A strain of Enterococcus faecalis producing pyrazine compounds and its application

Abstract

The present invention discloses a strain of Enterococcus faecalis producing pyrazine compounds and its application. The Enterococcus faecalis is classified as Enterococcus faecalis, the strain name is LZLJ2-6, and the deposit number is CGMCC No. 26363. The Enterococcus faecalis in the present invention can be used to produce pyrazine compounds, such as 2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isoamyl-2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine, etc.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and relates to a strain of Enterococcus faecalis producing pyrazine compounds separated from Luzhou-flavor medium-temperature Daqu and application thereof. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Pyrazines are a series of six-membered heterocyclic compounds containing symmetrical nitrogen at the 1 and 4 positions. They have unique sensory characteristics such as cocoa aroma, baking aroma, and nutty aroma. They contribute to the aroma and flavor of a variety of fermented foods and have important application value in the food industry. In addition, some pyrazine compounds are also used in the medical and health field due to their biological activity.

[0004] At present, there is a lack of research on enterococci that produce pyrazine compounds, and most studies focus on the Bacillus group. However, in addition to Bacillus, DNA sequencing analysis technology has found that enterococci are the dominant bacterial group in certain stages of the liquor brewing system. Considering the metabolic characteristics of Bacillus in the production of important pyrazine flavor substances during the brewing process, there may also be other microorganisms with the potential to produce pyrazine compounds in this brewing environment. Therefore, systematically exploring microorganisms that produce pyrazine compounds in the liquor brewing environment will provide more aroma-producing strains for future brewing, thereby promoting the upgrading of fermented foods. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Enterococcus faecalis producing pyrazine compounds and its application in view of the shortcomings of the prior art.

[0006] In order to solve the above technical problems, the present invention discloses the following solutions:

[0007] In a first aspect, the present invention discloses a strain of Enterococcus faecalis producing pyrazine compounds isolated and screened from koji, so as to solve the problem of developing functional bacterial strain resources.

[0008] Among them, the Enterococcus faecalis is classified and named Enterococcus faecalis, and the strain name is LZLJ2-6. It has been deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of December 30, 2022, a deposit number of CGMCC No. 26363, and an address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The 6S rDNA sequence of the Enterococcus faecalis LZLJ2-6 is shown in SEQ ID NO.1.

[0010] Wherein, the Enterococcus faecalis LZLJ2-6 is a Gram-positive bacterium, which is inverted and cultured on a PYG solid medium at 28°C for 20-24h. Figure 1 As shown, the colonies are white, opaque, round, with smooth surfaces and neat edges.

[0011] Among them, the enterococcus LZLJ2-6 is a well-characterized enterococcus strain that can metabolize and simultaneously produce pyrazine compounds such as 2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isopentyl-2,3-dimethylpyrazine, and 2,3,5-trimethylpyrazine.

[0012] In a second aspect, the present invention discloses the use of the Enterococcus faecalis described in the first aspect in the fermentation production of pyrazine compounds, specifically, the Enterococcus faecalis is activated and inoculated into a fermentation medium for fermentation to obtain a fermentation liquid containing pyrazine compounds.

[0013] The activation is to culture the Enterococcus faecalis in a culture medium at 28° C. for 24 hours and then inoculate the culture medium for fermentation.

[0014] Wherein, the activated culture medium is MRS liquid culture medium, comprising the following components in concentrations: glucose 18-22 g / L, peptone 9-11 g / L, yeast powder 3-5 g / L, beef extract powder 4-6 g / L, Tween 80 0.5-1.5 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, and dipotassium hydrogen phosphate 0.5-1.5 g / L. In some embodiments, the MRS liquid culture medium is 20 g / L glucose, 10 g / L peptone, 4 g / L yeast powder, 5 g / L beef extract powder, 1 g / L Tween 80, 2 g / L triammonium citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1 g / L potassium hydrogen phosphate, pH 7.0, and is sterilized by high pressure steam at 115°C for 20 minutes.

[0015] The activated Enterococcus faecalis is inoculated into the fermentation medium at a volume ratio of 0.5%-3.5%, preferably at a volume ratio of 1%-3%, such as 2%.

[0016] The fermentation medium comprises components with the following concentrations: glucose 14-18 g / L, tryptone 9-11 g / L, yeast powder 2-3 g / L, beef extract 4-6 g / L, bovine brain 3.55-4.15 g / L, bovine heart 4.4-5.4 g / L, Tween 80 0.4-0.6 g / L, ammonium citrate 0.8-1.2 g / L, sodium chloride 2-3 g / L, anhydrous sodium acetate 2-3 g / L, magnesium sulfate 0.03-0.07 g / L, manganese sulfate 0.02-0.03 g / L, disodium hydrogen phosphate 1-1.5 g / L, and dipotassium hydrogen phosphate 0.5-1.5 g / L. In some embodiments, the fermentation medium is 16 g / L glucose, 10 g / L tryptone, 2.5 g / L yeast powder, 5 g / L beef extract, 3.85 g / L bovine brain, 4.9 g / L bovine heart, 0.5 g / L Tween 80, 1 g / L ammonium citrate, 2.5 g / L sodium chloride, 2.5 g / L anhydrous sodium acetate, 0.05 g / L magnesium sulfate, 0.025 g / L manganese sulfate, 1.25 g / L disodium hydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, pH 6.0, and high-pressure steam sterilization at 115°C for 20 minutes.

[0017] Wherein, the fermentation temperature is 23-33°C, preferably 26-30°C, preferably 28°C.

[0018] Wherein, the fermentation time is 12-36h, preferably 18-30h, preferably 24h.

[0019] In a third aspect, the present invention discloses the use of the Enterococcus faecalis described in the first aspect in winemaking.

[0020] In a fourth aspect, the present invention discloses the use of the Enterococcus faecalis described in the first aspect as an edible flavor or flavor additive.

[0021] The pyrazine compounds described in the present invention include 2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isopentyl-2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine, pyrazine, 2-ethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, 3,5-diethyl-2-methyl-pyrazine, 2-acetylpyrazine, 2-acetyl-3-5-dimethylpyrazine, etc.

[0022] Beneficial effects:

[0023] (1) The Enterococcus faecalis that can produce pyrazine compounds (2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isopentyl-2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine) provided by the present invention is a strain with good flavor characteristics, which is derived from Luzhou-flavor medium-temperature Daqu and can improve the nutty and baked aroma characteristics of fermented foods, showing great potential for application in industries such as liquor brewing and food fermentation.

[0024] (2) The strain provided by the present invention produces pyrazine compounds, which account for 30.14% of the total volatile substances. The pyrazine substances produced by the strain can be used as food flavors or flavor additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0026] Figure 1 This is the colony morphology of Enterococcus faecalis LZLJ2-6.

[0027] Figure 2 This is the phylogenetic tree of strain LZLJ2-6.

[0028] Figure 3 This is the GC-MS spectrum of Enterococcus faecalis LZLJ2-6. DETAILED DESCRIPTION

[0029] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0030] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0031] The culture medium formula involved in the following examples is as follows:

[0032] PYG liquid culture medium: peptone 20g / L, yeast extract powder 10g / L, sodium chloride 0.08g / L, cysteine ​​hydrochloride 0.5g / L, calcium chloride 0.008g / L, magnesium sulfate 0.008g / L, sodium hydrogen phosphate 0.4g / L, potassium dihydrogen phosphate 0.04g / L, dipotassium hydrogen phosphate 0.04g / L, solvent is pure water, pH 6.0, high pressure steam sterilization at 121°C for 20 minutes.

[0033] PYG solid medium: Add 15 g / L agar to PYG liquid medium and sterilize with high pressure steam at 121°C for 20 minutes.

[0034] MRS liquid culture medium: glucose 20g / L, peptone 10g / L, yeast powder 4g / L, beef extract powder 5g / L, Tween 801g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, dipotassium hydrogen phosphate 1g / L, solvent is pure water, pH 7.0, high pressure steam sterilization at 115℃ for 20 minutes.

[0035] MRS solid medium: Add 15 g / L agar to MRS liquid medium and sterilize with high pressure steam at 115°C for 20 minutes.

[0036] Fermentation medium: glucose 16 g / L, tryptone 10 g / L, yeast powder 2.5 g / L, beef extract 5 g / L, bovine brain 3.85 g / L, bovine heart 4.9 g / L, Tween 80 0.5 g / L, ammonium citrate 1 g / L, sodium chloride 2.5 g / L, anhydrous sodium acetate 2.5 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.025 g / L, disodium hydrogen phosphate 1.25 g / L, dipotassium hydrogen phosphate 1 g / L, pH 6.0, high-pressure steam sterilization at 115°C for 20 min.

[0037] Example 1 Screening of strains producing pyrazine compounds

[0038] Separation method: Take 20g of medium-temperature Daqu sample and put it into a conical flask filled with 180mL of sterile distilled water. Oscillate on a constant temperature shaker for 10 minutes to fully disperse and mix the sample. Take 1mL of sample suspension and dilute it continuously with sterile water to 10 -2 ~10 -7 , take 0.1 mL of each gradient and evenly spread it on the PYG solid culture medium plate, prepare two plates in parallel, invert them, culture them at 28°C for 20-24h, and observe them in time.

[0039] Streak purification: Take out the plate with colonies, pick out single colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0040] Target strain selection: The purified colonies were inoculated into PYG liquid culture medium and cultured at 28°C for 24 hours. The strains with obvious nutty flavor in the fermentation liquid were selected as target strains through sensory evaluation.

[0041] Strain preservation: After purification, a single colony of each strain was picked into 5 mL of MRS liquid culture medium, and cultured at 37°C under anaerobic conditions for 20-24 hours. 1 mL of bacterial solution was pipetted into a bacterial preservation tube, and 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0042] Example 2 Molecular identification of target strains

[0043] Purify the strains obtained by screening, take fresh bacterial liquid in the logarithmic growth period, collect the bacteria by centrifugation, and extract genomic DNA using a bacterial genome extraction kit. Use bacterial universal primers 27F and 1492R for PCR amplification, and sequence to obtain the gene sequence of the 16S rDNA fragment. The details are as follows:

[0044] 27F(SEQ ID NO.2)(5′-AGAGTTTGATCCTGGCTCAG-3′)

[0045] 1492R(SEQ ID NO.3)(5′-GGTTACCTTGTTACGACTT-3′)

[0046] ①Reaction system (50μL)

[0047]

[0048] ②Reaction procedure

[0049]

[0050] According to the above process, 35 cycles of "denaturation-annealing-extension" PCR reaction were carried out, and the PCR products were separated and tested by 1.0% agarose gel electrophoresis with a voltage of about 11 V / cm and an electrophoresis time of 20 min.

[0051] The purification of PCR products was carried out according to the instructions of the mini-gel recovery PCR product purification kit of Shanghai Sangon Biotechnology Co., Ltd., and the sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0052] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST to determine the species information of the target strain, and the phylogenetic tree was constructed using MEGA 6.0. Figure 2 As shown, it was identified as Enterococcus faecalis and named Enterococcus faecalis LZLJ2-6.

[0053] Example 3 Detection experiment of volatile metabolites of strains

[0054] 1. Sample Preparation

[0055] The glycerol storage tube of Enterococcus faecalis LZLJ2-6 was dissolved and inoculated into MRS liquid medium, and then cultured at 28°C for 24 hours. After activation for three generations, the inoculation amount was inoculated into 400 mL fermentation medium at 2% (volume ratio) and cultured for 24 hours at 28°C. After the fermentation was completed, the bacterial liquid was obtained, and the supernatant was taken after centrifugation at 10000 rpm for 5 minutes.

[0056] 2. Volatile metabolite detection

[0057] The volatile products were determined by headspace solid phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS).

[0058] (1) HS-SPME extraction conditions

[0059] Take 200 μL of the supernatant and add it to the headspace bottle, add 5 mL of saturated NaCl solution, keep the prepared sample at 60 °C for 5 min, extract it with a 50 / 30 μm DVB / CAR / PDMS extraction head at 60 °C for 50 min, and desorb it at 250 °C at the GC injection port for 5 min after the extraction.

[0060] (2) GC-MS chromatographic conditions

[0061] Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: starting temperature 40°C, hold for 5min, increase to 100°C at 4°C / min, then increase to 230°C at 6°C / min, hold for 10min, carrier gas is high-purity helium (1.0mL / min); injection port temperature 250°C, no split.

[0062] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40-450 m / z.

[0063] Using 0.822 mg / mL 2-octanol as the internal standard, the compound search results were matched with the NIST standard library, and the target compound was confirmed when the similarity reached more than 80%. The culture solution without bacterial fermentation was used as the blank control group to calculate the relative content of each volatile substance.

[0064] Test results such as Figure 3As shown, pyrazine compounds include 2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isopentyl-2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine, pyrazine, 2-ethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, 3,5-diethyl-2-methyl-pyrazine, 2-acetylpyrazine, 2-acetyl-3-5-dimethylpyrazine and the like. The test results of the main pyrazine compounds produced by the fermentation of Enterococcus faecalis LZLJ2-6 are shown in Table 1. The contents of 2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isopentyl-2,3-dimethylpyrazine and 2,3,5-trimethylpyrazine are 379.99 μg / L, 330.10 μg / L, 131.79 μg / L and 102.84 μg / L respectively. The aforementioned main pyrazine compounds account for 83.78% of the total content of pyrazine compounds, and the produced pyrazine compounds account for 30.14% of the total volatile matter content.

[0065] Table 1 Detection results of pyrazine compound content in Enterococcus faecalis LZLJ2-6

[0066] Volatile metabolites Content (μg / L) 2,5-Dimethylpyrazine 379.99 3-Ethyl-2,5-methylpyrazine 330.10 5-Isopentyl-2,3-dimethylpyrazine 131.79 2,3,5-Trimethylpyrazine 102.84

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Enterococcus faecalis, which is classified as Enterococcus faecalis ( Enterococcus faecalis ), the strain is named LZLJ2-6, and has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is December 30, 2022, and the deposit number is CGMCC No. 26363.

2. The use of Enterococcus faecalis according to claim 1 in fermentation to produce pyrazine compounds, characterized in that: The pyrazine compounds are 2,5-dimethylpyrazine, 3-ethyl-2,5-methylpyrazine, 5-isopentyl-2,3-dimethylpyrazine and 2,3,5-trimethylpyrazine; the fermentation medium comprises the following components in concentrations: 14-18 g / L glucose, 9-11 g / L tryptone, 2-3 g / L yeast powder, 4-6 g / L beef extract, 3.55-4.15 g / L bovine brain, 4.4-5.4 g / L bovine heart, 0.4-0.6 g / L Tween 80, 0.8-1.2 g / L ammonium citrate, 2-3 g / L sodium chloride, 2-3 g / L anhydrous sodium acetate, 0.03-0.07 g / L magnesium sulfate, 0.02-0.03 g / L manganese sulfate, 1-1.5 g / L disodium hydrogen phosphate and 0.5-1.5 g / L dipotassium hydrogen phosphate.

3. The use according to claim 2, characterized in that: After being activated, the Enterococcus faecalis is inoculated into a fermentation medium at a volume ratio of 0.5%-3.5% for fermentation to obtain a fermentation liquid containing pyrazine compounds.

4. The use according to claim 3, characterized in that: The activated culture medium is an MRS liquid culture medium, which includes the following components in concentrations: 18-22 g / L glucose, 9-11 g / L peptone, 3-5 g / L yeast powder, 4-6 g / L beef extract powder, 0.5-1.5 g / L Tween 80, 1-3 g / L triammonium citrate, 4-6 g / L sodium acetate, 0.1-0.3 g / L magnesium sulfate, 0.04-0.06 g / L manganese sulfate, and 0.5-1.5 g / L dipotassium hydrogen phosphate.

5. The use according to claim 3, characterized in that: After being activated, the Enterococcus faecalis is inoculated into the fermentation medium at a volume ratio of 1%-3%.

6. The use according to claim 3, characterized in that: The fermentation temperature is 23-33°C.

7. The use according to claim 3, characterized in that: The fermentation temperature is 26-30°C.

8. The use according to claim 3, characterized in that: The fermentation temperature was 28°C.

9. The use according to claim 3, characterized in that: The fermentation time is 12-36h.

10. The use according to claim 3, characterized in that: The fermentation time is 18-30h.

11. The use according to claim 3, characterized in that: The fermentation time is 24h.

12. Use of the Enterococcus faecalis according to claim 1 in winemaking.

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