Bacillus cereus producing tetramethylpyrazine and application thereof

Abstract

This invention discloses a tetramethylpyrazine-producing Bacillus cereus and its applications. The name of this Bacillus cereus is *Bacillus cereus* (…). Bacillus cereus Lot A1030, with accession number GDMCC No: 66145, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 14, 2025. This Bacillus cereus exhibits strong resistance to high temperatures (50˚C) and ethanol (20%). It produces protease and amylase, with an amylase clear zone HC value of 2.19 and a protease clear zone HC value of 1.7. Liquid fermentation of this Bacillus cereus produces tetramethylpyrazine at a concentration of 0.8 g / L.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a tetramethylpyrazine-producing Bacillus cereus and its applications. Background Technology

[0002] Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine, abbreviated as TTMP) is a heterocyclic nitrogen-containing compound with significant biological activity and a unique flavor. Its chemical structure contains two nitrogen atoms and four methyl substituents, and its molecular formula is C8H12H2O. 12 N2, with a melting point of 82–86℃ and a boiling point of 190℃, is a white crystalline or powdery substance at room temperature. It possesses a complex aroma reminiscent of heated beef, roasted nuts, and fermented soybeans, and exhibits a milk chocolate flavor upon dilution. This compound is widely found in natural products and industrial applications, serving as an important flavor enhancer in the food industry and a core active ingredient in the traditional Chinese medicine Ligusticum chuanxiong, thus possessing dual value in functional food development and clinical treatment. Tetramethylpyrazine exhibits an extremely low flavor threshold in food systems, significantly enhancing the flavor profile of baked goods, dairy products, meat products, and beverages. Its formation pathways primarily include the Maillard reaction and microbial metabolism. As the main active ingredient of Ligusticum chuanxiong, tetramethylpyrazine's pharmacological effects encompass multi-system regulation, such as cardiovascular protection, neuroprotection, anti-inflammation, and immune modulation.

[0003] 3-hydroxy-2-butanone, with the chemical formula C4H8O2, is an α-hydroxy ketone containing adjacent carbonyl (C=O) and hydroxyl (-OH) functional groups. At room temperature, it is a colorless to pale yellow liquid with a unique aroma similar to butter, cream, or yogurt; when diluted, it exhibits a nutty or caramel flavor. 3-hydroxy-2-butanone is an intermediate product of microbial glycolysis and a precursor to tetramethylpyrazine.

[0004] Bacillus cereus ( Bacillus cereus Bacillus is a spore-forming, Gram-positive bacterium. (Bacillus spp.) Bacillus It has strong environmental adaptability and can tolerate some adverse environments. There are currently no reports of tetramethylpyrazine-producing Bacillus cereus. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a tetramethylpyrazine-producing Bacillus cereus.

[0006] Another object of the present invention is to provide the application of the above-mentioned tetramethylpyrazine-producing Bacillus cereus.

[0007] The objective of this invention is achieved through the following technical solution: a tetramethylpyrazine-producing Bacillus cereus, named Bacillus cereus (… Bacillus cereusAccession number A1030, with accession number GDMCC No: 66145, was deposited on April 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The aforementioned tetramethylpyrazine-producing Bacillus cereus possesses the ability to produce protease, amylase, acetoin, and tetramethylpyrazine, and is resistant to ethanol and high temperatures.

[0009] The application of the above-mentioned tetramethylpyrazine-producing Bacillus cereus in the preparation of acetoin and / or tetramethylpyrazine.

[0010] The application of the above-mentioned tetramethylpyrazine-producing Bacillus cereus in the brewing of baijiu; preferably, its application in the koji-making stage of baijiu brewing.

[0011] A method for preparing acetoin and / or tetramethylpyrazine involves inoculating the above-mentioned tetramethylpyrazine-producing Bacillus cereus into a liquid culture medium for fermentation culture, wherein the supernatant of the culture medium contains acetoin and tetramethylpyrazine.

[0012] The above method for preparing acetoin and / or tetramethylpyrazine further includes the following step: separating and purifying the culture medium to obtain acetoin and / or tetramethylpyrazine.

[0013] The liquid culture medium is a culture medium suitable for the growth of tetramethylpyrazine-producing Bacillus cereus; its preferred composition is as follows: 80 g / L sucrose, 12 g / L peptone, 24 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 30 g / L diammonium hydrogen phosphate, pH 7.5, and distilled water as the solvent.

[0014] The preferred fermentation conditions are as follows: temperature 35-40℃, shaking speed 160-200 r / min, and culture time 2-7 days; more preferably, the conditions are as follows: temperature 37-38℃, shaking speed 180-200 r / min, and culture time 3-4 days.

[0015] The purification process is preferably carried out using liquid chromatography.

[0016] The present invention has the following advantages and effects compared with the prior art: (1) The Bacillus cereus provided by the present invention has strong resistance to high temperature and ethanol growth. This strain can grow normally in 20% ethanol without inhibition and can grow well at 50˚C.

[0017] (2) The Bacillus cereus protease and amylase provided by the present invention have an amylase clear zone HC value of 2.19 and a protease clear zone HC value of 1.7.

[0018] (3) The liquid fermentation of Bacillus cereus provided by the present invention can produce tetramethylpyrazine with a content of 0.8 g / L. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0020] Example 1 The strains isolated in this study were obtained from the high-temperature koji of Jiujiang Winery. The specific isolation and screening process was as follows: First, rice was steamed for 50 minutes, cooled, and then inoculated with high-temperature koji. It was then cultured at 30℃ for 24 hours, followed by a second culture at 40℃ for 24 hours. After the culture, sterile water and glass beads were added to the fermented mash, and the mixture was shaken for 1 hour to allow for the full release of microorganisms. After serial dilution, the mash was spread onto LB broth solid medium, and single colonies were isolated, yielding a total of 67 strains. Subsequently, 55 strains with relevant enzyme activities were screened using amylase and protease clear zone experiments. Further screening using solid-state wheat koji culture yielded 22 target strains. A VP test was then used for initial screening to identify 10 strains with strong acetoin production capabilities. Finally, a liquid fermentation method was used for secondary screening, confirming strain A1030 as the target functional strain.

[0021] The LB broth solid culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, and deionized water to a final volume of 1 L, with the pH adjusted to 7.0.

[0022] The 10 strains obtained from the initial screening were subjected to a tetramethylpyrazine production test, as follows: (1) The bacterial strains were inoculated into 20 mL of seed culture medium and cultured at 37˚C and 200 rpm for 12 h to obtain seed culture. The seed culture medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, prepared with distilled water and the pH was adjusted to 7.0.

[0023] (2) The seed culture was inoculated into the fermentation medium at a volume percentage of 5% and cultured at 37˚C and 200rpm for 72h to obtain the fermentation broth. The fermentation medium consisted of the following components: 80g / L sucrose, 12g / L peptone, 24g / L yeast extract, 3g / L dipotassium hydrogen phosphate, 30g / L diammonium hydrogen phosphate, pH 7.5, and prepared with distilled water.

[0024] (3) The fermentation broth was centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 µm filter membrane and then subjected to HPLC-PDA detection. The HPLC method conditions for the determination of tetramethylpyrazine were as follows: the instrument was a Waters 2695 high performance liquid chromatograph and a 2998 PDA detector; the chromatographic column was a Sunfire C18 reversed-phase column (250 × 4.6 mm, 5 µm); the mobile phase A was ultrapure water (containing 0.05% v / v, trifluoroacetic acid); the mobile phase B was methanol; the mobile phase volume ratio was 3:7; the column temperature was 45˚C; the flow rate was 1 mL / min; the detection wavelength was 190-400 nm; 294 nm was the extraction wavelength of tetramethylpyrazine; the injection volume was 10 µL; and the detection time was 8 min. A standard curve of tetramethylpyrazine was prepared by using tetramethylpyrazine standard, and the result was Y = 42717819*X + 60352, R 2 =0.9986. Using this tetramethylpyrazine standard curve, the tetramethylpyrazine content in the fermentation broth can be calculated.

[0025] Ten strains were screened again using liquid fermentation, and the content of tetramethylpyrazine (TTMP) was determined by HPLC. The results are shown in Table 1.

[0026] Table 1. Tetramethylpyrazine content produced by liquid fermentation of 10 bacterial strains .

[0027] Example 2 The culture medium for the protease clear zone assay was a protease qualitative solid medium, which consisted of 15.0 g / L skim milk powder, 20 g / L agar powder, and distilled water to a final volume of 1 L, pH 7.0, and was sterilized at 115˚C for 20 min.

[0028] The culture medium for the amylase clear zone assay was a qualitative solid medium for amylase, consisting of 10.0 g / L soluble starch, 5.0 g / L bacteriological peptone, 1.0 g / L yeast extract, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.5 g / L sodium chloride, 20.0 g / L agar powder, and distilled water to a final volume of 1 L. The medium was pH 7.0 and sterilized at 121˚C for 20 min.

[0029] Strains A1030 were inoculated onto protease and amylase qualitative media by spot inoculation and cultured at 37˚C for 48 h.

[0030] After the culture was completed, the diameter of the transparent zone D and the diameter of the bacterial strain d were recorded using a marker on the solid culture medium.

[0031] In this invention, strain A1030 has an amylase clear zone HC ratio of 2.19 and a protease clear zone HC ratio of 1.7.

[0032] Example 3 The culture medium used for the VP test was glucose-peptone water, which consisted of 5 g / L glucose, 5 g / L peptone, and 5 g / L disodium hydrogen phosphate, with a pH of 7.2, and was prepared with distilled water.

[0033] The colorimetric reagent A used in the VP test is 40% w / v potassium hydroxide solution; the colorimetric reagent B is 6% v / v naphthol ethanol solution.

[0034] The procedure was to culture strain A1030 in LB broth medium at 37˚C for 24 hours, and then inoculate it into VP test medium at an inoculation rate of 5% v / v and culture it at 37˚C for 48 hours.

[0035] Take 0.5 mL of the culture medium after 48 hours of incubation and place it in a 1.5 mL centrifuge tube. Add 0.2 mL of chromogenic reagent A and 0.5 mL of chromogenic reagent B, mix well, and let stand at room temperature for 5 minutes. Simultaneously, prepare a blank control by adding the chromogenic reagent to 0.5 mL of sterile LB broth after 48 hours of incubation. If the color turns red after standing, indicating a positive VP test, it means the strain has the ability to produce acetoin.

[0036] The results showed that strain A1030 was positive in the VP test, compared with the negative result of the blank control.

[0037] Example 4 (1) Determination of high temperature resistance: The seed culture of strain A1030 was inoculated into LB broth medium and cultured at 37℃, 40℃, 45℃ and 50℃ respectively, with a shaking speed of 200 rpm for 48 h. The OD was then measured. 600 Three parallels are set for each temperature.

[0038] The results are shown in Table 2. It can be seen that strain A1030 has a certain degree of heat resistance; at 37℃, the OD600 value is 1.678±0.023. As the temperature increases to 50℃, the OD600 value decreases. 600 The value dropped to 1.033±0.221, indicating that it grows best at 37℃ and can maintain a certain growth activity at higher temperatures such as 50℃.

[0039] Table 2. Heat resistance of Bacillus cereus A1030 .

[0040] (2) Determination of ethanol tolerance: LB broth medium containing different concentrations of ethanol was prepared, with ethanol concentrations of 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20% by volume. Seed culture of strain A1030 was inoculated into the above-mentioned LB broth medium containing different concentrations of ethanol and cultured at 37℃ and 200 rpm for 48 h. Its OD was then measured. 600 Three replicates were prepared for each concentration of LB broth containing ethanol.

[0041] The results are shown in Table 3. It can be seen that strain A1030 exhibits a certain degree of tolerance to ethanol. The OD600 value was highest at 0% ethanol concentration (1.678±0.087). With increasing ethanol concentration, the OD600 value generally fluctuated. Relatively high growth was maintained at concentrations between 11% and 17% (e.g., OD600 was 1.654±0.163 at 14% ethanol). However, growth was significantly inhibited at ethanol concentrations of 18% and above (e.g., OD600 was 1.002±0.051 at 18% ethanol). This indicates that strain A1030 can maintain growth within a certain ethanol concentration range, possessing a certain degree of ethanol tolerance, and can tolerate 20% ethanol.

[0042] Table 3. Ethanol resistance of Bacillus cereus A1030 .

[0043] Example 5 Strawberry strain A1030 was streaked onto LB broth plates and incubated at 37°C for 18–36 hours. Initially (after 18 hours), the colonies were milky white, round, and smooth with a glossy surface. Later (after 36 hours), the colonies became irregularly round, flat, and grayish-white, with larger colonies. Microscopic examination revealed short rod-shaped colonies that produced spores.

[0044] The culture media and experimental methods for physiological and biochemical tests were based on those in *Handbook of Systematic Identification of Common Bacteria*, edited by Dong Xiuzhu, Cai Miaoying, et al., Science Press, 2001, 1st edition, pp. 353-398. The test results indicated that this strain is a Gram-positive, aerobic, chemoheterotrophic bacterium.

[0045] Example 6 In this example, strain A1030 was identified, and the specific process is as follows: DNA was extracted from strain A1030 using a bioengineering kit, and PCR amplification was performed using universal primers (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R: 5'GGTTACCTTGTTACGACTT-3').

[0046] The PCR reaction system consisted of: 10 µL 2× Taq Mix, 2 µL DNA template, 1 µL primer 27F (24.1 nmol concentration), 1 µL primer 1492R (22.8 nmol concentration), and 6 µL ddH2O.

[0047] The PCR amplification program was as follows: pre-denaturation at 94˚C for 5 minutes; denaturation at 95˚C for 30 seconds, annealing at 56˚C for 1 minute, extension at 72˚C for 30 seconds, for 35 cycles; and finally extension at 72˚C for 7 minutes.

[0048] The sequencing was performed by Suzhou Genewiz. The PCR amplification products were sequenced. Generally, the sequence quality at both ends of the sequencing is poor. The low-quality sequences at both ends were removed by quality cutting. The quality-controlled paired-end sequencing results were then assembled to obtain the 16S rDNA sequence shown in SEQ ID NO.1.

[0049] The 16S rDNA sequence is as follows:

[0050] The assembled 16S rDNA sequence was compared with the NCBI database. Based on the coverage and similarity of the comparison results, the sample with the highest comparison score was selected, confirming the species as Bacillus cereus. Bacillus cereus A1030 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 14, 2025, with accession number GDMCC No: 66145.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A tetramethylpyrazine-producing Bacillus cereus, characterized in that: The name of the tetramethylpyrazine-producing Bacillus cereus is Bacillus cereus (Bacillus cereus). Bacillus cereus Accession number A1030, with accession number GDMCC No: 66145, was deposited on April 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The use of the tetramethylpyrazine-producing Bacillus cereus according to claim 1 in the preparation of acetoin and / or tetramethylpyrazine.

3. The application of the tetramethylpyrazine-producing Bacillus cereus as described in claim 1 in the brewing of Baijiu (Chinese liquor).

4. The application according to claim 3, characterized in that: The baijiu brewing process mentioned refers to the koji-making stage in baijiu brewing.

5. A method for preparing acetoin and / or tetramethylpyrazine, characterized in that: The tetramethylpyrazine-producing Bacillus cereus of claim 1 is inoculated into a liquid culture medium for fermentation culture, wherein the supernatant of the culture medium contains acetoin and / or tetramethylpyrazine.

6. The method according to claim 5, characterized in that... It also includes the following steps: separating and purifying the culture medium to obtain acetoin and / or tetramethylpyrazine.

7. The method according to claim 5 or 6, characterized in that: The liquid culture medium is composed of the following: 80 g / L sucrose, 12 g / L peptone, 24 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 30 g / L diammonium hydrogen phosphate, pH 7.5, and distilled water as the solvent.

8. The method according to claim 5 or 6, characterized in that: The fermentation conditions are as follows: temperature 35-40℃, shaking speed 160-200 r / min, and culture time 2-7 days.

9. The method according to claim 6, characterized in that: The purification process described herein is a preparation of liquid chromatography.