Pediococcus acidilactici capable of producing various pyrazines and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly relates to pediococcus acidilactici capable of producing various pyrazines and application of the pediococcus acidilactici. The strain is separated from distiller's yeast of Baijiu, and the preservation number of the strain is GDMCC NO: 65814. Volatile flavor substances in fermentation liquid of the strain are detected through GC-MS, it is found that more than ten kinds of pyrazine compounds can be generated, and compared with existing strains, more kinds of pyrazine compounds can be generated. Therefore, the strain disclosed by the invention has a relatively high application value and can be used for fermenting and brewing wine.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and specifically relates to a strain of Pediococcus acidilactici ( Pediococcus acidilactici ) and its applications. Background Art

[0002] Pyrazine is a six-membered ring compound containing nitrogen at the 1,4 positions, with a distinct aroma of burnt or roasted nuts or sesame. Common microorganisms that can produce pyrazines are mainly bacteria, including Bacillus and Mycobacterium. As a class of aromatic compounds, pyrazines have very important application value in the food industry. Because of their flavors similar to roasted meat, roasted peanuts, toasted bread, and cocoa aroma, pyrazines are widely used in the production of liquor, coffee, or tea. In addition to being used as flavor ingredients in the food industry, pyrazines also have multiple functions such as inhibiting tumors, dilating blood vessels, anti-inflammatory, anti-oxidation, and treating diabetes. Studies have shown that 2-methylpyrazine has antioxidant, triglyceride-decomposing and α-glucosidase inhibitory effects, which is of great significance for improving hyperglycemia and providing energy to the body; tetramethylpyrazine can be applied to the central nervous system to improve learning disabilities; in addition, trimethylpyrazine (TMP) also has very important applications in the medical field, with antioxidant, anti-inflammatory and nerve protection effects, and plays a very important role in the treatment of ischemic stroke, Alzheimer's disease, spinal cord injury and other aspects.

[0003] Chinese liquor has a long history. It is made of cereals as the main raw materials, with Daqu, Xiaoqu or bran qu as saccharification and fermentation agents, using solid, semi-solid or liquid fermentation methods, and through cooking, saccharification, fermentation, distillation, aging, blending and other processes. "Qu is the soul of liquor", the fragrance and various flavors in liquor mostly come from qu. Pyrazine compounds in liquor have the characteristics of low threshold and high odor intensity, which play an important role in the formation of caramel aroma. They are important flavor substances in liquor and important components of volatile aroma substances in qu. At present, 29 pyrazine compounds have been detected in Chinese liquor, mainly alkyl pyrazines, including tetramethylpyrazine, trimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-methylpyrazine, 2,3-dimethylpyrazine, etc. Therefore, it is of great significance to the liquor production industry to explore strains that can produce a variety of pyrazine substances. Summary of the invention

[0004] The present invention obtains a strain of Pediococcus acidilactici Q2 that can produce multiple pyrazines through screening. Through GC-MS detection and analysis, Q2 can produce more than ten pyrazine compounds, including pyrazine, 2-methylpyrazine, 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, 2-methyl-5-isopropylpyrazine, 2-vinylpyrazine, 3-ethyl-2,5-methylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 3,5-diethyl-2-methyl-pyrazine, and 2-ethyl-3,5,6-trimethylpyrazine. It has been reported that 4-methylpyrazine can be produced in Pediococcus acidilactici, and other pyrazine substances have not been reported, so it has great application value in brewing or other food industries.

[0005] Therefore, the present invention provides a strain of Pediococcus acidilactici that produces multiple pyrazines ( Pediococcus acidilactici ), its deposit number is GDMCC NO:65814.

[0006] The present invention also provides a method for culturing the Pediococcus acidilactici, characterized in that it adopts TSB liquid culture medium for culturing, the culture temperature is 30-45° C., the pH is 4-8, and the sodium chloride concentration in the culture medium is 0.3-0.8%.

[0007] Preferably, the culture temperature is 36-38°C, the pH is 4-6, and the sodium chloride concentration in the culture medium is 0-0.4%.

[0008] Specifically, the formula of TSB liquid culture medium is: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L.

[0009] The present invention provides a culture of the Pediococcus acidilactici obtained by the culture method.

[0010] The present invention further provides application of the Pediococcus acidilactici in fermentation and winemaking.

[0011] Specifically, it is used to produce liquor flavor substances, and the liquor flavor substances are selected from pyrazine, 2-methylpyrazine, 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, 2-methyl-5-isopropylpyrazine, 2-vinylpyrazine, 3-ethyl-2,5-methylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 3,5-diethyl-2-methyl-pyrazine, and 2-ethyl-3,5,6-trimethylpyrazine.

[0012] The invention provides an additive for fermentation and winemaking, which is prepared from the Pediococcus acidilactici and is used for producing flavor substances in fermentation and winemaking.

[0013] Specifically, it is prepared by the culture.

[0014] Biomaterial deposit information: The strain Q2 of the present invention belongs to Pediococcus acidilactici ( Pediococcus acidilactici ), deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on January 16, 2025, the address of the depository unit is: 5th Floor, Dayuan Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the deposit number is GDMCCNO.65814, and the classification name is Pediococcus acidilactici ( Pediococcus acidilactici ). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Colony morphology of Pediococcus acidilactici Q2.

[0016] Figure 2 16S rRNA phylogenetic tree of Pediococcus acidilactici Q2 and its similar species. DETAILED DESCRIPTION

[0017] The present invention is further described below through specific examples in order to provide a better understanding of the present invention, but the present invention is not limited thereto.

[0018] Among them, the basic components of the culture medium used in the embodiment are as follows: TSA medium: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L, agar 15 g / L.

[0019] TSB fluid medium: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L.

[0020] Enrichment medium: yeast extract 3 g / L, peptone 10 g / L, starch 3 g / L, MgSO4·7H2O 0.01 g / L, KH2PO4 0.2 g / L, Na2HPO4 2 g / L.

[0021] Fermentation medium: peptone 20 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate 3 g / L, glucose 100 g / L, diammonium hydrogen phosphate 30 g / L.

[0022] Example 1: Isolation of Q2 strain The source of the wine koji used in the example is a winery in Sichuan. Weigh 10 g of Daqu and place it in 90 ml of physiological saline (0.9% NaCl) and shake it at room temperature at 180 r / min for 30 min. Heat it in a water bath at 85 °C for 30 min, take 5.00 mL of the supernatant and add it to 95 mL of enrichment medium, and shake it at 37 °C and 200 r / min for 24 h. Take the enrichment solution for gradient dilution, spread it on a TSA plate, and culture it in a 37 °C incubator for 1 to 2 days. Pick single colonies of different morphologies on the plate for separation and purification, and a total of 89 colonies are isolated. Purify it three times by three-zone line to obtain purified cultured bacteria. The Q2 strain obtained by the above separation and purification is stored by (1) TSA plate / test tube slant; (2) after mixing the Q2 bacterial suspension with 50% glycerol in a ratio of 1:1, it is placed in liquid nitrogen for quick freezing, and then placed in a -80 °C refrigerator for cryopreservation.

[0023] Example 2: Identification of Q2 strain The species identification of Q2 strain was mainly carried out through morphology and molecular biology.

[0024] 1. Morphological characteristics The Q2 strain was inoculated into TSA medium and the colony morphology was observed after 48 h of culture. Figure 1 ). It was observed that the Q2 strain was round, milky white, with neat edges, and was a Gram-positive bacterium.

[0025] 2. Molecular Biology Identification The purified Q2 strain single colony was inoculated into TSB liquid medium and cultured at 37°C for 48h. The bacterial broth was cultured using the Tiangen Bacterial Genome Kit according to the instructions for bacterial genome extraction. The whole genome DNA was used as the PCR template, 2×Taq PCR Mix product, 25μL PCR reaction system, and universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene sequence. The PCR amplification program was 94°C pre-denaturation for 3min, 30 cycles including 94°C denaturation for 30s, 55°C annealing for 30s, and 72°C extension for 1min. Finally, 72 degrees extension for 10min. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0026] The obtained 16S rRNA sequence of the Q2 strain is as follows (SEQ ID NO: 1):

[0027] Then, the Q2 16S rRNA gene sequence obtained by sequencing was used for NCBI Blast search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the comparison results showed that Q2 was similar to Pediococcus acidilactici ( Pediococcus acidilactici ) had the highest similarity of 99.39%. In order to further clarify the taxonomic status of Q2, strains with close genetic relationship with Q2 16S rRNA sequence were selected to construct phylogenetic tree and perform phylogenetic analysis. Figure 2 As shown, Q2 is most closely related to Pediococcus acidilactici ( Figure 2 ).

[0028] Combining morphological and molecular biological characteristics, Q2 was preliminarily identified as Pediococcus acidilactici. The strain was submitted to the Guangdong Microbiological Culture Collection Center for preservation on January 16, 2025, with the preservation number: GDMCC NO: 65814.

[0029] 3. Study on biological characteristics of Q2 Growth temperature range: Q2 was inoculated into TSB medium and cultured at 15, 20, 30, 37, 45 and 50°C for 48 h, and OD was measured by spectrophotometer. 600 Absorbance value, analyze the growth range of Q2.

[0030] Growth pH range: Inoculate Q2 into TSB medium, adjust pH to 3, 4, 5, 6, 7, 8, 9, culture at 37°C for 48 h, and measure OD by spectrophotometer 600 The absorbance values ​​were used to analyze the pH growth range of Q2.

[0031] NaCl tolerance range: Inoculate Q2 into TSB medium, adjust the NaCl concentration to (%, w / v) 0, 2, 4, 6, 8, 9, 10, 11, 12, culture at 37°C for 48 h, and measure OD by spectrophotometer. 600 Absorbance value, analyze the NaCl tolerance range of Q2.

[0032] The results showed that the Q2 strain could grow at 30-45°C, with the optimum growth temperature being 37°C. The Q2 strain could grow at a pH value of 4-8, with the optimum growth pH being 5. The Q2 strain could grow at a NaCl concentration of 0-4%, with the optimum NaCl concentration being 0%.

[0033] Example 3: Analysis of flavor substances produced by Q2 strain Q2 was inoculated into the fermentation medium and cultured at 37 °C and 200 r / min for 48 h to obtain the fermentation broth of the strain. 5.0 mL of the culture broth was placed in a 20 mL headspace bottle, 2.0 g of NaCl was added, 20.0 μL of 99.07 mg / L internal standard 2-octanol was added, and the sample bottle was screwed tight. The extraction head was aged at 250 °C for 30 min and then inserted into the sample bottle, equilibrated at 40 °C for 30 min, extracted for 40 min, and the extraction head was desorbed at 250 °C for 3 min at the GC injection port.

[0034] Chromatographic conditions: The chromatographic column was an HP-WAX elastic quartz fiber capillary column (30 m×0.25 mm, 0.25 μm); the temperature program was: 40 °C for 3 min, then increased to 150 °C at 4 °C / min, then maintained for 3 min, then increased to 240 °C at 9 °C / min, then maintained for 9 min; the carrier gas (He, 99.999%) flow rate was 1.0 mL / min, and the sample was injected without splitting. Mass spectrometry conditions: electron bombardment ion source; electron bombardment energy was 70 eV; transfer line temperature was 230 °C; quadrupole temperature was 150 °C; ion source temperature was 230 °C; mass scanning range was m / z 35-600.

[0035] After GC-MS analysis, it was found that the Q2 strain could produce more than ten pyrazine compounds (Table 1).

[0036] Table 1 Pyrazine flavor substances produced during the fermentation of Pediococcus acidilactici

[0037] These pyrazine compounds are important flavor substances in liquor. It has been reported that 4-methylpyrazine can be produced in Pediococcus acidilactici, and other pyrazine substances have not been reported. Pediococcus acidilactici Q2 in the present invention can produce more types of pyrazine substances. Therefore, the Pediococcus acidilactici screened in the present invention has great application value in winemaking or other food industries.

Claims

1. A strain of Pediococcus acidilactici producing multiple pyrazines ( Pediococcus acidilactici ), characterized in that Its deposit number is GDMCC NO:65814.

2. The method for culturing Pediococcus acidilactici according to claim 1, characterized in that: It is cultured in TSB liquid culture medium, with a culture temperature of 30-45°C, a pH of 4-8, and a sodium chloride concentration of 0.3-0.8% in the culture medium.

3. The method for culturing Pediococcus acidilactici according to claim 2, characterized in that: The culture temperature is 36-38°C, the pH is 4-6, and the sodium chloride concentration in the culture medium is 0-0.4%.

4. The method for culturing Pediococcus acidilactici according to claim 2, characterized in that: The formula of TSB liquid culture medium is: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L.

5. The culture of Pediococcus acidilactici obtained by the culturing method according to any one of claims 2 to 4.

6. Use of Pediococcus acidilactici as claimed in claim 1 in fermentation and winemaking.

7. The use according to claim 6, characterized in that The invention is used for producing liquor flavor substances, wherein the liquor flavor substances are selected from pyrazine, 2-methylpyrazine, 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, 2-methyl-5-isopropylpyrazine, 2-vinylpyrazine, 3-ethyl-2,5-methylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 3,5-diethyl-2-methyl-pyrazine and 2-ethyl-3,5,6-trimethylpyrazine.

8. An additive for fermentation and winemaking, which is prepared from the Pediococcus acidilactici as claimed in claim 1 and is used to produce flavor substances in fermentation and winemaking.

9. The additive according to claim 7, characterized in that It is prepared by the culture according to claim 4.

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