ​​of 37 strains at 560 nm

[0069]

[0070] Example 2

[0071] This example screened 36 bacterial strains (1 to 36#) for the best TTMP production capacity. The specific process is as follows:

[0072] Dissolve 10g of sodium chloride (NaCl), 10g of peptone, and 5g of yeast extract in 1000mL of water to prepare LB medium. The pH of the medium is 7.4. Thirty-seven potential high-TTMP-producing strains were inoculated into this liquid medium and cultured at 37°C and 150 rpm for 24 hours to activate the strains.

[0073] 30g peptone, 100g glucose (C6H 12 O6·H2O) and 10g yeast powder were dissolved in 1000mL of water to prepare a liquid fermentation medium. The pH of the medium was 7.5. Thirty-seven strains were inoculated into the liquid fermentation medium and cultured at 37°C and 150 rpm for 48 hours. 3mL of the culture broth was transferred to a 10mL centrifuge tube and centrifuged at 8000 rpm for 5 minutes. The supernatant was collected and filtered through a 0.22μm aqueous filter. Approximately 1mL of the filtered sample was collected and placed in a sample bottle. TTMP production by the strains under liquid conditions was measured using HPLC.

[0074] Activated strains were selected from LB culture medium and inoculated into liquid fermentation medium. After static culture at 37°C for 48 hours, the amount of TTMP produced by the strains was determined by HPLC. 36 strains were screened for TTMP production. The test results are shown in Table 1.

[0075] Table 2 TTMP content produced by liquid fermentation of 36 strains

[0076]

[0077]

[0078] As shown in Table 2, HPLC results indicate that all strains (1-36#) can produce TTMP in liquid culture. Among them, strains 4#, 9#, 10#, 11#, 13#, 14#, 15#, and 29# produced relatively high yields. These 36 strains were used in subsequent experiments.

[0079] Example 3

[0080] This example screened 36 strains producing TTMP by liquid fermentation to identify the strain with the best TTMP production capacity under solid-state fermentation conditions. The specific process is as follows:

[0081] 100g glucose (C6H 12 A liquid fermentation medium was prepared by dissolving 1000 mL of water (1000 mL of 1% CO₂O₆·H₂O), 30 g of peptone, 10 g of yeast extract, and 30 g of diammonium phosphate ((NH₄)₂HPO₄). The pH of the medium was 7.5. Thirty-six bacterial strains were inoculated into this liquid fermentation medium and incubated at 37°C and 150 rpm for 48 hours to complete fermentation.

[0082] Liquid fermentation broth was used as seed liquid. An 8% (v / w) inoculum was added to bran and soybean meal powder in an 8:2 ratio. After mixing thoroughly, an appropriate amount of water was added to adjust the humidity to 30% for incubation. 50g of the culture was placed in a 250mL conical flask to prepare the solid-state fermentation medium. After incubation at 37°C for 5 days, an appropriate amount of the culture was weighed and dried at 105°C to constant weight. The water content was calculated. A 5g dry weight sample was added to 50mL of 40% ethanol and sonicated for 1 hour. 2mL of the supernatant was centrifuged at 8000 rpm for 10 minutes, filtered through a 0.22μm organic filter membrane, and analyzed by HPLC.

[0083] The strains that had completed fermentation in the liquid fermentation medium were inoculated into solid-state fermentation medium. After 5 days of static culture at 37°C, the TTMP production by the strains was determined by HPLC to screen for the strain with the highest TTMP production. The results are shown in Table 3.

[0084] Table 3 TTMP content produced by solid-state fermentation of 36 strains

[0085]

[0086]

[0087] As shown in Table 3, the HPLC results showed that only 18 strains could produce TTMP in solid-state culture medium. Among them, strain 6# produced the highest TTMP yield under solid-state fermentation conditions, at 1.366 mg / g, and was used for subsequent experiments.

[0088] Example 4

[0089] In this example, strain 6# was subjected to plasma mutagenesis and the TTMP content was detected under solid-state fermentation conditions. The specific process is as follows:

[0090] Strain 6# was inoculated into LB medium and cultured for activation at 37°C and 200 rpm for 24 h. 1 mL of bacterial solution was transferred to an EP tube and centrifuged at 12,000 rpm for 2 min. The supernatant was discarded and 500 μL of sterile water was added to the bacterial solution and shaken evenly to prepare a new bacterial suspension.

[0091] 20 μL of bacterial suspension was aspirated onto a sterilized metal sheet for ARTP mutagenesis. The irradiation time was 0s-120s, with a detection point every 20s. (i.e., the mutagenesis time was: 0, 20, 40, 60, 80, 100, 120s). After the mutagenesis, the metal sheet was transferred to a tube containing 1 mL of 25% glycerol using sterile tweezers and shaken to make a new bacterial suspension. The bacterial suspension was diluted 10 -6 Spread onto LB medium and incubate at 37°C for 24 hours. Inoculate the strain into liquid fermentation medium to produce a seed solution. Inoculate the seed solution at an 8% (v / w) inoculum into a 250 mL shake flask containing 50 g of bran solid medium. Incubate at 37°C for 5 days. Weigh an appropriate amount of the culture, dry it at 105°C to constant weight, and calculate its water content. Take a 5 g dry weight sample, add 50 mL of 40% ethanol, and sonicate for 1 hour. Take 2 mL of the supernatant, centrifuge at 8000 rpm for 10 minutes, filter through a 0.22 μm organic filter, and analyze by HPLC.

[0092] The strains that had completed fermentation in the liquid fermentation medium were inoculated into solid-state fermentation medium. After static culture at 37°C for 5 days, the TTMP production by the strains was determined by HPLC to screen for the strain with the highest TTMP production. The test results are shown in Table 4.

[0093] Table 4 TTMP content produced by solid-state fermentation of strains

[0094]

[0095]

[0096] As shown in Table 4, the HPLC results showed that strain 13 produced the highest TTMP yield under solid-state fermentation conditions, at 2.859 mg / g. Therefore, it was named YB13 and used in subsequent experiments.

[0097] Example 5

[0098] In this example, the YB13 strain was identified, and the specific process is as follows:

[0099] DNA of the HBSD09 strain was extracted and PCR amplified using universal primers (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3').

[0100] The PCR reaction system was: 2.0 μL of 10×Ex Taq buffer, 0.2 μL of 5U Ex Taq, 1.6 μL of 2.5 mM dNTP Mix, 1 μL of primer 1 / primer 2 (sequence 1 and sequence 2), 0.5 μL of DNA template, and 13.7 μL of ddH2O.

[0101] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min, entering the cycle, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1.5 min, 35 cycles, and a final extension at 72°C for 1.5 min.

[0102] The amplified products were sequenced using the next-generation sequencing platform 3730. The sequence quality at both ends of the sequencing is generally poor. Low-quality sequences were removed by mass shearing. The paired-end sequencing results after quality control were assembled to obtain the 16S rRNA sequence shown in SEQ ID NO. 1.

[0103] The sequence of SEQ ID NO.1 is as follows:

[0104] GCGGTGGGGGCGTGCTATACATGCAGTCGAGCGGACAGATGGGAGC

[0105] TTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCT

[0106] GCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGC

[0107] TTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTT

[0108] ACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACC

[0109] AAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGG

[0110] ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC

[0111] CGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG

[0112] TTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAA

[0113] TAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACG

[0114] TGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATT

[0115] GGGCGTAAAGGGCTCGCAGGCGGTTCMTTAAGTCTGATGTGAAAGCCCC

[0116] CGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGA

[0117] AGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGG

[0118] AGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGA

[0119] GGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC

[0120] GCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCT

[0121] GCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTG

[0122] AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG

[0123] TTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGA

[0124] CAATCCTAGAGATAGGACGTCCCTCTCGGGGGCAGAGTGACAGGTGGTG

[0125] CATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC

[0126] GAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGG

[0127] TGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCAT

[0128] CATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAA

[0129] AGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAG

[0130] TTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTA

[0131] ATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACAC

[0132] CGCCCGTCACAACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAAC

[0133] CTTTAGGAGCCAGCCGCCGAAGGGACCGT

[0134] The assembled 16S rRNA sequence was compared to the NCBI database. The alignment results were judged based on coverage and similarity. The highest score was selected to confirm that the sample species was halodurable Bacillus. Halodurable Bacillus YB13 was deposited in the China Center for Microbiological Culture Collection, with the accession number CGMCC No. 28161.

[0135] Comparison analysis of the strain sequence with the database showed that it was in the same branch as Bacillus halotolerans with the highest similarity (99.52%). Strain YB13 was preliminarily identified as Bacillus halotolerans and submitted to NCBI with accession number PQ303489.

[0136] Example 4

[0137] YB13 was applied to the brewing of liquor. The strain was inoculated into LB medium and cultured at 180rpm for 48h to prepare a seed liquid. The solution was sent to the koji making workshop for expansion and then mixed evenly with other bacterial seed liquids. A bacterial bran was prepared using a disc. The prepared bacterial bran was mixed with yeast bran and white koji in a certain proportion to form a combined bran and grain mash. The mixture was evenly piled for 3 days and then put into the pool for fermentation after the pile was completed. The seed liquid prepared without adding YB13 was used as the control group. The grain mash and wine mash were tested for TTMP content during the brewing process, and the brewed original liquor was evaluated. The specific process is as follows:

[0138] HS-SPME-GC-MS was used to determine the TTMP in grain mash and wine mash. The HS-SPME conditions were as follows: the extraction head was 50 / 30DVB / CAR / PDMS (2 cm), equilibrated at 50°C for 10 min, then extracted at 50°C for 40 min, and the stirring rate of the magnetic stirrer was 250 r / min. After the extraction, the extraction was completed and the GC-MS analysis was performed.

[0139] GC conditions: The chromatographic column was a DB-5MS quartz capillary column (30 m × 0.25 mm, 0.25 μm); the temperature program was an initial temperature of 50°C, held for 1 min, then increased at 8°C / min to 100°C, held for 5 min, then increased at 15°C / min to 235°C, held for 4 min. The carrier gas was high-purity helium (He) (purity >99.999%) at a flow rate of 2 μL / min; split injection was used with a split ratio of 5:1.

[0140] MS conditions: electron ionization (EI) source; electron energy 70 eV; quadrupole temperature 150°C, ion source temperature 230°C, and transfer line temperature 245°C. Selected ion monitoring (SIM) mode was used, monitoring ions at 136 m / z, 54 m / z, and 42 m / z, with a quantification ion at 136 m / z. Solvent delay time was 5 min.

[0141] Qualitative and quantitative analysis: qualitative analysis was performed using retention time combined with mass spectrometry, and quantitative analysis was performed using the external standard method.

[0142] The winery organized four rating experts, Huang, Gong, Gao, and Liang, to evaluate the original wine. The wine samples evaluated included the upper, middle, lower, and three-layer mixed samples of the experimental and control groups. The wine samples were named: A upper, A middle, A lower, and A mixed. The results are as follows Figure 4 、 5 shown.

[0143] from Figure 2 、3 It can be seen that the content of TTMP in the stacking and brewing process shows a trend of gradual increase, and after the high-TTMP-producing YB13 strain is added, the content of TTMP produced in the brewing process is higher than that in the control group. Figure 4 、 5 Experts evaluated the lower layer of raw liquor as having the best quality. Furthermore, the scores of the upper, middle, lower, and mixed liquor samples in the experimental group were all higher than those in the control group.

[0144] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A high-yield tetramethylpyrazine strain, named Bacillus halotolerans YB13, was deposited in the China General Microbiological Culture Collection Center on January 12, 2024, with a deposit number of CGMCC No. 28161.

2. A solid-state fermentation medium for white wine lees of halotolerant Bacillus YB13, characterized in that: It includes the following components: bran, soybean meal powder (solid culture medium).

3. The solid-state fermentation medium of halodurable Bacillus YB13 white wine lees according to claim 2, characterized in that The bran and soybean meal powder are evenly mixed in a ratio of 8:2, and then an appropriate amount of water is added. The humidity is adjusted to 30% for culturing, and each 50 g is placed in a 250 mL conical flask.

4. A strain screened from the solid-state fermentation medium of white wine lees by salt-tolerant Bacillus YB13 was optimized by plasma mutagenesis.

5. A method for screening high-yielding tetramethylpyrazine strains, characterized in that: The specific steps include: (1) The high-temperature koji in the koji room was randomly collected at 5 sampling points, crushed and sieved for use; (2) Place the crushed koji powder in LB liquid culture medium for enrichment culture; (3) Dilute the enriched sample and spread it on LB solid medium, incubate at 37°C for 12-24 hours to isolate single colonies; (4) The single colony strains isolated at room temperature were prepared into seed liquid in batches and spread on LB solid medium and cultured at 50°C for 48 hours to isolate single colonies; (5) The purified strains were inoculated into VP liquid culture medium and cultured on a shaking platform; (6) Add O'Meara reagent to the culture solution, shake evenly, and place it in a constant temperature incubator for incubation. After the culture solution is placed in a photometer to measure the absorbance value; (7) Inoculate the strain with the higher absorbance value into liquid fermentation medium and culture overnight; (8) The culture medium was centrifuged, the supernatant was collected, and the supernatant was filtered through an aqueous phase filter membrane. The filtered sample was placed in a sample bottle, and the TTMP content in the sample was determined by HPLC. The strain with higher TTMP production was selected for solid-state fermentation rescreening; (9) The strain with the highest TTMP production content during solid-state fermentation was inoculated into LB medium and cultured overnight; (10) Take 1 mL of bacterial suspension in an EP tube, centrifuge and discard the supernatant, add 500 μL of sterile water to the bacterial suspension and shake evenly to prepare a new suspension; (11) Pipette 20 μL of the suspension onto a quenched metal sheet for ARTP mutagenesis. The irradiation time is 0-120 s, with one monitoring point every 20 s. (12) After the mutagenesis is completed, use sterile tweezers to move the metal piece to a tube containing 1 mL of 25% glycerol, shake it evenly to make a new bacterial suspension, and dilute the bacterial suspension by 10 -6 Spread onto LB medium and culture at 37°C overnight; (13) Single colonies were randomly selected from the plates, and the TTMP content was detected after fermentation culture. The strain with the highest yield was molecularly identified and determined to be halotolerant Bacillus subtilis YB13.

6. The method for screening a high-yield tetramethylpyrazine strain according to claim 5, characterized in that: The sieve aperture in step (1) is 100 mesh, and the enrichment culture time in step (2) is 1 hour.

7. The method for screening a high-yield tetramethylpyrazine strain according to claim 5, characterized in that: The screened strains were cultured at room temperature of 37°C for 24 h and at high temperature of 50°C for 48 h.

8. The method for screening a high-yield tetramethylpyrazine strain according to claim 5, characterized in that: The TTMP production of the screened strain under solid-state fermentation conditions is between 0.021 and 1.366 mg / g (dry basis), that is, the TTMP production per gram of dry weight culture medium is between 0.021 and 1.366 mg.

9. The method for screening a high-yielding tetramethylpyrazine strain according to claim 5, characterized in that: After plasma mutagenesis, the TTMP production of the strain under solid-state fermentation conditions was as high as 2.859 mg / g (dry basis), that is, the production of TTMP per gram of dry weight culture medium was 2.859 mg.

10. An application of a high-yield tetramethylpyrazine strain, characterized in that: Bacterial seed liquid is prepared with salt-tolerant Bacillus YB13, which is evenly mixed with other bacterial seed liquids. Bacterial bran koji is prepared using a disc, and is mixed with yeast bran koji and white koji in a certain proportion to form a fusion bran koji, which is finally used in the brewing of fusion-fragrant liquor.

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