Pediococcus pentosaceus capable of producing various pyrazines and application of Pediococcus pentosaceus

Abstract

The invention belongs to the field of microorganisms, and particularly relates to pediococcus pentosaceus capable of producing various pyrazines and application of the pediococcus pentosaceus. The strain is separated from distiller's yeast of Baijiu, and the preservation number of the strain is GDMCC NO: 65815. Tests prove that the strain can generate special pyrazine compounds, such as 2-methylpyrazine, other strains do not generate the special pyrazine compounds, and the 2-methylpyrazine has typical nut and baked food fragrance and can be used in the field of food processing of wine brewing, spices and the like.

Description

Technical Field

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

[0002] Chinese liquor has a long history. It is made from cereals as the main raw materials, with Daqu, Xiaoqu or Fuqu as saccharification and fermentation agents, using solid, semi-solid or liquid fermentation methods, and through processes such as cooking, saccharification, fermentation, distillation, aging, and blending. "Qu is the soul of liquor", and the aroma and various flavors of liquor mostly come from qu. Qu is made from rice, wheat, barley, peas and other raw materials, and is cultivated and enriched with various microorganisms under a certain temperature and humidity environment. It promotes the normal fermentation of distiller's grains during the brewing process, and provides the necessary enzyme system, functional bacteria system and flavor precursor substances for the fermentation process. Its quality directly affects the flavor and quality of liquor.

[0003] Pyrazine is a six-membered ring compound containing nitrogen at the 1,4 positions. It has a distinct aroma of burnt aroma or roasted nuts or sesame seeds and is widely used in the production of liquor, coffee, or tea. Pyrazine compounds in liquor have the characteristics of low threshold and high odor intensity. They play an important role in the formation of burnt aroma. They are important flavor substances in liquor and an important component of volatile aroma substances in Daqu. 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. 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, antioxidant, and treating diabetes. Studies have shown that pyrazines in liquor are mainly produced by microorganisms, most of which are reported to be Bacillus (such as Bacillus licheniformis). Therefore, discovering strains that can produce a variety of pyrazine substances is of great significance for enhancing the flavor of liquor during production. Summary of the invention

[0004] The present invention obtains a strain of Pediococcus pentosaceus Q1 which can produce multiple pyrazines through screening. Through GC-MS detection and analysis, Q1 can produce four pyrazine compounds, including 2-methylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine and 2,3,5,6-tetramethylpyrazine.

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

[0006] The present invention also provides a method for culturing the Pediococcus pentosaceus, 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 0.25-4.5%.

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

[0008] More 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 also provides a culture of the Pediococcus pentosaceus obtained by the culture method.

[0010] The present invention further provides the use of the Pediococcus pentosaceus in fermentation and winemaking 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, and 2-ethyl-3,5,6-trimethylpyrazine.

[0012] The present invention also provides an additive for fermentation and winemaking, which is prepared from the Pediococcus pentosaceus and is used to produce flavor substances in fermentation and winemaking. More specifically, it is prepared from the culture.

[0013] Biomaterial deposit information: The strain Q1 of the present invention belongs to Pediococcus pentosaceus ( Pediococcus pentosaceus ), deposited in Guangdong Microbial 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.65815, and the classification name is Pediococcus pentosaceus ( Pediococcus pentosaceus ). BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 Phylogenetic tree of 16S rRNA of Pediococcus pentosaceus Q1 and related species. DETAILED DESCRIPTION

[0016] The basic components of the culture medium used in the examples are as follows: TSA medium: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L, agar 15 g / L.

[0017] TSB liquid medium: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L.

[0018] Enrichment medium: yeast extract 3 g / L, peptone 10 g / L, starch 3 g / L, MgSO 4 7H 2 O 0.01 g / L, KH 2 PO 4 0.2 g / L, Na 2 HPO 4 2 g / L.

[0019] 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.

[0020] Example 1: Isolation of Q1 strain Daqu comes from a winery in Sichuan. Daqu was placed in physiological saline (0.9% NaCl), shaken and cultured at room temperature for 30 minutes, heated in a water bath at 85°C for 30 minutes, and the supernatant was added to the enrichment medium, and shaken and enriched at 37°C for 24 hours. The enrichment solution was spread on TSA medium, cultured at 37°C for 1-2 days, and a total of 89 strains were isolated. The isolated strains were streaked and purified. The purified strains were inoculated into the fermentation medium respectively, cultured at 37°C for 12 hours, and the strain seed liquid was obtained. The seed liquid was inoculated into a fermentation medium with a liquid volume of 5 mL / 45 mL according to the inoculation volume of 4%, and cultured at 37°C for 36 hours to obtain the strain fermentation liquid. Take 1mL of the strain fermentation liquid, add VP (Voges-Proskauer) reagent, shake and mix, place for 5 minutes, observe the color change, and select the strain whose color turns red after the reaction during the screening process. A total of 30 strains were screened. Among them, the Q1 strain is one of them. The Q1 strain was preserved by test tube slant preservation and glycerol freezing.

[0021] Example 2: Identification of Q1 strain 1. Morphological identification The Q1 strain was inoculated into TSA medium and cultured at 37°C for 48 h before observing the morphological characteristics of the colonies ( Figure 1 ). It was observed that the colony of Q1 was round, milky white, smooth and opaque, with neat edges, and was a Gram-positive bacterium.

[0022] 2. Identification of physiological characteristics The Q1 strain was inoculated into TSB liquid culture medium to study the tolerance of the Q1 strain to temperature, pH and salt concentration as well as the optimal culture conditions.

[0023] Growth temperature range: Q1 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 Q1.

[0024] Growth pH range: Inoculate Q1 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 Q1.

[0025] 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. OD was measured by spectrophotometer. 600 Absorbance value, analyze the NaCl tolerance range of Q1.

[0026] Studies have shown that the Q1 strain can grow at 15-45°C, with an optimal growth temperature of 37°C. The Q1 strain can grow at a pH value of 4-8, with an optimal growth pH of 7. The Q1 strain can grow at a NaCl concentration of 0.5-4.5%, with an optimal NaCl concentration of 0.5%.

[0027] 3. Molecular Biology Identification After the Q1 strain was inoculated into TSB liquid medium and cultured at 37℃ for 48 h, the bacteria were collected and the whole genome DNA of the Q1 strain was extracted using a bacterial genome 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') of the bacterial 16S rRNA gene. The PCR amplification program was: 94℃ pre-denaturation for 3min, 30 cycles including 94℃ denaturation for 30s, 55℃ annealing for 30s, and 72℃ extension for 1min. Finally, 72 degrees extension for 10min. After amplification, the PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0028] The obtained Q1 strain 16S rRNA sequence (SEQ ID NO: 1):

[0029] The sequences obtained by sequencing were compared on NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the Q1 strain was most closely related to Pediococcus pentosaceus, with a similarity of 99.59%. The Q1 strain and its closely related strains were selected based on sequence similarity to construct a phylogenetic tree. The results showed that the Q1 strain was most closely related to Pediococcus pentosaceus ( Figure 2 ).

[0030] Combining the results of morphological, physiological and biochemical, and molecular biological identification, Q1 was identified as Pediococcus pentosaceus ( Pediococcus pentosaceus ). The strain was submitted to Guangdong Microbiological Collection Center for preservation on January 16, 2025, with the preservation number GDMCC NO:65815.

[0031] Example 3: Analysis of pyrazines produced by Q1 strain After inoculating the Q1 strain into the fermentation medium, shake and culture at 37℃ for 48 h. Take 5.0 mL of the mixed sample in a 20 mL headspace bottle, add 2.0 g NaCl, add the internal standard 2-octanol, and tighten the sample bottle. After aging the extraction head at 250℃ for 30 min, insert it into the sample bottle, balance at 40℃ for 30 min, extract for 40 min, and desorb the extraction head at 250℃ for 3 min in the GC injection port.

[0032] 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 ℃ for 3 min, increased to 150 ℃ at 4 ℃ / min, maintained for 3 min, increased to 240 ℃ at 9 ℃ / min, maintained for 9 min; the carrier gas (He, 99.999%) flow rate was 1.0 mL / min, and the injection was non-split.

[0033] Mass spectrometry conditions: electron bombardment ion source; electron bombardment energy 70 eV; transfer line temperature 230 °C; quadrupole temperature 150 °C; ion source temperature 230 °C; mass scan range m / z 35-600.

[0034] GC-MS analysis revealed that the Q1 strain can produce many flavor substances, including a variety of pyrazine compounds (Table 1).

[0035] Table 1 Statistics of pyrazine substances in Pediococcus pentosaceus Q1

[0036] Among them, no other strains of Pediococcus pentosaceus have been reported to produce 2-methylpyrazine, which has a typical nutty and baked food aroma and can be used in food processing fields such as brewing and spices.

Claims

1. A strain of Pediococcus pentosaceus that produces multiple pyrazines ( Pediococcus pentosaceus ), characterized in that Its deposit number is GDMCC NO:65815.

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

3. The method for culturing 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 culture medium is 0.5%.

4. The method for culturing Pediococcus pentosaceus 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 pentosaceus obtained by the culturing method according to any one of claims 2 to 4.

6. Use of the Pediococcus pentosaceus according to claim 1 in fermentation and winemaking or in the preparation of spice products.

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 pentosaceus 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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