Fusospirochetes and their applications

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a 2-acetylfuran-producing fusostreptococcus and application thereof. In view of the fact that no 2-acetylfuran-producing fusostreptococcus has been found in existing research, a 2-acetylfuran-producing fusostreptococcus new strain is isolated and screened from distiller's yeast, and the preservation number of the strain is CGMCC No. 26364. The strain can be applied to the fermented food industry, and can also be applied to biological source essence and perfume and medical industry, so as to promote the development of the industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to 2-acetylfuran-producing Weissella confusa and application thereof. BACKGROUND

[0002] 2-acetylfuran is a yellow-brown liquid, and has an almond, nut, yeast, milk and sweet caramel-like aroma. Natural products exist in volatile aroma components of coffee, tomatoes, beer, green tea, raisins and potato chips. It can be used as an organic synthesis raw material, a pharmaceutical intermediate and a food flavor.

[0003] In existing research, it is found that the content of 2-acetylfuran in volatile substances of fermented food can be increased by adjusting microorganisms. For example, Xiao Hong Tang et al. (Xiao Hong Tang, Wen Xie Hu, Yan Feng Wei, et al. Screening of wild wine yeast from grape and study on its biological characteristics [J]. Journal of Shandong University: Science Edition, 2014, 49(3): 7.) obtained a strain of Saccharomyces cerevisiae PJ16 from grape skin and soil in a vineyard, and the concentrations of n-hexanol, 1-nonanol, 2-nonanol, ethyl lactate, acetophenone, benzothiazole and 2-acetylfuran in the wine brewed by the strain were much higher than those in the wine brewed by a commercial yeast. Zeng Deyong (Zeng Deyong. Optimization of functional microorganisms in production of Maotai-flavor liquor and application research [D]. Guizhou University.) prepared a reinforced Daqu by optimizing the ratio of functional bacteria (Bacillus amyloliquefaciens and Bacillus licheniformis), yeast (Saccharomyces cerevisiae and Pichia pastoris) and aspergillus (Aspergillus niger, Mucor, and Aspergillus flavus), and the volatile substances such as n-propanol, acetic acid, ethyl lactate, 2,3,5,6-tetramethylpyrazine and 2-acetylfuran in the wine sample were obviously improved.

[0004] Weissella confusa is a lactic acid bacteria widely existing in fermented food, meat, milk, vegetables and soil, and is often used in the food industry to improve the properties of food. However, no 2-acetylfuran-producing Weissella confusa has been found in existing research. SUMMARY

[0005] In view of the fact that no 2-acetylfuran-producing Weissella confusa has been found in existing research, a new strain of 2-acetylfuran-producing Weissella confusa is isolated and screened from Daqu, and the strain can be applied not only to the fermented food industry but also to the medical industry and the production of biologically derived flavors and fragrances, thereby promoting the development of the industry.

[0006] The present application provides a 2-acetylfuran-producing Weissella confusa, and the preservation number of the strain is CGMCC No. 26364.

[0007] The preservation time is December 30, 2022, and the preservation unit is China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Beichen West Road, Yard 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, with a postal code of 100101. The name is: fusion Weissella LZLJ2-4 (Weissella confusa).

[0008] The 16S rDNA sequence of the above-mentioned fusion Weissella LZLJ2-4 is shown as SEQ ID NO: 1:

[0009]

[0010] The present application also provides a microbial agent, which takes the fusion Weissella as an active ingredient.

[0011] The present application also provides the application of the above-mentioned fusion Weissella in the production of 2-acetylfuran.

[0012] The present application also provides the application of the above-mentioned fusion Weissella in the field of liquor brewing.

[0013] The present application also provides the application of the above-mentioned fusion Weissella in the preparation of organic synthesis raw materials, pharmaceutical intermediates, fragrances and spices, and fermented foods by using 2-acetylfuran.

[0014] Specifically, in the application, the method for fermentation of 2-acetylfuran by the fusion Weissella includes the following steps: after the fusion Weissella is inoculated in the MRS liquid medium for culture and activation, the fusion Weissella is inoculated in the fermentation medium for fermentation, and the fermentation culture is completed.

[0015] The MRS liquid medium includes the following components: glucose 20 g / L, peptone 10 g / L, yeast powder 4 g / L, beef infusion powder 5 g / L, Tween 80 1 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, and dipotassium hydrogen phosphate 1 g / L.

[0016] The liquid medium culture condition is static culture at 30-37℃ for 20-24h.

[0017] The inoculation amount of the fusion Weissella to the fermentation medium is 2v / v%.

[0018] The fermentation culture condition is static culture at 30-37℃ for 24-48h.

[0019] The fermentation medium includes the following components: glucose 16 g / L, tryptone 10 g / L, yeast powder 2.5 g / L, beef extract 5 g / L, beef brain 3.85 g / L, beef 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, and dipotassium hydrogen phosphate 1 g / L.

[0020] By using the above-mentioned fermentation process, the content of 2-acetylfuran produced by the fusion Weissella is 0.096 mg / L.

[0021] Beneficial effects: the application separates and screens a strain of 2-acetyl furan-producing Weissella confusa from commercially available wine starter, and the preservation number of the strain is: CGMCC No. 26364. The strain is named Weissella confusa LZLJ2-4. The strain has stable performance, simple fermentation process, does not destroy the balance of microorganisms in the original fermentation process, and produces 2-acetyl furan with a content of 0.096 mg / L, and can be widely applied in the preparation of various fermented foods such as wine, soy sauce, vinegar and bread. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is the colony morphology chart of the Weissella confusa in Example 1.

[0023] Figure 2 It is the GC-MS molecular fragment mass spectrum chart of 2-acetyl furan produced by the Weissella confusa in Example 2.

[0024] Figure 3 It is the GC-MS molecular fragment mass spectrum chart of 2-acetyl furan standard.

[0025] The Weissella confusa of the application was preserved on December 30, 2022, and the preservation number thereof is: CGMCC No. 26364. The preservation unit is China General Microbiological Culture Collection Center (CGMCC), which is located at No. 1, Beichen West Road, Haidian District, Beijing, China, and the postal code thereof is 100101. The classification and naming is: Weissella confusa. DETAILED DESCRIPTION

[0026] The scheme of the application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the application, and should not be regarded as limiting the scope of the application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product instruction is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained from the market.

[0027] The medium formula involved in the examples is as follows:

[0028] MRS liquid medium: glucose 20 g / L, peptone 10 g / L, yeast powder 4 g / L, beef infusion powder 5 g / L, Tween 80 1 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, potassium phosphate dibasic 1 g / L, pH 7.0, high-pressure steam sterilization at 115℃ for 20 minutes.

[0029] MRS solid medium: 15 g / L agar was added to the MRS liquid medium, and it was sterilized by high-pressure steam at 115°C for 20 minutes.

[0030] Fermentation medium: glucose 16 g / L, tryptone 10 g / L, yeast extract 2.5 g / L, beef extract 5 g / L, beef brain 3.85 g / L, beef 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, potassium hydrogen phosphate 1 g / L, pH 6.0, sterilized by high-pressure steam at 115°C for 20 minutes.

[0031] Example 1: Strain isolation and purification

[0032] 1) Isolation method: 20 g of koji sample was taken and placed in a conical flask containing 180 mL of sterile distilled water, and the sample was fully dispersed and mixed by constant temperature shaking for 10 min. 1 mL of the sample suspension was taken and diluted by the double dilution method to 10 -2 ~10 -7 100 μL was taken from each gradient dilution and evenly spread on MRS solid medium plates, two plates were prepared in parallel, inverted, and incubated at 37°C under anaerobic conditions for 36-48 h, and observed in time.

[0033] 2) Streaking purification: the plate with grown colonies was taken out, and single colonies with different colony morphologies were picked and subjected to secondary streaking until all single colonies were purified. The colonies on the MRS agar medium were small, moist, white, raised in the middle, and smooth on the surface, as shown in Figure 1 .

[0034] 3) Strain preservation: the single colonies of each strain after purification were picked into 5 mL of MRS liquid medium and incubated at 37°C under anaerobic conditions for 20-24 h, 1 mL of the bacterial solution was taken and added to a 0.5 mL 60% sterile glycerol solution, resuspended, and stored at -80°C.

[0035] Example 2: Detection of 2-acetylfuran-producing ability

[0036] 1) Preparation of test bacterial solution:

[0037] After the glycerol preservation tube of the screened strain was dissolved, it was inoculated into MRS liquid medium and incubated at 37°C for 24 h, and after three generations of activation, it was inoculated into 50 mL of MRS liquid medium at a 2% (v / v) inoculation amount and incubated for 24 h to obtain the test bacterial solution.

[0038] (2) HS-SPME / GC-MS detection method for 2-acetylfuran:

[0039] Headspace solid-phase microextraction / gas chromatography-mass spectrometry (HS-MS / MS) was used: the supernatant was collected and added to a headspace vial, along with saturated NaCl solution. The prepared sample was equilibrated at 60°C for 5 min, then extracted at 60°C for 50 min using a 50 / 30 μm DVB / CAR / PDMS extraction head. After extraction, desorption was performed at 250°C for 5 min at the GC inlet. Compound search results were matched with the NIST standard spectral library; compounds with a similarity of over 80% were confirmed as target compounds.

[0040] GC-MS detection chromatographic conditions:

[0041] Gas chromatography conditions: HP-INNOWAX column (60m × 0.25mm × 0.25μm); Temperature program: initial temperature 40℃, hold for 5 min, increase to 100℃ at 4℃ / min, then increase to 230℃ at 6℃ / min.

[0042] Hold for 10 min with high-purity helium as the carrier gas (1.0 mL / min); injector temperature 250℃, no splitting.

[0043] 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.

[0044] Through the above detection methods, such as Figure 2 , 3 As shown, a strain capable of producing 2-acetylfuran was screened from the isolated strains, and this strain was selected for further research.

[0045] Example 3: Method for identifying strains

[0046] The purified and screened strains were collected by centrifugation of fresh bacterial culture in the logarithmic growth phase, and genomic DNA was extracted using a bacterial genome extraction kit. The full-length 16S rDNA sequence was amplified using universal lactic acid bacteria primers 27F / 1492R, as follows:

[0047] SEQ ID NO: 2: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

[0048] SEQ ID NO:3:1492R(5′-GGTTACCTTGTTACGACTT-3′)

[0049] Reaction system (50 μL)

[0050]

[0051] Reaction Procedure

[0052]

[0053] The PCR products were separated and detected by 1.0% agarose gel electrophoresis at a voltage of about 11 V / cm for 20 min.

[0054] The sequencing of the PCR products was completed by Shanghai Shenguo Biotechnology Co., Ltd. The gene sequence of the 16S rDNA fragment obtained by sequencing was compared by BLAST of NCBI, and the 16S rDNA sequence is shown as SEQ ID NO: 1. According to the morphological characteristics of the strain, the strain species information was determined, and it was identified as Weissella confusa, and named as Weissella confusa LZLJ2-4.

[0055] Example 4: Fermentation culture of Weissella confusa

[0056] The glycerol preservation tube of Weissella confusa LZLJ2-4 was dissolved and inoculated into MRS liquid medium, and incubated at 37°C for 24 h. After three generations of activation, it was inoculated into 300 mL of fermentation medium at a 2% (volume ratio) inoculation amount and cultured for 24 h. After fermentation, the bacterial liquid was obtained, and the supernatant was taken after centrifugation at 10,000 rpm for 5 min. The detection method was the same as the GC-MS detection method in Example 2. 0.822 mg / mL of 2-octanol was used as an internal standard, and the original culture medium was used as a blank control. The content of 2-acetylfuran was calculated. After 24 h of static culture of Weissella confusa, the yield of 2-acetylfuran was 0.096 mg / L.

[0057] It should be noted that the specific features, structures, materials or characteristics described in the specification can be combined in any appropriate manner in any one or more embodiments. In addition, those skilled in the art can combine and combine the different embodiments and the features of the different embodiments described in the specification without contradiction.

Claims

1. Fusion Weissella ( Weissella confusa ), characterized by: The accession number is: CGMCC No.26364.

2. A microbial inoculant, characterized in that: The active ingredient is the Fusion Weissella bacterium as described in claim 1.

3. The use of the Fusion Weissella as described in claim 1 in the production of 2-acetylfuran.

4. The application of the Fusion Weissella as described in claim 1 in the field of Baijiu brewing.

5. The application of the Fusion Weissella as described in claim 1 in the preparation of organic synthesis raw materials, pharmaceutical intermediates or flavorings using 2-acetylfuran.

6. The application according to any one of claims 3-5, characterized in that, The method for producing 2-acetylfuran by fermentation of *Westernia fusionis* includes the following steps: *Westernia fusionis* is inoculated into MRS liquid medium for culture and activation, then inoculated into fermentation medium for fermentation, and fermentation culture is carried out.

7. The application according to claim 6, characterized in that: The MRS liquid culture medium consists of: glucose 20 g / L, peptone 10 g / L, yeast extract 4 g / L, beef extract 5 g / L, Tween 80 1 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, and dipotassium hydrogen phosphate 1 g / L.

8. The application according to claim 6, characterized in that: The liquid culture medium is cultured at 30℃-37℃ for 20-24 hours under static conditions.

9. The application according to claim 6, characterized in that: The inoculation amount of the fused Weissella into the fermentation medium was 2 v / v%. The fermentation conditions were: static incubation at 30℃-37°C for 24-48 hours.

10. The application according to claim 6, characterized in that: The fermentation medium consisted of 16 g / L glucose, 10 g / L tryptone, 2.5 g / L yeast extract, 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, and 1 g / L dipotassium hydrogen phosphate.

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