Bacillus dasycarpus and its applications

Abstract

This invention belongs to the field of microbial technology, specifically relating to *Bacillus daquensis*, a thermoresistant microorganism producing 2,3,5-trimethylpyrazine, and its applications. Addressing the issue of the limited number of reported microorganisms capable of producing pyrazine compounds, this invention provides a novel thermoresistant *Bacillus daquensis* strain producing 2,3,5-trimethylpyrazine, with accession number CGMCC No. 26367. The *Bacillus daquensis* LZLJ2-3 provided by this invention is thermoresistant and possesses the ability to ferment and produce 2,3,5-trimethylpyrazine, which can be used in the preparation of koji (fermentation starter) for baijiu (Chinese liquor) brewing, enriching the aroma compounds in the liquor. Simultaneously, its short fermentation cycle and low cost provide a possibility for the microbial preparation of 2,3,5-trimethylpyrazine and can also serve as a research subject for recombinant organisms, which is of great significance for promoting the industrial production of 2,3,5-trimethylpyrazine.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Bacillus dauricum, a heat-resistant bacterium that produces 2,3,5-trimethylpyrazine, and its applications. Background Technology

[0002] Pyrazine compounds are important intermediates in the synthesis of pharmaceuticals and pesticides, and are also used as food flavorings. 2,3,5-Trimethylpyrazine is a high-end food flavoring that has been developed in recent years. It has a rich aroma of cocoa beans, nuts, roasted potatoes, and roasted peanuts. Naturally occurring pyrazine is found in foods such as roasted barley, black tea, cheese, cocoa products, coffee, and rum. It is an important raw material for flavorings and tobacco flavorings, and can be directly used in the food industry to flavor various foods such as bread, pudding, and soft drinks.

[0003] Existing research indicates that pyrazine compounds are the material basis for the roasted aroma of baijiu. There are few reports on microorganisms that can produce 2,3,5-trimethylpyrazine. For example, Guo Chengshuan et al. (Guo Chengshuan, Ouyang Puyue, Xie He. GC / MS analysis of volatile flavor components produced by Bacillus subtilis E20 fermentation [J]. China Brewing, 2010(9):3.) found that the main metabolites of solid-state fermentation of Bacillus subtilis E20 include phenylacetaldehyde, 4-methyl-2,6-di-tert-butylphenol, indole, α-furan methanol, 2,3,5-trimethylpyrazine, 2-acetylthiazole, etc. Liang Huizhen et al. (Liang Huizhen, Lu Yanxiang, Liu Zheng, et al. Screening and application of high-pyrazine-producing Bacillus strains in high-temperature Daqu [J]. China Brewing, 2022, 41(1):7.) used high-temperature culture and solid-state fermentation to screen high-pyrazine-producing strains from high-temperature Daqu. The metabolites of the strains were detected by headspace solid-phase microextraction and gas chromatography-mass spectrometry. The strains obtained were Sonora desert Bacillus, Bacillus subtilis, Bacillus licheniformis, Bacillus cereus and Bacillus amyloliquefaciens. They had the ability to produce 2,3,5-trimethylpyrazine in solid-state fermentation, with the highest being 130.28 μg / kg.

[0004] Studies have found that thermoresistant bacteria can metabolize aroma compounds such as 3-hydroxy-2-butanone, 2,3-butanediol, and pyrazines, and can produce various enzymes such as amylases and proteases, driving various biochemical reactions. The formation of baijiu flavor is closely related to thermoresistant bacteria, and the typical style and characteristics of baijiu are important manifestations of the role of thermoresistant bacteria in daqu (fermentation starter). Therefore, obtaining a heat-resistant strain that produces 2,3,5-trimethylpyrazine is of great significance for the research of baijiu daqu production technology. Summary of the Invention

[0005] In response to the problem that there are few microorganisms that can produce pyrazine compounds in existing reports, this invention provides a new heat-resistant Bacillus dauricum that produces 2,3,5-trimethylpyrazine and its applications.

[0006] The present invention first provides a strain of 2,3,5-trimethylpyrazine-producing *Scopulibacillus daqui*, with accession number CGMCC No. 26367.

[0007] The deposit was made on December 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The yeast strain was screened from the yeast starter and is named: *Scopulibacillus daqui* LZLJ2-3.

[0008] The 16S rDNA sequence of the above-mentioned Bacillus daquica is shown in SEQ ID NO:1:

[0009]

[0010] The present invention also provides a microbial agent, which uses the aforementioned Bacillus daquica as its active ingredient.

[0011] The present invention also provides the application of the above-mentioned Bacillus dahurica in the production of 2,3,5-trimethylpyrazine.

[0012] This invention also provides the application of the above-mentioned Bacillus rock in the field of Baijiu brewing.

[0013] The present invention also provides the application of the above-mentioned Bacillus rock in increasing the content of pyrazine compounds in fermented products or flavorings.

[0014] Specifically, in the aforementioned application, the following fermentation process is used to prepare pyrazine compounds, including the following steps: inoculating Bacillus daquis into LB liquid medium to prepare seed culture, inoculating the seed culture into solid fermentation medium, and then fermenting.

[0015] The seed culture conditions for the Bacillus daquis seed liquid are: 50℃-60℃, 120r / min shaker culture for 18h-24h.

[0016] The LB liquid culture medium formula is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, autoclaved at 115°C for 20 min.

[0017] The inoculation volume of the seed culture into the solid fermentation medium was 5-10 v / v.

[0018] The solid fermentation medium is prepared by the following method: soaking wheat in 10wt% pure water for 20h, adding 40wt% water to wet-crush the wheat grains, dispensing, and autoclaving at 121℃ for 20min.

[0019] The fermentation culture conditions are as follows: culture at 50℃ for 1 day, then culture at 55℃ for 1 day, then culture at 60℃ for 1 day.

[0020] Furthermore, using the above fermentation process, the 2,3,5-trimethylpyrazine content produced by Bacillus daquis is 0.362 mg / kg.

[0021] Beneficial Effects: This invention isolates and screens a strain of *Scopulibacillus daqui* capable of producing 2,3,5-trimethylpyrazine from Luzhou Laojiao medium-temperature Daqu (a type of starter culture). Its preservation number is CGMCC No. 26367, and it is named *Scopulibacillus daqui* LZLJ2-3. The *Scopulibacillus daqui* LZLJ2-3 provided by this invention is heat-resistant, growing at 37℃-65℃, with an optimal culture temperature of 50℃-55℃, making it suitable for the high-temperature fermentation process in the preparation of medium- and high-temperature Daqu. It possesses the ability to ferment and produce 2,3,5-trimethylpyrazine, which can be used in the preparation of starter culture for Baijiu (Chinese liquor) brewing, enriching the aroma compounds in the liquor. Simultaneously, its short fermentation cycle and low cost provide a possibility for the microbial preparation of 2,3,5-trimethylpyrazine, and it can also serve as a research object for recombinant organisms, which is of great significance for promoting the industrial production of 2,3,5-trimethylpyrazine. Attached Figure Description

[0022] Figure 1 Example 1: Bacillus daquis colony morphology;

[0023] Figure 2 This is a GC-MS molecular fragment mass spectrum of 2,3,5-trimethylpyrazine produced by Bacillus daquensis in Example 2;

[0024] Figure 3 This is a GC-MS molecular fragment mass spectrum of 2,3,5-trimethylpyrazine standard.

[0025] This invention provides *Scopulibacillus daqui*, a 2,3,5-trimethylpyrazine-producing bacterium, deposited on December 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The accession number is CGMCC No. 26367. The classification name is *Scopulibacillus daqui*. Detailed Implementation

[0026] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0027] The culture medium formulations involved in the examples are as follows:

[0028] R2A solid culture medium: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, pH 7.2 ± 0.2, autoclaved at 115°C for 20 minutes.

[0029] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, autoclaved at 115°C for 20 minutes.

[0030] Solid-state fermentation medium: Wheat grains were soaked in 10wt% pure water for 20h, then 40wt% water was added and the mixture was wet-crushed using a crusher. The mixture was dispensed into 250mL Erlenmeyer flasks, 100g / flask, and autoclaved at 121℃ for 20min.

[0031] Example 1: Isolation and purification of strains

[0032] (1) Separation method: Take 20g of Luzhou Laojiao medium-temperature Daqu sample and place it in a conical flask containing 180mL of sterile distilled water. Shake on a constant temperature shaker for 10min to thoroughly disperse and mix the sample. Take 1mL of the sample suspension and dilute it to 10 using the serial dilution method. -2 ~10 -7 Take 100 μL of each dilution and spread it evenly on an R2A solid medium plate. Prepare two parallel plates, invert them, and incubate them in a 50°C incubator for 36-48 h, observing them regularly.

[0033] (2) Streak purification: Remove the plate from which colonies have grown, pick single colonies with different morphologies, and streak them a second time until all single colonies are purified. Colonies on R2A solid medium are moist, translucent white, and have a smooth surface. Figure 1 As shown.

[0034] (3) Preservation of strains: After purification, single colonies of each strain were picked into 5 mL of LB liquid medium and incubated at 50 °C for 24 h. 1 mL of the bacterial solution was then transferred to a preservation tube, 0.5 mL of 60% sterile glycerol solution was added, the culture was resuspended, and the culture was stored at -80 °C.

[0035] Example 2: Determination of the ability to produce 2,3,5-trimethylpyrazine

[0036] (1) Prepare the bacterial suspension to be tested:

[0037] After the glycerol preservation tubes of the selected strains were dissolved, they were inoculated into LB liquid medium and cultured at 50°C for 24 h. After three generations of activation, they were inoculated into 50 mL of LB liquid medium at a 2% (volume ratio) inoculation rate and cultured for 24 h to obtain the bacterial solution to be tested.

[0038] (2) HS-SPME / GC-MS method for the detection of 2,3,5-trimethylpyrazine:

[0039] Headspace solid-phase microextraction / gas chromatography-mass spectrometry (HS-MS / MS) was used: the supernatant was collected and added to a headspace vial, along with saturated NaCl solution. 2-Octanol (0.822 mg / ml) was used as an internal standard. The prepared sample was equilibrated at 60°C for 5 min, followed by extraction at 60°C for 50 min using a 50 / 30 μm DVB / CAR / PDMS extraction head. After extraction, desorption was performed at 250°C for 5 min at the GC inlet. Compound search results were matched with the NIST standard spectral library; compounds with a similarity of over 80% were confirmed as target compounds. A culture broth without added bacteria was used as a blank control group, and the content of each volatile substance was calculated.

[0040] GC-MS detection chromatographic conditions:

[0041] Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: initial temperature 40℃, hold for 5 min, increase to 100℃ at 4℃ / min, then increase to 230℃ at 6℃ / min, hold for 10 min; carrier gas is high-purity helium (1.0mL / min); injection port temperature 250℃, splitless.

[0042] 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.

[0043] Through the above detection methods, such as Figure 2 , 3 As shown, a strain with a strong ability to produce 2,3,5-trimethylpyrazine was screened from the isolated strains, and the content of 2,3,5-trimethylpyrazine produced was 0.143 mg / L. This strain was selected for further research.

[0044] Example 3: Morphological and Molecular Identification of Bacteria

[0045] After amplification of the target strain, fresh bacterial culture in the logarithmic growth phase was collected by centrifugation, and genomic DNA was extracted using a bacterial genome extraction kit. The full-length 16S rDNA sequence was amplified using universal bacterial primers 27F / 1492R, as follows:

[0046] SEQ ID NO: 2: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

[0047] SEQ ID NO:3:1492R(5′-GGTTACCTTGTTACGACTT-3′)

[0048] ①Reaction system (50μL)

[0049]

[0050] ②Reaction Procedure

[0051]

[0052] Following the above procedure, perform 35 cycles of "denaturation-annealing-extension" PCR reaction. The PCR products are separated and tested by 1.0% agarose gel electrophoresis at a voltage of approximately 11V / cm for 20 minutes.

[0053] The PCR products were purified according to the instructions of the small-volume gel recovery PCR product purification kit from Shanghai Sangon Biotech Co., Ltd., and the sequencing was performed by Shanghai Sangon Biotech Co., Ltd.

[0054] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI BLAST, and the species information of the strain was determined based on the morphological characteristics of the strain. It was identified as *Scopulibacillus daqui*, and its 16S rDNA is shown in SEQ ID NO:1. It was named *Scopulibacillus daqui* LZLJ2-3 and deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 30, 2022, with the accession number CGMCC No. 26367.

[0055] Example 4: Solid-state fermentation of Bacillus rock spp. LZLJ2-3

[0056] After dissolving the obtained Bacillus daquica LZLJ2-3 glycerol preservation tubes, 1% of the inoculum was inoculated into 10 mL of LB liquid medium. After incubation at 50°C for 20 h, 1% of the inoculum was transferred to 150 mL of LB liquid medium and cultured on a shaker at 50°C and 120 rpm for 1 day to prepare a seed culture. 5% of the seed culture was then inoculated into solid-state fermentation medium, with the solid medium containing LB liquid medium serving as a blank control. The culture was incubated at 50°C for 1 day, then transferred to 55°C for 1 day, and finally to 60°C for 1 day. Samples were collected after fermentation, and the volatile component determination method was the same as in Example 2. The results showed that the 2,3,5-trimethylpyrazine produced by solid-state fermentation of Bacillus daquica LZLJ2-3 was 0.362 mg / kg.

[0057] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in one or more embodiments. Furthermore, those skilled in the art can combine and integrate the different embodiments described in this specification and the features of those embodiments without contradiction.

Claims

1. Bacillus daqueri ( Scopulibacillus daqui ), characterized by: The accession number is CGMCCNo.26367.

2. A microbial inoculant, characterized in that: The active ingredient is Bacillus daquensis as described in claim 1.

3. The use of the Bacillus daquensis of claim 1 in the production of 2,3,5-trimethylpyrazine.

4. The application of Bacillus rock spores as described in claim 1 in the field of Baijiu brewing.

5. The use of Bacillus rockellarium as described in claim 1 in increasing the content of 2,3,5-trimethylpyrazine in fermented products or flavorings.

6. The application according to any one of claims 3-5, characterized in that: 2,3,5-Trimethylpyrazine was prepared using the following fermentation process, which included the following steps: Bacillus daquis was inoculated into LB liquid medium to prepare a seed culture, the seed culture was inoculated into a solid fermentation medium, and fermentation was carried out.

7. The application according to claim 6, characterized in that: The seed culture conditions for the Bacillus daquis seed culture were 50°C-60°C, 120 r / min, and shaker culture for 18-24 h.

8. The application according to claim 6, characterized in that: The LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, autoclaved at 115°C for 20 min.

9. The application according to claim 6, characterized in that: The seed culture was inoculated into the solid fermentation medium at a rate of 5-10 v / v %; the fermentation conditions were as follows: cultured at 50℃ for 1 day, then at 55℃ for 1 day, then at 60℃ for 1 day.

10. The application according to claim 6, characterized in that: The solid fermentation medium was prepared by the following method: wheat grains were moistened with 10wt% pure water for 20h, then wet-crushed with 40wt% water, packaged, and sterilized at 121℃ for 20min.

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