Bacillus cereus for producing tetramethylpyrazine and application thereof

Abstract

The invention discloses a bacillus cereus strain for producing tetramethylpyrazine and application of the bacillus cereus strain. The name of the bacillus cereus is bacillus cereus A1030, the preservation number of the bacillus cereus is GDMCC No: 66145, and the bacillus cereus is preserved in Guangdong Microbial Culture Collection Center on April 14, 2025. The bacillus cereus has very strong high temperature (50C) resistant and ethanol (20%) resistant growth capability; the bacillus cereus produces protease and amylase, the HC value of the amylase transparent circle is 2.19, and the HC value of the protease transparent circle is 1.7; the liquid state fermentation of the bacillus cereus can produce tetramethylpyrazine, and the content of tetramethylpyrazine is 0.8 g / L.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Bacillus cereus strain producing tetramethylpyrazine and its application. Background Art

[0002] Tetramethylpyrazine (2,3,5,6 - tetramethylpyrazine, abbreviated as TTMP) is a heterocyclic nitrogen - containing compound with significant biological activity and unique flavor. Its chemical structure contains two nitrogen atoms and four methyl substituents, with the molecular formula C8H 12 N2, melting point of 82 - 86 °C, boiling point of 190 °C, presenting as white crystals or powder at room temperature, having a compound aroma of beef heating, nut baking and fermented soybean, and showing milk chocolate flavor after dilution. This compound widely exists in natural products and industrial fields. It is not only an important flavor enhancer in the food industry but also the core active ingredient of traditional Chinese medicine Ligusticum chuanxiong, with dual values in functional food development and clinical treatment. Tetramethylpyrazine shows a very low flavor threshold in food systems and can significantly improve the flavor levels of baked foods, dairy products, meat products, and beverages. Its formation pathways mainly include the Maillard reaction and microbial metabolism. As the main active ingredient of Ligusticum chuanxiong, the pharmacological effects of tetramethylpyrazine cover multi - system regulation, such as cardiovascular protection, neuroprotection, anti - inflammation, and immune regulation, etc.

[0003] Acetoin (3 - hydroxy - 2 - butanone), with the chemical formula C4H8O2, is an α - hydroxy ketone, containing adjacent carbonyl (C = O) and hydroxyl (-OH) functional groups in the molecule. It is a colorless to light yellow liquid at room temperature, having a unique aroma similar to butter, cream or yogurt, and showing nut or caramel flavor after dilution. Acetoin is an intermediate product of microbial glycolysis and also a precursor substance of tetramethylpyrazine.

[0004] Bacillus cereus ( Bacillus cereus ) is a spore - forming Gram - positive bacterium. The genus Bacillus ( Bacillus ) has strong environmental adaptability and can tolerate some adverse environments. There is currently no report on Bacillus cereus strains producing tetramethylpyrazine. Summary of the Invention

[0005] The primary objective of the present invention is to overcome the drawbacks and deficiencies of the prior art and provide a Bacillus cereus strain producing tetramethylpyrazine.

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

[0007] The objective of the present invention is achieved by the following technical solutions: A Bacillus cereus strain producing tetramethylpyrazine, named Bacillus cereus ( Bacillus cereusA1030, with the preservation number of GDMCC No: 66145, was preserved on April 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, 100 Xianlie Middle Road, Guangzhou.

[0008] The above-mentioned Bacillus cereus producing tetramethylpyrazine has the ability to produce protease, amylase, acetoin, and tetramethylpyrazine, and can tolerate ethanol and high temperatures.

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

[0010] Use of the above-mentioned Bacillus cereus producing tetramethylpyrazine in Baijiu brewing; preferably in the koji-making stage of Baijiu brewing.

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

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

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

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

[0015] The purification is preferably preparative 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 growth abilities at high temperatures and in the presence of ethanol. This strain can grow normally in 20% ethanol without inhibition and can grow well at a high temperature of 50 °C.

[0017] (2) The Bacillus cereus provided by the present invention produces protease and amylase. The HC value of the amylase clear zone is 2.19, and the HC value of the protease clear zone is 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 manners

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

[0020] Example 1 The strain isolation material for this study was taken from the high-temperature koji of Jiujiang Winery. The specific isolation and screening process is as follows: First, steam the rice in a rice cooker for 50 minutes, cool it, inoculate it with high-temperature koji, and culture it at 30 °C for 24 hours, then transfer it to 40 °C for 24 hours. After the culture is completed, take the fermented grains, add sterile water and glass beads, shake for 1 hour to fully release the microorganisms, and coat them on the solid LB broth medium after gradient dilution to obtain single colonies. A total of 67 strains were obtained. Subsequently, 55 strains with relevant enzyme activities were screened out through the amylase and protease transparent circle experiments, and 22 target strains were further screened out through solid-state wheat koji culture. Then, 10 strains with stronger acetoin-producing ability were initially screened through the V-P test, and finally, the strain A1030 was determined as the target functional strain through re-screening by the liquid fermentation method.

[0021] The composition of the solid LB broth medium is 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 20 g / L of agar powder, made up to 1 L with deionized water, and the pH value is adjusted to 7.0.

[0022] The 10 strains preliminarily screened were tested for tetramethylpyrazine production, and the steps are as follows: (1)Inoculate the strains into 20 mL of seed medium respectively, and culture them in a shaker at 37 °C and 200 rpm for 12 h to obtain seed liquid. The composition of the seed medium is 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, prepared with distilled water, and the pH value is adjusted to 7.0.

[0023] (2)Inoculate the seed liquid into the fermentation medium at a volume percentage of 5%, and culture it in a shaker at 37 °C and 200 rpm for 72 h to obtain the fermentation broth. Among them, the composition of the fermentation medium is as follows: 80 g / L of sucrose, 12 g / L of peptone, 24 g / L of yeast powder, 3 g / L of dipotassium hydrogen phosphate, 30 g / L of diammonium hydrogen phosphate, pH 7.5, prepared with distilled water.

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

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

[0026] Table 1 Content of tetramethylpyrazine produced by liquid fermentation of 10 strains .

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

[0028] The medium for the amylase clear zone experiment was an amylase qualitative solid medium, which consisted of 10.0 g / L soluble starch, 5.0 g / L bacteriological peptone, 1.0 g / L yeast powder, 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, made up to 1 L with distilled water, pH = 7.0, and sterilized at 121˚C for 20 min.

[0029] Strain A1030 was inoculated onto the protease qualitative medium and the amylase qualitative medium in the form of spotting, and cultured at 37˚C for 48 h.

[0030] After the cultivation, the diameter D of the clear zone and the diameter d of the strain on the solid medium were recorded with a marker pen.

[0031] In the present invention, the HC ratio of the amylase clear zone of strain A1030 was 2.19, and the HC ratio of the protease clear zone was 1.7.

[0032] Example 3 The medium used for the V-P test is glucose peptone water, and its composition is 5 g / L glucose, 5 g / L peptone, 5 g / L disodium hydrogen phosphate, pH 7.2, prepared with distilled water.

[0033] The developer solution A for the V-P test: 40% w / v potassium hydroxide solution; the developer solution B: 6% v / v naphthol ethanol solution.

[0034] The implementation steps are to culture strain A1030 in LB broth medium at 37˚C for 24 h, inoculate it into the V-P test medium at an inoculation amount of 5% v / v, and culture it at 37˚C for 48 h.

[0035] Take 0.5 mL of the culture solution cultured for 48 h above into a 1.5 mL centrifuge tube, add 0.2 mL of the developer solution A and 0.5 mL of the developer solution B, mix well, and let it stand at room temperature for 5 min. At the same time, make a blank control, take 0.5 mL of sterile LB broth cultured for 48 h, and add the developer as a negative control. If the color turns red after standing, that is, the V-P test is positive, indicating that the strain has the ability to produce acetoin.

[0036] The results show that the V-P test result of strain A1030 is positive, compared with the negative of the blank control.

[0037] Example 4 (1) Determination of high temperature resistance: Inoculate the seed solution of strain A1030 into LB broth medium, and culture it at 37°C, 40°C, 45°C and 50°C respectively, with the shaker speed of 200 rpm, culture for 48 h, and measure its OD 600 . Set three parallels for each temperature.

[0038] The results are shown in Table 2. It can be seen that strain A1030 has a certain high temperature resistance. At 37°C, the OD 600 value is 1.678±0.023. As the temperature rises to 50°C, the OD 600 value drops to 1.033±0.221, indicating that its growth condition is the best at 37°C and it can maintain a certain growth activity at higher temperatures such as 50°C.

[0039] Table 2 High temperature resistance of Bacillus cereus A1030 .

[0040] (2)Determination of ethanol tolerance: Prepare LB broth media containing different concentrations of ethanol, with the ethanol concentrations being 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20% by volume percentage. Inoculate the seed solution of strain A1030 into the above-mentioned LB broth media containing different concentrations of ethanol, and culture it in a shaker at 37 °C and 200 rpm for 48 h, and measure its OD 600 . Set three parallels for each concentration of LB broth media containing ethanol.

[0041] The measurement results are shown in Table 3. It can be seen that strain A1030 has a certain tolerance to ethanol. When the ethanol concentration is 0%, the OD600 value is the highest (1.678 ± 0.087). As the ethanol concentration increases, the OD600 value generally shows a fluctuating change. It can still maintain a relatively high growth rate at some concentrations from 11% to 17% (for example, when the ethanol concentration is 14%, OD600 is 1.654 ± 0.163). However, when the ethanol concentration reaches 18% and above (for example, when the ethanol concentration is 18%, OD600 is 1.002 ± 0.051), the growth is significantly inhibited. This indicates that strain A1030 can maintain growth within a certain ethanol concentration range, has certain ethanol tolerance characteristics, and can tolerate 20% ethanol.

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

[0043] Example 5 Streak strain A1030 on an LB broth media plate and culture it at 37 °C for 18 - 36 h. The colony morphology in the initial stage (the initial stage is 18 h of culture) is milky white, round, with a smooth and shiny surface. In the later stage of culture (the later stage is after 36 h of culture), the colonies are irregularly round, with a flat surface, grayish white, and relatively large colonies. Microscopic examination shows short rod-shaped and spore-forming.

[0044] For the physiological and biochemical test media and test methods, refer to "Common Bacterial Systematic Identification Manual" edited by Dong Xiuzhu, Cai Miaoying, etc., Science Press, 2001, the first edition, p353 - 398. The detection results show 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: Use a Sangon kit to extract DNA from strain A1030, and perform PCR amplification using universal primers (upstream primer 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’; downstream primer 1492R: 5’GGTTACCTTGTTACGACTT-3’).

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

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

[0048] The sequencing work was completed by Genewiz (Suzhou) Inc. The products after PCR amplification were sequenced. Generally, the sequence quality at both ends of the sequencing is poor. The low-quality sequences at both ends of the sequencing were removed by quality trimming, and the double-ended sequencing results after quality control were assembled. The assembled 16S rDNA sequence is shown as SEQ ID NO.1.

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

[0050] The assembled 16S rDNA sequences were compared with the NCBI database, and based on the coverage and similarity of the comparison results, the one with the highest comparison score was selected to determine that the species of the sample was *Bacillus cereus*. *Bacillus cereus* A1030 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on April 14, 2025, with the deposit 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 by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

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

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

3. Use of the Bacillus cereus producing tetramethylpyrazine according to claim 1 in the brewing of Chinese liquor.

4. The application according to claim 3, wherein: The brewing of Chinese liquor mentioned refers to the koji-making stage in the brewing of Chinese liquor.

5. A method for preparing acetoin and / or tetramethylpyrazine, characterized in that: Inoculate the Bacillus cereus producing tetramethylpyrazine according to claim 1 into a liquid medium for fermentation culture, and the supernatant of the culture solution contains acetoin and tetramethylpyrazine.

6. The method according to claim 5, wherein It further includes the following steps: separating and purifying the culture solution to obtain acetoin and / or tetramethylpyrazine.

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

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

9. The method according to claim 6, wherein: The purification is preparative liquid chromatography.