A strain of Pediococcus acidilactici producing multiple pyrazines and its application

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a strain of Pediococcus acidilactici ( Pediococcus acidilactici ) and its application. The strain was isolated from liquor koji, and its deposit number is GDMCC NO: 65814. The volatile flavor substances in the fermentation broth of the strain were detected by GC-MS, and it was found that more than ten kinds of pyrazine compounds were produced, which is more than that produced by existing strains. Therefore, the strain of the present invention has great application value and can be used for fermentation and winemaking.

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] Pyrazines are six-membered ring compounds containing nitrogen at the 1,4 positions, with a distinct aroma of burnt nuts or sesame seeds. Common microorganisms that produce pyrazines are primarily bacteria, including Bacillus and Mycobacterium. As a class of aromatic compounds, pyrazines have significant application value in the food industry. Due to their flavor profiles reminiscent of roasted meat, roasted peanuts, toasted bread, and cocoa, pyrazines are widely used in the production of liquor, coffee, and tea. In addition to their use as flavoring ingredients in the food industry, pyrazines also possess numerous benefits, including tumor suppression, vasodilator activity, anti-inflammatory and antioxidant properties, and diabetes treatment. Studies have shown that 2-methylpyrazine has antioxidant, triglyceride-decomposing and α-glucosidase inhibitory effects, which are of great significance for improving hyperglycemia and providing energy to the body; tetramethylpyrazine can be applied to the central nervous system and can improve learning disabilities; in addition, trimethylpyrazine (TMP) also has very important applications in the medical field, with antioxidant, anti-inflammatory and nerve-protecting effects, and plays a very important role in the treatment of ischemic stroke, Alzheimer's disease, spinal cord injury and other aspects.

[0003] Chinese baijiu (white liquor) has a long history. Made primarily from grains, it uses daqu (big qu), xiaoqu (small qu), or gluten qu (gluten fermentation agent) as a saccharifying and fermenting agent. It uses solid, semi-solid, or liquid fermentation methods, and undergoes a series of processes, including steaming, saccharification, fermentation, distillation, aging, and blending. Qu (koji) is the "soul" of liquor, and the aroma and various flavors of baijiu (white liquor) largely originate from the koji. Pyrazine compounds in baijiu (white liquor) have low threshold values and high odor intensity, playing a crucial role in the formation of burnt aromas. They are important flavor compounds in baijiu and a key component of the volatile aroma compounds in koji. Currently, 29 pyrazine compounds have been detected in Chinese baijiu (white liquor), primarily alkylpyrazines, including tetramethylpyrazine, trimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-methylpyrazine, and 2,3-dimethylpyrazine. Therefore, identifying strains capable of producing a variety of pyrazine compounds is of great significance to the baijiu (white liquor) industry. Summary of the Invention

[0004] The present invention screened and obtained a strain of Pediococcus acidilactici Q2 that can produce multiple pyrazines. GC-MS analysis revealed that 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. Furthermore, 4-methylpyrazine has been reported to be produced in Pediococcus acidilactici, while other pyrazines have not been reported. Therefore, the strain has significant 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 the use of the Pediococcus acidilactici in fermentation 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 Provincial Microbial Culture Collection Center (GDMCC) on January 16, 2025, the deposit address is: 5th Floor, Dayuan Laboratory 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 with reference to 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:

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

[0020] TSB fluid medium: tryptone 15 g / L, soytone 5 g / L, sodium chloride 5 g / L.

[0021] 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.

[0022] 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.

[0023] Example 1: Isolation of Q2 strain

[0024] The koji used in the examples was sourced from a winery in Sichuan. 10 g of koji was weighed and placed in 90 ml of physiological saline (0.9% NaCl) and shaken at room temperature at 180 r / min for 30 min. The mixture was heated in a water bath at 85°C for 30 min, and 5.00 mL of the supernatant was added to 95 mL of enrichment medium. The mixture was enriched at 37°C and 200 r / min for 24 h. The enrichment solution was diluted gradiently, spread on TSA plates, and cultured in a 37°C incubator for 1 to 2 days. Single colonies of different morphologies on the plates were picked for isolation and purification, and a total of 89 colonies were isolated. The bacteria were purified three times by three-zone striping to obtain purified cultured bacteria. The Q2 strain obtained by the above isolation and purification was stored by (1) TSA plate / test tube slant; (2) the Q2 bacterial suspension was mixed with 50% glycerol in a ratio of 1:1, quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

[0025] Example 2: Identification of Q2 strain

[0026] The species identification of Q2 strain was mainly carried out through morphology and molecular biology.

[0027] 1. Morphological characteristics

[0028] The Q2 strain was inoculated into TSA medium and the colony morphology was observed after culturing for 48 h. Figure 1 ). Observation revealed that strain Q2 was round, milky white, with neat edges, and was a Gram-positive bacterium.

[0029] 2. Molecular Biology Identification

[0030] A single colony of the purified Q2 strain was inoculated into TSB liquid medium and cultured at 37°C for 48 hours. The bacterial genome was extracted from the culture using the Tiangen Bacterial Genomics Kit according to the manufacturer's instructions. Whole-genome DNA was used as a PCR template using 2× Taq PCR Mix in a 25 μL PCR reaction system, using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The PCR amplification protocol consisted of a 3-minute initial denaturation at 94°C, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. Finally, an extension at 72°C for 10 minutes was performed. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0031] The obtained 16S rRNA sequence of the Q2 strain is as follows (SEQ ID NO: 1):

[0032]

[0033] Then, the sequenced Q2 16S rRNA gene sequence was used to perform NCBI Blast search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the alignment results showed that Q2 was closely related to Pediococcus acidilactici ( Pediococcus acidilactici ) has 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 ).

[0034] Combining morphological and molecular biological characteristics, Q2 was preliminarily identified as Pediococcus acidilactici. This strain was submitted to the Guangdong Provincial Microbiological Culture Collection for preservation on January 16, 2025, with the deposit number: GDMCC NO: 65814.

[0035] 3. Study on biological characteristics of Q2

[0036] Growth temperature range: Q2 was inoculated into TSB medium and cultured at 15, 20, 30, 37, 45 and 50°C for 48 h, and the OD was measured by spectrophotometer. 600 The absorbance value was used to analyze the growth range of Q2.

[0037] Growth pH range: Inoculate Q2 into TSB medium, adjust the pH to 3, 4, 5, 6, 7, 8, 9, culture at 37 ° C for 48 h, and measure OD by spectrophotometer. 600 The absorbance value was used to analyze the pH growth range of Q2.

[0038] NaCl tolerance range: Q2 was inoculated into TSB medium and the NaCl concentration was adjusted to (%, w / v) 0, 2, 4, 6, 8, 9, 10, 11, 12. The culture was carried out at 37°C for 48 h and the OD was measured by spectrophotometer. 600 The absorbance value was used to analyze the NaCl tolerance range of Q2.

[0039] The results showed that the Q2 strain can grow at 30-45°C, with an optimal growth temperature of 37°C. The Q2 strain can grow at a pH of 4-8, with an optimal growth pH of 5. The Q2 strain can grow at a NaCl concentration of 0-4%, with an optimal NaCl concentration of 0%.

[0040] Example 3: Analysis of flavor substances produced by Q2 strain

[0041] Q2 was inoculated into the fermentation medium and cultured at 37°C and 200 rpm for 48 hours to obtain the fermentation broth. 5.0 mL of the culture broth was transferred to a 20 mL headspace vial, and 2.0 g of NaCl and 20.0 μL of 99.07 mg / L internal standard 2-octanol were added. The vial was then screwed securely. The extraction tip was conditioned at 250°C for 30 minutes, then inserted into the vial. The sample was then equilibrated at 40°C for 30 minutes, extracted for 40 minutes, and desorbed from the GC inlet at 250°C for 3 minutes.

[0042] 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, held for 3 min, then increased to 240°C at 9°C / min, held for 9 min; the carrier gas (He, 99.999%) was injected at a flow rate of 1.0 mL / min, splitless. Mass spectrometry conditions included an electron impact ion source with an electron impact energy of 70 eV, a transfer line temperature of 230°C, a quadrupole temperature of 150°C, and an ion source temperature of 230°C; and a mass scan range of m / z 35 to 600.

[0043] GC-MS analysis revealed that the Q2 strain could produce more than ten pyrazine compounds (Table 1).

[0044] Table 1 Pyrazine flavor compounds produced during the fermentation of Pediococcus acidilactici

[0045]

[0046] These pyrazine compounds are important flavor compounds in liquor. Currently, 4-methylpyrazine has been reported to be produced in Pediococcus acidilactici, but no other pyrazines have been reported. Pediococcus acidilactici Q2, the strain used in the present invention, can produce a wider variety of pyrazines. Therefore, the strain screened in the present invention has significant application value in winemaking and other food industries.

Claims

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

2. The method for culturing Pediococcus acidilactici according to claim 1, wherein The method adopts TSB liquid culture medium for culture, the culture temperature is 30-45 DEG C, the pH value is 4-8, and the concentration of sodium chloride in the culture medium is 0-4%.

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

4. The method for culturing Pediococcus acidilactici according to claim 2, wherein The formula of TSB liquid culture medium is: tryptone 15g / L, soytone 5g / L, sodium chloride 5g / L. 5 . A culture containing Pediococcus acidilactici obtained by the culture method according to claim 2 .

6. Use of the Pediococcus acidilactici as claimed in claim 1 in fermentation 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, comprising the Pediococcus acidilactici according to claim 1 and used for producing flavor substances in fermentation and winemaking.

9. The additive according to claim 8, characterized in that It is prepared by the culture according to claim 5.

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