Fibrian seraberlindnerella vaginalis, which produces organic acids and acetates, and its applications

Abstract

This invention discloses a *Fibrians cerebelli* strain capable of producing organic acids and acetates, and its applications, belonging to the field of biology. This invention screened a *Fibrians cerebelli* strain capable of producing organic acids and acetates from brewing yeast, with the preservation number CGMCC No. 26368. The strain provided by this invention can produce a variety of organic acids and acetates, and can be applied to traditional fermented foods, broadening the selection of yeast strains for food fermentation and promoting the standardized industrial production of traditional fermented foods.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a yeast strain that can produce organic acids and acetate compounds, obtained by screening, separating and purifying yeast from brewing yeast. Specifically, it relates to a strain of *Fibrian seraberlindnerella* that can produce organic acids and acetate compounds and its applications. Background Technology

[0002] Traditional fermented foods include baijiu (Chinese liquor), soy sauce, vinegar, and pickles. The fermentation process involves a variety of microorganisms. Through enzymes produced by these microorganisms, such as oxidoreductases, hydrolases, isomerases, lyases, and transferases, the raw materials (carbohydrates, proteins, lipids, and other macromolecular nutrients) are hydrolyzed to produce primary metabolites such as monosaccharides, free fatty acids, and free amino acids. These microorganisms further metabolize these substances to produce various secondary metabolic flavor compounds such as organic acids, alcohols, esters, and aldehydes. Dominant strains strongly correlated with flavor formation can be broadly classified into three categories: bacteria, yeasts, and molds. Yeasts are divided into *Saccharomyces cerevisiae* and non-*Saccharomyces cerevisiae*. *Saccharomyces cerevisiae* is the main microorganism producing ethanol in various fermented wines such as baijiu, huangjiu, and fruit wines. Non-*Saccharomyces cerevisiae* primarily regulate the formation of flavor compounds.

[0003] Organic acids and acetates are characteristic flavor components of many fermented foods. For example, the characteristic volatile flavor compounds in red yeast rice wine include isobutanol, isoamyl alcohol, ethyl acetate, ethyl propionate, ethyl butyrate, and isoamyl acetate; the main flavor compounds in glutinous rice wine are ethyl acetate, isoamyl acetate, ethyl octanoate, ethyl decanoate, ethyl 2-methylpropionate, and butyl acetate; the formation of sweet, bitter, umami, and astringent flavors in Shaoxing wine is mainly related to organic acids, primarily acetic acid, propionic acid, butyric acid, valeric acid, and hexanoic acid.

[0004] The source of microorganisms in traditional fermented foods is shifting from relying on natural colony succession to artificial control, thereby reducing the uncertainty of the microbial community and achieving standardized production. Existing research on ester-producing yeasts in fermented foods has largely focused on ethyl ester-producing aroma-producing yeasts, with no research on non-Saccharomyces cerevisiae that produce multiple organic acids and acetate compounds. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to fill the gap in existing research by screening out a yeast strain that can produce organic acids and acetates from brewing yeast, which can be used as a fermentation agent for food to increase the content and variety of organic acids and acetates in fermented foods.

[0006] The technical solution adopted by this invention to solve its technical problem is as follows: This invention first provides a strain of *Fibrian seraphim* that can produce organic acids and acetate compounds, the strain being named *Fibrian seraphim*.Cyberlindnera fabianii It was deposited on December 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26368.

[0007] The ITS sequence of *Fibrian sericulture*, which can produce organic acids and acetate compounds, is shown in SEQ ID NO:1.

[0008] The ITS sequence of *Fibrian cerinensis*, SEQ ID NO:1, which produces organic acids and acetates: .

[0009] Among them, the organic acids produced by *Fibrian sericulture*, which can produce organic acids and acetate compounds, include acetic acid, hexanoic acid, isobutyric acid, and octanoic acid.

[0010] Among them, the acetate compounds produced by *Fibrian sericulture*, which can produce organic acids and acetate compounds, include ethyl acetate, isoamyl acetate, phenylethyl acetate, propyl acetate, and isobutyl acetate.

[0011] The organic acid production of *Fibrian sera Lindnerella vaginalis*, which can produce organic acids and acetates, is as follows: acetic acid 0.524 mg / L, hexanoic acid 0.544 mg / L, isobutyric acid 0.281 mg / L, and octanoic acid 0.124 mg / L; the acetate production is as follows: ethyl acetate 71.796 mg / L, isoamyl acetate 3.682 mg / L, phenethyl acetate 3.836 mg / L, propyl acetate 0.200 mg / L, and isobutyl acetate 0.657 mg / L.

[0012] Among them, the biological characteristics of the above-mentioned *Fibrian seraberlindnerella* that can produce organic acids and acetate compounds are as follows: when cultured on YPD solid medium at 30°C for 2-3 days, the colonies are round, milky white, with neat edges, smooth and moist surface, and are sticky and easy to pick up.

[0013] Among them, the YPD solid culture medium in the above-mentioned Fibrian Seberlindnerella vaginalis that can produce organic acids and acetate compounds is: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar 15g / L, pH 7.0, autoclaved at 115℃ for 20 minutes.

[0014] The present invention also provides the application of the above-mentioned Fibrian seraberlindnerella vaginalis, which is capable of producing organic acids and acetates, in the preparation of organic acids and acetates.

[0015] The process for preparing organic acids and acetates using the aforementioned *Fibrian sera Lindnerella* yeast, which can produce organic acids and acetates, is as follows: After activating *Fibrian sera Lindnerella*, it is inoculated into a fermentation medium, cultured at 30°C, with a shaking speed of 120 rpm, aerobic culture for 72 h, and a pH of 7.0. After fermentation, it is centrifuged and the supernatant is collected.

[0016] In the above process, the activation operation is as follows: Fibrian Seberlindnerella vaginalis is inoculated into YPD medium and cultured at 20-30℃ for 12-20h, and activated for three generations.

[0017] In the above process, the YPD culture medium consists of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, pH 7.0, and is autoclaved at 115°C for 20 minutes.

[0018] The beneficial effects of this invention are: This invention screened out a non-brewing yeast strain capable of producing various organic acids and acetate compounds from brewing yeast, named *Saccharomyces cerevisiae*. Cyberlindnera fabianii The non-Saccharomyces cerevisiae strains screened in this invention are designated LZLJ1-2. These strains can be applied to traditional fermented foods, broadening the selection of yeast strains for food fermentation and promoting the standardized industrial production of traditional fermented foods.

[0019] The *Fibriania cerevisiae* LZLJ1-2 of this invention has the accession number CGMCC No. 26368. The accession date was December 30, 2022. The accession center is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. It is classified and named accordingly. Cyberlindnera fabianii LZLJ1-2. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of *Fibrian sacchariformis* LZLJ1-2. Detailed Implementation

[0021] The technical solution of the present invention can be implemented in the following manner.

[0022] This invention first provides a strain of *Fébien Seberlindnerella vaginalis* capable of producing organic acids and acetate compounds, named *Fébien Seberlindnerella vaginalis* LZLJ1-2 (… Cyberlindnera fabianii It was deposited on December 30, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.26368.

[0023] The ITS sequence of *Fibrian sericulture*, which produces organic acids and acetate compounds according to the present invention, is shown in SEQ ID NO:1.

[0024] Among them, the organic acids produced by *Fibrian sericulture*, which can produce organic acids and acetate compounds, include acetic acid, hexanoic acid, isobutyric acid, and octanoic acid; the acetate compounds produced include ethyl acetate, isoamyl acetate, phenethyl acetate, propyl acetate, and isobutyl acetate.

[0025] The organic acid production of *Fibrian sera Lindnerella vaginalis*, which can produce organic acids and acetates, is as follows: acetic acid 0.524 mg / L, hexanoic acid 0.544 mg / L, isobutyric acid 0.281 mg / L, and octanoic acid 0.124 mg / L; the acetate production is as follows: ethyl acetate 71.796 mg / L, isoamyl acetate 3.682 mg / L, phenethyl acetate 3.836 mg / L, propyl acetate 0.200 mg / L, and isobutyl acetate 0.657 mg / L.

[0026] Among them, the biological characteristics of the above-mentioned *Fibrian seraberlindnerella* that can produce organic acids and acetate compounds are as follows: when cultured on YPD solid medium at 30°C for 2-3 days, the colonies are round, milky white, with neat edges, smooth and moist surface, and are sticky and easy to pick up.

[0027] Among them, the YPD solid culture medium in the above-mentioned Fibrian Seberlindnerella vaginalis that can produce organic acids and acetate compounds is: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar 15g / L, pH 7.0, autoclaved at 115℃ for 20 minutes.

[0028] Based on the above findings, the present invention also provides the application of the above-mentioned *Fibrian seraberlindnerella*, which can produce organic acids and acetates, in the preparation of organic acids and acetates.

[0029] The process for preparing organic acids and acetates using the aforementioned *Fibrian sera Lindnerella* yeast, which can produce organic acids and acetates, is as follows: After activating *Fibrian sera Lindnerella*, it is inoculated into a fermentation medium, cultured at 30°C, with a shaking speed of 120 rpm, aerobic culture for 72 h, and a pH of 7.0. After fermentation, it is centrifuged and the supernatant is collected.

[0030] In the above process, the activation operation is as follows: Fibrian Seberlindnerella vaginalis is inoculated into YPD medium and cultured at 20-30℃ for 12-20h, and activated for three generations.

[0031] In the above process, the YPD culture medium consists of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, pH 7.0, and is autoclaved at 115°C for 20 minutes.

[0032] The technical solution and effects of the present invention will be further explained below through practical examples. Example

[0033] The culture medium formulations involved in the examples are as follows: YPD medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, pH 7.0, autoclaved at 115℃ for 20 minutes.

[0034] YPD solid medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar 15g / L, pH 7.0, autoclaved at 115℃ for 20 minutes.

[0035] Example 1: Screening of yeast strains that produce organic acids and acetates Take 1.5 g of yeast sample and place it into a 250 mL Erlenmeyer flask containing glass beads and 50 mL of sterile water. Shake overnight at 115 r / min on a shaker at 37℃. After natural settling, take 1 mL of the supernatant and continuously dilute it to 10 with sterile water. -5 For each dilution gradient, 0.2 mL was spread onto Bengal Red solid medium plates and incubated at 30°C for 2-3 days. Then, vigorous single colonies were picked, isolated and purified by multiple streaking tests, and inoculated into YPD medium. The culture temperature was 30°C, the shaking speed was 120 rpm, and the culture time was 2-3 days. The volatile products were qualitatively determined by headspace solid phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS).

[0036] The specific methods for HS-SPME / GC-MS are as follows: (1) Inoculate the seed culture into YPD medium with 1% inoculation amount, 10 mL of sample bottle solution in 15 mL sample bottle, culture temperature 30℃, shaker speed 120 rpm, culture for 48 hours.

[0037] (2) HS-SPME extraction conditions: Insert the extraction head into the headspace of the sample vial and adsorb at 60℃ for 60 min. After adsorption, remove the extraction head and insert it into the gas chromatograph injection port and desorb at 230℃ for 5 min.

[0038] (3) GC analysis conditions: Gas chromatography conditions: HP-INNOWAX column (60 m × 0.25 mm × 0.25 μm); Temperature program: Initial temperature 40℃, hold for 5 min, increase to 100℃ at 4℃ / min, then increase to 230℃ at 6℃ / min, hold for 10 min, carrier gas is high-purity helium (1.0 mL / min); Injector temperature 250℃, splitless.

[0039] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230℃; transfer line temperature 250℃; mass range 40-450 m / z.

[0040] Based on the HS-SPME / GC-MS results, a yeast strain capable of producing organic acids and acetates was obtained and preserved at -80°C with 30% glycerol for further analysis.

[0041] Example 2 Molecular identification of yeasts producing organic acids and acetates Biological characteristics of the target yeast: Incubate on YPD solid medium at 30℃ for 2 days and observe colony morphology, such as... Figure 1 As shown, the colonies are round, milky white, with neat edges, smooth and moist surfaces, and are sticky and easy to pick up.

[0042] Molecular biological identification method (ITS sequence amplification): Take 1 mL of bacterial culture, centrifuge at 10000 rpm for 5 min, remove the supernatant to obtain bacterial sludge, add CTAB solution and phenol-chloroform-isoamyl alcohol (25:24:1) to extract nucleic acid DNA, centrifuge at 12000 rpm for 5 min to obtain the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1), mix well, centrifuge at 12000 rpm for 5 min to obtain the supernatant, wash twice with ethanol solution, centrifuge at 12000 rpm for 5 min to remove the supernatant, blow the precipitate dry, add 50 μL of sterile water to resuspend, and obtain the bacterial DNA template.

[0043] Primers were used for PCR amplification: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') with ITS sequence as shown in SEQ ID NO:2 and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') with ITS sequence as shown in SEQ ID NO:3.

[0044] ①Reaction system (20 μL) ②Reaction Procedure Pre-denaturation: 95℃, 5 min; denaturation: 95℃, 30 s; annealing: 56℃, 30 s; extension: 72℃, 1 min 30 s; repeat denaturation, annealing and extension for 25 cycles; final extension: 72℃, 10 min.

[0045] Sequencing was performed by Shanghai Sangon Biotech Co., Ltd. The gene sequence of the ITS fragment obtained from sequencing was compared with NCBI's BLAST algorithm to determine the strain species information, identifying it as *Saccharomyces cerevisiae*. Cyberlindnera fabianii It was named *Fibrian cyperrindnerella vaginalis* LZLJ1-2.

[0046] Example 3: Experiment on the detection of volatile metabolites in yeast (1) Sample preparation: Febian Seberlindnerella vaginalis LZLJ1-2 ( Cyberlindnerafabianii After dissolving the glycerol in the preservation tube, inoculate it into 5 mL of YPD medium and incubate at 30°C for 12-20 h. After three generations of activation, inoculate it into 400 mL of fermentation medium at a 1% (v / v) inoculation rate, incubate at 30°C and 120 rpm for 72 h. After fermentation, obtain the fermentation broth, centrifuge at 10000 rpm for 5 min, and collect the supernatant.

[0047] (2) Detection of volatile metabolites Volatile metabolites in the samples were detected using the HS-SPME / GC-MS method described in Example 1. 2-Octanol (0.822 mg / mL) was used as an internal standard. Compound search results were matched with the NIST standard spectral library; compounds with a similarity of over 80% were confirmed as target compounds. The culture broth without added bacteria served as a blank control group, and the content of each volatile substance was calculated.

[0048] The test results are shown in Table 1. The organic acids produced by fermentation of *Saccharomyces cerevisiae* LZLJ1-2 include acetic acid, hexanoic acid, isobutyric acid, and octanoic acid, with contents of 0.524 mg / L, 0.544 mg / L, 0.281 mg / L, and 0.124 mg / L, respectively. The acetate compounds produced include ethyl acetate, isoamyl acetate, phenethyl acetate, propyl acetate, and isobutyl acetate, with contents of 71.796 mg / L, 3.682 mg / L, 3.836 mg / L, 0.200 mg / L, and 0.657 mg / L, respectively.

[0049] Table 1. Results of detection of volatile metabolites in yeast

Claims

1. A type of *Fibrian seraberlindnerella* capable of producing organic acids and acetate compounds, characterized in that: The strain is named as Fabienne Cephaloascus weberi (C. weberi) Cyberlindnera fabianii ) LZLJ1-2, which was preserved in China General Microbiological Culture Collection Center on December 30, 2022, and the preservation number is CGMCC No. 26368.

2. The *Fibrian sera berry* species capable of producing organic acids and acetate compounds according to claim 1, characterized in that: The ITS sequence of *Fibrian sera lindnerella vaginalis*, which produces organic acids and acetates, is shown in SEQ ID NO:

1.

3. The *Fibrian sera berry* species capable of producing organic acids and acetate compounds according to claim 1, characterized in that: The organic acids produced by *Fibrian sericulture* include acetic acid, hexanoic acid, isobutyric acid, and octanoic acid; the acetates produced include ethyl acetate, isoamyl acetate, phenylethyl acetate, propyl acetate, and isobutyl acetate.

4. The *Fibrian sera berry* species capable of producing organic acids and acetate compounds according to claim 3, characterized in that: The organic acid production of *Fibrian sera lindnerella*, which produces organic acids and acetates, was as follows: acetic acid 0.524 mg / L, hexanoic acid 0.544 mg / L, isobutyric acid 0.281 mg / L, and octanoic acid 0.124 mg / L. The acetate production was as follows: ethyl acetate 71.796 mg / L, isoamyl acetate 3.682 mg / L, phenethyl acetate 3.836 mg / L, propyl acetate 0.200 mg / L, and isobutyl acetate 0.657 mg / L.

5. The *Fibrian sera berry* capable of producing organic acids and acetate compounds according to any one of claims 1-4, characterized in that: The biological characteristics of *Fibrian sera lindnerella*, which can produce organic acids and acetate compounds, are as follows: when cultured on YPD solid medium at 30°C for 2-3 days, the colonies are round, milky white, with neat edges, smooth and moist surfaces, and are sticky and easily picked up.

6. The *Fibrian sera berry* species capable of producing organic acids and acetate compounds according to claim 5, characterized in that: The YPD solid culture medium consisted of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 15 g / L agar, pH 7.0, and was autoclaved at 115°C for 20 minutes.

7. The use of *Fibrian sera lindnerella vaginalis*, as described in any one of claims 1-6, capable of producing organic acids and acetates, in the preparation of organic acids and acetates, characterized in that: The organic acids include acetic acid, hexanoic acid, isobutyric acid, and octanoic acid; the acetate compounds include ethyl acetate, isoamyl acetate, phenethyl acetate, propyl acetate, and isobutyl acetate.

8. The application of *Fibrian seralinedensis*, which is capable of producing organic acids and acetates according to claim 7, in the preparation of organic acids and acetates, characterized in that: After activating the *Fibrian sacchariformis*, it was inoculated into the fermentation medium and cultured at 30°C with a shaking speed of 120 rpm for 72 hours under aerobic conditions at a pH of 7.

0. After fermentation, the mixture was centrifuged and the supernatant was collected.

9. The application of *Fibrian seraphim*, capable of producing organic acids and acetates according to claim 8, in the preparation of organic acids and acetates, characterized in that: The activation process involves inoculating *Fibrian sera lindnerella vaginalis* into YPD medium and culturing it at 20-30℃ for 12-20 hours, followed by three generations of activation. The YPD medium consists of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, pH 7.0, and is autoclaved at 115℃ for 20 minutes.

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