A Pediococcus pentosaceus strain producing multiple pyrazines and its application

Abstract

The present invention belongs to the field of microorganisms, and particularly relates to a strain of *Pediococcus pentosaceus* that can produce various pyrazines ( Pediococcus pentosaceus ) and its applications. This strain was isolated from Baijiu koji, and its preservation number is GDMCC NO: 65815. Tests have proven that this strain can produce special pyrazine compounds, such as 2-methylpyrazine, which is not produced in other strains. 2-Methylpyrazine has a typical aroma of nuts and baked foods and can be used in food processing fields such as winemaking and spices.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Pediococcus pentosaceus strain ( Pediococcus pentosaceus ) capable of producing multiple pyrazines and its applications. Background Art

[0002] Chinese Baijiu has a long history. It is mainly made from cereal grains, using daqu, xiaoqu or bran koji as saccharifying and fermenting agents, and adopting solid-state, semi-solid-state or liquid fermentation methods. It is made through processes such as steaming, saccharification, fermentation, distillation, aging, and blending. "Koji is the soul of liquor", and the aroma and various flavors in Baijiu mostly come from koji. Koji is made from raw materials such as rice, wheat, barley, and peas. It is enriched with various microorganisms through cultivation under certain temperature and humidity conditions, promoting the normal fermentation of fermented grains during the brewing process, providing necessary enzyme systems, functional bacterial systems, and flavor precursor substances for the fermentation process, and its quality directly affects the flavor and quality of Baijiu.

[0003] Pyrazine is a six-membered ring compound containing nitrogen at the 1,4 positions, with obvious caramel or roasted nut and roasted sesame aromas. It is widely used in the production processes of Baijiu, coffee, or tea. Pyrazine compounds in Baijiu have the characteristics of low threshold and high odor intensity, playing an important role in the formation of caramel aroma. They are important flavor substances in Baijiu and also important components of the volatile flavor substances in daqu. Currently, 29 kinds of pyrazine compounds have been detected in Chinese Baijiu, mainly alkyl pyrazines, including tetramethylpyrazine, trimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-methylpyrazine, 2,3-dimethylpyrazine, etc. In addition to being used as flavor components in the food industry, pyrazine substances also have various effects such as inhibiting tumors, dilating blood vessels, anti-inflammatory, antioxidant, and treating diabetes. Research shows that pyrazine substances in Baijiu are mainly produced by microorganisms, and most of the reported ones are Bacillus species (such as Bacillus licheniformis). Therefore, exploring strains that can produce multiple pyrazine substances is of great significance for enhancing the flavor during the production process of Baijiu. Summary of the Invention

[0004] A Pediococcus pentosaceus strain Q1 capable of producing multiple pyrazines was obtained through screening in the present invention. Through GC-MS detection and analysis, Q1 can produce 4 kinds of pyrazine compounds, including 2-methylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2,3,5,6-tetramethylpyrazine.

[0005] The present invention provides a Pediococcus pentosaceus strain ( Pediococcus pentosaceus ) capable of producing multiple pyrazines, characterized in that its preservation number is GDMCC NO: 65815.

[0006] The present invention also provides a method for culturing the Pediococcus pentosaceus as described above, characterized in that it is cultured using a TSB liquid medium, the culture temperature is 30 - 45 °C, the pH is 4 - 8, and the sodium chloride concentration in the medium is 0.25 - 4.5%.

[0007] Specifically, the culture temperature is 36 - 38 °C, the pH is 7 - 8, and the sodium chloride concentration in the medium is 0.5%.

[0008] More specifically, the formulation of the TSB liquid medium is: tryptone 15 g / L, soy peptone 5 g / L, sodium chloride 5 g / L.

[0009] The present invention also provides a culture of the Pediococcus pentosaceus obtained by the above-described culture method.

[0010] The present invention further provides the application of the Pediococcus pentosaceus in fermented wine-making or in the preparation of spice products.

[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, 2-ethyl-3,5,6-trimethylpyrazine.

[0012] The present invention also provides an additive for fermented wine-making, which is prepared from the Pediococcus pentosaceus as described above and is used to produce flavor substances in fermented wine-making. More specifically, it is prepared from the above-described culture.

[0013] Biological material preservation information: The strain Q1 of the present invention belongs to Pediococcus pentosaceus( Pediococcus pentosaceus ), and was deposited at the Guangdong Provincial Microbial Culture Collection Center (abbreviated as GDMCC) on January 16, 2025. The address of the deposit unit is: 5th Floor, Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; the deposit number is GDMCC NO.65815, and the taxonomic name is Pediococcus pentosaceus( Pediococcus pentosaceus ). Description of the Drawings

[0014] Figure 1 Colony morphology diagram of Pediococcus pentosaceus Q1.

[0015] Figure 2 Phylogenetic evolution tree of 16S rRNA of Pediococcus pentosaceus Q1 and related strains. Detailed Description of the Invention

[0016] The basic components of the culture media used in the examples are as follows:

[0017] TSA medium: Tryptone 15 g / L, Soytone 5 g / L, Sodium chloride 5 g / L, Agar 15 g / L.

[0018] TSB liquid medium: Tryptone 15 g / L, Soytone 5 g / L, Sodium chloride 5 g / L.

[0019] 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.

[0020] 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.

[0021] Example 1: Isolation of Strain Q1

[0022] The Daqu was from a distillery in Sichuan. The Daqu was placed in physiological saline (0.9% NaCl), shaken at room temperature for 30 min, heated in a water bath at 85 °C for 30 min, and then the supernatant was taken and added to the enrichment medium, and shaken for enrichment treatment at 37 °C for 24 h. The enriched solution was spread on the TSA medium by gradient coating, and cultured at 37 °C for 1 - 2 d, and a total of 89 strains were isolated. The isolated strains were purified by streaking. The purified strains were respectively inoculated into the fermentation medium and cultured at 37 °C for 12 h to obtain the strain seed liquid. The seed liquid was inoculated into the fermentation medium with a loading volume of 5 mL / 45 mL according to an inoculation amount of 4% and cultured at 37 °C for 36 h to obtain the strain fermentation liquid. Take 1 mL of the strain fermentation liquid, add V-P (Voges-Proskauer) reagent, shake well, let stand for 5 min, observe the color change, and select the strains whose color turns red after the reaction during the screening process. A total of 30 strains were screened. Among them, Strain Q1 was one of them. Strain Q1 was preserved by test tube slant preservation and glycerol cryopreservation.

[0023] Example 2: Identification of Strain Q1

[0024] 1. Morphological identification

[0025] Inoculate Strain Q1 into the TSA medium and observe the colony morphological characteristics after culturing at 37 °C for 48 h ( Figure 1 ). It was observed that the Q1 colony was round, milky white, smooth and opaque, with neat edges, and was a Gram-positive bacterium.

[0026] 2. Physiological characteristic identification

[0027] The Q1 strain was inoculated into TSB liquid medium to study the tolerance of the Q1 strain to temperature, pH, and salt concentration, as well as the optimal culture conditions.

[0028] Growth temperature range: Q1 was inoculated into TSB medium and cultured at 15, 20, 30, 37, 45, and 50 °C for 48 h, and the OD 600 absorbance value was detected by a spectrophotometer to analyze the growth range of Q1.

[0029] Growth pH range: Q1 was inoculated into TSB medium, and the pH value was adjusted to 3, 4, 5, 6, 7, 8, 9, and cultured at 37 °C for 48 h. The OD 600 absorbance value was detected by a spectrophotometer to analyze the pH growth range of Q1.

[0030] NaCl tolerance range: Q1 was inoculated into TSB medium, and the NaCl concentration was adjusted to (% w / v) 0.5, 2.5, 4.5, 6.5, 8.5, 9.5, 10.5, 11.5, 12.5, and cultured at 37 °C for 48 h. The OD 600 absorbance value was detected by a spectrophotometer to analyze the NaCl tolerance range of Q1.

[0031] The research shows that the Q1 strain can grow at 15 - 45 °C, and the optimal growth temperature is 37 °C. The Q1 strain can grow at a pH value of 4 - 8, and the optimal growth pH is 7. The Q1 strain can grow at an NaCl concentration of 0.5 - 4.5%, and the optimal NaCl concentration is 0.5%.

[0032] 3. Molecular biology identification

[0033] After the Q1 strain was inoculated into TSB liquid medium and cultured at 37 °C for 48 h, the cells were collected. The genomic DNA of the Q1 strain was extracted using a bacterial genomic DNA extraction kit. The 16S rRNA sequence of the Q1 strain was amplified using the universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACGGCTACCTTGTTACGACTT-3') for bacterial 16S rRNA gene. The PCR amplification program was: pre-denaturation at 94 °C for 3 min, 30 cycles including denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min. Finally, extension at 72 °C for 10 min. After amplification, the PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0034] The obtained 16S rRNA sequence of the Q1 strain (SEQ ID NO: 1):

[0035]

[0036] The sequences obtained by sequencing were aligned on NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that strain Q1 had the closest genetic relationship with Pediococcus pentosaceus, with a similarity of 99.59%. Based on sequence similarity, strain Q1 and its closely related strains were selected to construct a phylogenetic tree, and the results showed that strain Q1 had the closest genetic relationship with Pediococcus pentosaceus ( Figure 2 ).

[0037] Combined with the results of morphological identification, physiological and biochemical identification, and molecular biological identification, Q1 was identified as Pediococcus pentosaceus( Pediococcus pentosaceus ). And this strain was deposited in Guangdong Microbial Culture Collection Center on January 16, 2025, with the deposit number GDMCC NO:65815.

[0038] Example 3: Analysis of pyrazine substances produced by strain Q1

[0039] Strain Q1 was inoculated into the fermentation medium and cultured with shaking at 37 °C for 48 h. 5.0 mL of the mixed sample was taken into a 20 mL headspace vial, 2.0 g of NaCl was added, the internal standard 2-octanol was added, and the sample vial was tightened. After the extraction head was aged at 250 °C for 30 min, it was inserted into the sample vial, equilibrated at 40 °C for 30 min, extracted for 40 min, and the extraction head was desorbed at 250 °C in the GC injection port for 3 min.

[0040] Chromatographic conditions: The chromatographic column was an HP-WAX elastic quartz fiber capillary column (30 m×0.25 mm, 0.25 μm); the temperature programming: hold at 40 °C for 3 min, increase to 150 °C at a rate of 4 °C / min, hold for 3 min, increase to 240 °C at a rate of 9 °C / min, hold for 9 min; the carrier gas (He, 99.999%) flow rate was 1.0 mL / min, and the injection was splitless.

[0041] Mass spectrometry conditions: Electron impact ion source; electron impact energy 70 eV; transfer line temperature 230 °C; quadrupole temperature 150 °C; ion source temperature 230 °C; mass scanning range m / z 35-600.

[0042] Through GC-MS analysis, it was found that strain Q1 could produce many flavor substances, including various pyrazine compounds (Table 1).

[0043] Table 1 Statistical table of pyrazine substances of Pediococcus pentosaceus Q1

[0044]

[0045] Among them, the production of 2-methylpyrazine has not been reported in other strains of Pediococcus pentosaceus, and 2-methylpyrazine has a typical aroma of nuts and baked foods and can be used in food processing fields such as winemaking and spices.

Claims

1. Pediococcus pentosaceus producing multiple pyrazines( Pediococcus pentosaceus) , characterized in that Its preservation number is GDMCC NO: 65815.

2. The culturing method of Pediococcus pentosaceus according to claim 1, characterized in that, It is cultured using TSB liquid medium, the culture temperature is 30 - 45 °C, the pH is 4 - 8, and the sodium chloride concentration in the medium is 0.25 - 4.5%.

3. The culturing method of Pediococcus pentosaceus according to claim 2, characterized in that, The culture temperature is 36 - 38 °C, the pH is 7 - 8, and the sodium chloride concentration in the medium is 0.5%.

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

5. The culture of Pediococcus pentosaceus obtained by the culture method according to any one of claims 2 to 4, characterized in that it contains Pediococcus pentosaceus as described in claim 1.

6. The application of Pediococcus pentosaceus as described in claim 1 in fermenting wine-making.

7. The application according to claim 6, characterized in that, It is used to produce liquor flavor substances, and the liquor flavor substances are ethyl acetate, 2 - methylpyrazine, acetoin, 2,6 - dimethylpyrazine, 2,3,5 - trimethylpyrazine and 2,3,5,6 - tetramethylpyrazine.

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