Phaffia rhodozyma, its inoculant and use

Abstract

The application discloses a strain of Scheffersomyces fibuliger and a microbial agent and application thereof, and belongs to the field of biology.The Scheffersomyces fibuliger (Scheffersomyces fibuliger) Cyberlindnera fabianii ) LZLJ1-6 has a preservation number of CCTCC NO: M 2025590. Pure-strain fermentation of the strain can metabolically produce a plurality of flavor substances such as secondary octanone, nonanal and heptyl alcohol. Application of a microbial agent containing the strain to rice wine fermentation can significantly increase the richness of flavor substances in the rice wine, including a plurality of alcohol, ester, ketone, acid and phenolic compounds, and meanwhile, the content of some original flavor substances is increased. The strain provided by the application has characteristics in flavor metabolism, is particularly outstanding in synthesis of characteristic aroma components such as secondary octanone, and has important significance for enriching flavor levels and improving aroma complexity in wine production, especially rice wine fermentation, and also provides high-quality strain resources for development of new fermented foods.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a strain of *Fibrian cyperrindnerella vaginalis* (…). Cyberlindnera fabianii ) and its inoculants and applications. Background Technology

[0002] Sweet rice wine is a traditional Chinese alcoholic beverage with a long history. It is made primarily from high-quality glutinous rice through natural fermentation using a mixture of various microorganisms. Its smooth and mellow flavor, along with its delicate and pleasant aroma, makes it an indispensable part of Chinese culinary culture. Furthermore, the fermentation process generates various active substances such as oligosaccharides, polypeptides, amino acids, and vitamins, giving it rich nutritional value.

[0003] Rice wine starter (koji) is a crucial saccharification and fermentation agent in the rice wine brewing process. Its microbial community structure directly affects the quality of the starter and ultimately shapes the flavor and style of the rice wine. Traditional starter has a complex microbial composition, making it susceptible to various factors during production, such as raw materials, temperature, humidity, and environmental microorganisms. This results in poor batch-to-batch stability, affecting the controllability and consistency of production. To improve fermentation stability and reduce production fluctuations, commercially available rice wine starters are often prepared using single or a few pure microbial strains (such as highly saccharifying Rhizopus and highly alcohol-producing Saccharomyces cerevisiae). While this process improves controllability, it also results in a relatively simple flavor profile and aroma complexity in the brewed rice wine, lacking the rich taste characteristic of traditional rice wine.

[0004] Studies have shown that the rich aroma of traditional rice wine originates from the synergistic effect of various flavor compounds such as alcohols, esters, and ketones, with compounds like octanone playing a significant role in the formation of the wine's complex flavor profile. However, existing commercially available yeast starters, due to their limited microbial composition, often struggle to effectively generate these characteristic aroma components during fermentation, leading to severe homogenization of product flavors. Therefore, screening yeast strains with excellent aroma-producing characteristics, especially those capable of metabolizing and producing a variety of unique characteristic flavor compounds, from traditional rice wine starters not only helps to elucidate the microbial functions of traditional starters but also provides important microbial resources for developing innovative rice wine products with diverse flavors and prominent aromas. Summary of the Invention

[0005] To develop yeasts with unique flavor metabolism capabilities to effectively enrich the flavor compounds and enhance the aroma complexity of rice wine, this invention isolates and screens a new strain of *Saccharomyces cerevisiae* LZLJ1-6 from traditional rice wine yeast. Its accession number is CCTCC NO: M 2025590. The accession date is March 27, 2025. The depositary institution is the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The classification name is: Cyberlindnera fabianii When this strain is fermented in pure YPD liquid and malt extract liquid media, it can metabolize and produce a variety of aroma compounds, such as ethanol, nonanol, 2-ethylhexanol, isobutanol, isoamyl alcohol, n-heptanol, n-octanol, phenethyl alcohol, acetic acid, isobutyric acid, nonanal, phenylacetaldehyde, phenethyl acetate, ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl butyrate, 2-octanone, 3-ethyl-2,5-methylpyrazine, etc.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a strain of *Fibrian cytomegalovirus* (Fibrian cytomegalovirus). Cyberlindnera fabianii LZLJ1-6, its accession number is: CCTCC NO: M 2025590.

[0007] The ITS nucleotide sequence of the aforementioned *Fibrian cytomegalovirus* LZLJ1-6 is shown in SEQ ID NO:1.

[0008] SEQ ID NO:1: ITS nucleotide sequence of *Saccharomyces cerevisiae* strain LZLJ1-6: .

[0009] The biological characteristics of the aforementioned *Fibrian cytomegalovirus* LZLJ1-6 are as follows: the colonies are milky white, round with neat edges, smooth and moist surface, and easily picked up.

[0010] Secondly, the present invention provides a microbial inoculant containing live cells or dry cells of the aforementioned *Fibrian cytomegalovirus LZLJ1-6*.

[0011] Furthermore, the preparation method of the above-mentioned microbial inoculant includes the following steps: S1-1. Inoculate Febian Seberlindnerella vaginalis LZLJ1-6 into YPD liquid medium and culture to obtain activated bacterial solution; S1-2. Inoculate the activated bacterial solution into YPD liquid medium and culture to obtain seed culture; S1-3. Inoculate the seed liquid into the culture medium, and obtain the microbial agent through fermentation, drying, and pulverization.

[0012] Furthermore, in step S1-1 above, the inoculation amount of the *Fibriania cerevisiae* LZLJ1-6 is 5%~10% (v / v).

[0013] Furthermore, in steps S1-2 above, the inoculation amount of the activated bacterial solution is 5%~10% (v / v).

[0014] Furthermore, in steps S1-3 above, the inoculation amount of the seed liquid is 2% to 5% (v / m) of the dry weight of the culture medium.

[0015] Furthermore, in steps S1-3 above, the culture medium includes at least one of wheat bran, wheat, rice, and glutinous rice.

[0016] Furthermore, in steps S1-3 above, the fermentation temperature is 28~30℃ and the fermentation time is 2~3 days.

[0017] Furthermore, in steps S1-3 above, the drying temperature is 40~45℃ and the drying time is 12~24h.

[0018] Thirdly, the present invention provides the application of the above-mentioned *Fibrian sacchariformis* LZLJ1-6 or the above-mentioned microbial inoculant in the production of flavor substances, characterized in that the flavor substances include at least one of the following: ethanol, n-hexanol, 3-nonen-1-ol, benzyl alcohol, phenethyl alcohol, n-heptanol, n-octanol, nonanol, isobutanol, isoamyl alcohol, linalool, 2-ethylhexanol, dodecanol, ethyl acetate, isoamyl acetate, phenethyl acetate, isoamyl propionate, ethyl butyrate, ethyl hexanoate, propyl octyl lactone, propyl nonyl lactone, diisobutyl phthalate, acetic acid, butyric acid, isobutyric acid, 3-methylvaleric acid, nonanal, phenylacetaldehyde, 2,3-dihydro-2,2,6-trimethylbenzaldehyde, 2-octanone, methylheptenone, acetophenone, geranylacetone, 4-vinyl-2-methoxyphenol, 1,3-di-tert-butylbenzene, and 3-ethyl-2,5-methylpyrazine.

[0019] Fourthly, the present invention provides the application of the above-mentioned *Fibrian Seberlindnerella vaginalis* LZLJ1-6 or the above-mentioned microbial agents in fermented wine brewing, fermented food or yeast.

[0020] Furthermore, the aforementioned fermented wine is at least one of rice wine, yellow wine, sake, beer, wine, or fruit wine.

[0021] Furthermore, the aforementioned fermented food is at least one of sourdough, enzymes, soy sauce, or fermented soybean paste.

[0022] Furthermore, the aforementioned yeast is at least one of bran yeast, small yeast, medium-temperature large yeast, or high-temperature large yeast.

[0023] Fifthly, the present invention provides the application of the above-mentioned *Fibrians cerevisiae* LZLJ1-6 or the above-mentioned microbial inoculant in the preparation of rice wine.

[0024] Furthermore, the above applications include increasing the richness of flavor compounds in rice wine; said flavor compounds include at least one of 2-undecen-1-ol, trans-2-octen-1-ol, 3-nonen-1-ol, nonanol, ethyl 3-phenylpropionate, ethyl 3-hydroxyhexanoate, ethyl 3-hydroxyoctanoate, ethyl acetate, phenethyl butyrate, 3-methylvaleric acid, acetophenone, geranylacetone, 4-vinyl-2-methoxyphenol, and 4-ethyl-2-methoxyphenol.

[0025] Furthermore, the above applications include increasing the content of flavor substances in rice wine; said flavor substances include at least one of 2-ethylhexanol, benzyl alcohol, isoamyl acetate, phenethyl acetate, acetic acid, octanone, and 4-vinylphenol.

[0026] Furthermore, the method described above includes the following steps: S2-1. Wash, soak, steam, and cool the glutinous rice, then inoculate it with commercial rice wine yeast for the first fermentation. S2-2. After the initial fermentation is complete, centrifuge to collect the supernatant and filter to remove bacteria; S2-3. Inoculate the supernatant after step S2-2 with microbial inoculum for further fermentation to obtain rice wine.

[0027] Furthermore, in step S2-1 above, the inoculation amount of the commercial rice wine starter is 0.2%~0.8% (v / m) of the dry weight of the glutinous rice.

[0028] Furthermore, in step S2-1 above, the temperature of the initial fermentation is 25~30℃, and the fermentation time is 2~3 days.

[0029] Furthermore, in step S2-2 above, a 0.22μm filter membrane is used for filtration and sterilization.

[0030] Furthermore, in steps S2-3 above, the inoculation amount of the microbial agent is 0.2%~0.5% (v / m) of the dry weight of the glutinous rice.

[0031] Furthermore, in steps S2-3 above, the concentration of the microbial agent is 10. 8 ~10 9 CFU / g.

[0032] Furthermore, in steps S2-3 above, the temperature for the second fermentation is 25~30℃, and the fermentation time is 2~3 days.

[0033] The beneficial effects of this invention are as follows: This invention isolates, screens, and identifies a strain of *Saccharomyces cerevisiae* LZLJ1-6 from traditional rice wine yeast, with the preservation number CCTCC NO: M 2025590. Under pure culture fermentation conditions, this strain can metabolize and produce abundant flavor compounds, including various alcohols, esters, ketones, acids, and lactones such as n-hexanol, phenethyl alcohol, isoamyl acetate, and nonyl lactone. Among these, the synthesis capacity of 2-octanone is significant, reaching 1.6031 mg / L in YPD liquid medium, accounting for 28.35% of the total flavor compounds. During the fermentation of glutinous rice juice, 2-octanone accounts for up to 50.07% of the total flavor compounds, and the fermented glutinous rice juice exhibits a rich, complex floral and fruity aroma, demonstrating excellent aroma-producing characteristics.

[0034] The strain was prepared into a microbial inoculant and applied to rice wine fermentation, which significantly improved the flavor profile and aroma complexity of the rice wine. Experimental results showed that after adding *Saccharomyces cerevisiae* LZLJ1-6, the content of octanone in rice wine increased from 0.0849 mg / L to 0.1333 mg / L, an increase of 57%. Simultaneously, various flavor compounds were newly added, including 2-undecen-1-ol, ethyl 3-phenylpropionate, ethyl 3-hydroxyhexanoate, ethyl 3-hydroxyoctanoate, ethyl acetate, 3-methylvaleric acid, acetophenone, 4-vinyl-2-methoxyphenol, and 4-ethyl-2-methoxyphenol. Furthermore, the contents of 2-ethylhexanol, benzyl alcohol, isoamyl acetate, phenylethyl acetate, and acetic acid were further increased. These flavor compounds encompass alcohols, esters, ketones, acids, and phenols, collectively contributing to a richer fruity, floral, and complex aroma in the rice wine, significantly improving its sensory quality.

[0035] The *Fibriania cerevisiae* LZLJ1-6 strain provided in this invention is derived from traditional yeast starter and exhibits excellent food safety and fermentation adaptability. This strain possesses metabolic characteristics that synthesize substances such as 2-octanone, providing a key microbial resource for enhancing the complexity and layering of flavor in rice wine. Furthermore, this strain not only contributes to a deeper understanding of the complex microbial interaction mechanisms in traditional yeast starter but also provides crucial support for the stable improvement of yeast starter performance, the systematic optimization of rice wine flavor, and the differentiated development of specialty products, thus holding potential significance for promoting technological upgrading and quality improvement in the rice wine industry. Attached Figure Description

[0036] Figure 1 This is a colony morphology diagram of *Fibrian cytomegalovirus* LZLJ1-6. Figure 2 GCMS image of fermented glutinous rice juice by pure strain of *Fibrian cyperrindnerella vaginalis* LZLJ1-6. Figure 3GCMS image of pure culture of *Nardella ferruginis* LZLJ1-6 fermented on YPD liquid medium; Figure 4 GCMS image of pure culture of *Fibrian cytomegalovirus LZLJ1-6* fermented malt extract liquid medium; Figure 5 GCMS image of fermented rice wine using *Fibrian cyperinae* LZLJ1-6.

[0037] Preservation instructions for *Fibrian sacchariformis* LZLJ1-6 of this invention: The accession number is CCTCC NO: M 2025590. The accession date is March 27, 2025. The depositary institution is the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The classification name is: Cyberlindnera fabianii . Detailed Implementation

[0038] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0039] In one embodiment of the present invention, a strain of *Saccharomyces cerevisiae* LZLJ1-6 was isolated and screened from traditional rice wine yeast, with the preservation number CCTCC NO: M 2025590. *Saccharomyces cerevisiae* LZLJ1-6 was identified through a combination of morphological characteristics and molecular biological analysis. Its morphological characteristics are: milky white, round colonies with neat edges, smooth and moist surface, and easily picked up. Its ITS nucleotide sequence is shown in SEQ ID NO:1.

[0040] In one embodiment of the present invention, a microbial inoculant is provided, the microbial inoculant containing live cells or dry cells of *Neptunia fusbeckia orientalis* LZLJ1-6, with a live cell count of 10-1. 8 ~10 9 CFU / g.

[0041] In some specific embodiments of the present invention, the preparation method of the microbial agent is as follows: *Fibrian Seberlindnerella vaginalis* LZLJ1-6 is inoculated into YPD liquid medium at 5%~10% (v / v) and cultured to obtain an activated bacterial solution; the activated bacterial solution is inoculated into YPD liquid medium at 5%~10% (v / v) and cultured to obtain a seed culture; the seed culture is inoculated into at least one culture medium of wheat bran, wheat, rice, or glutinous rice at 2%~5% (v / m) of the dry weight of the culture medium, and fermented at 28~30℃ for 2~3 days; the fermentation product is dried at 40~45℃ for 12~24 hours and then pulverized to obtain the final product.

[0042] In one embodiment of the present invention, the use of *Fibrian sacchariformis* LZLJ1-6 or microbial agents containing therein in the production of flavor substances is provided.

[0043] In some specific embodiments of the present invention, the application involves inoculating activated *Fibrian sacchariformis* LZLJ1-6 into YDP liquid medium, glutinous rice juice fermentation medium, or malt juice liquid medium, and culturing at 28-30°C for 3-6 days. The resulting metabolic products include ethanol, n-hexanol, 3-nonen-1-ol, benzyl alcohol, phenethyl alcohol, n-heptanol, n-octanol, nonanol, isobutanol, isoamyl alcohol, linalool, 2-ethylhexanol, dodecanol, ethyl acetate, and isopentyl acetate. At least one flavoring substance selected from the following: ester, phenethyl acetate, isoamyl propionate, ethyl butyrate, ethyl hexanoate, octyl lactone, nonyl lactone, diisobutyl phthalate, acetic acid, butyric acid, isobutyric acid, 3-methylvaleric acid, nonanal, phenylacetaldehyde, 2,3-dihydro-2,2,6-trimethylbenzaldehyde, octanone, methylheptenone, acetophenone, geranylacetone, 4-vinyl-2-methoxyphenol, 1,3-di-tert-butylbenzene, and 3-ethyl-2,5-methylpyrazine.

[0044] In one embodiment of the present invention, the application of *Fibrian sacchariformis* LZLJ1-6 or a microbial agent containing therein in the brewing of fermented beverages is provided. The fermented beverage is at least one of rice wine, yellow wine, sake, beer, wine, or fruit wine; preferably rice wine.

[0045] In some specific embodiments of the present invention, the application method is as follows: Glutinous rice is washed and soaked for 4-8 hours, steamed for 30-40 minutes, then mixed with 30% (v / m) of cooled boiled water by dry weight of the glutinous rice and spread out to cool to 28-35°C. 0.2%-0.8% (v / m) of commercial rice wine koji (a formula consisting of Rhizopus and Saccharomyces cerevisiae) is inoculated based on the dry weight of the glutinous rice, and fermented at 25-30°C for 2-3 days. After fermentation, the supernatant is centrifuged and filtered through a 0.22μm filter membrane for sterilization. 0.2%-0.5% (v / m) of microbial inoculant based on the dry weight of the glutinous rice is added to the supernatant, the inoculant having a viable count of 10-1. 8 ~10 9CFU / g, continue fermentation at 25~30℃ for 2~3 days to obtain rice wine.

[0046] In some specific embodiments of the present invention, the rice wine prepared by the above application method has a variety of added flavor substances, specifically including at least one of the following: 2-undecen-1-ol, trans-2-octen-1-ol, 3-nonen-1-ol, nonanol, ethyl 3-phenylpropionate, ethyl 3-hydroxyhexanoate, ethyl 3-hydroxyoctanoate, ethyl acetate, phenylethyl butyrate, 3-methylvaleric acid, acetophenone, geranylacetone, 4-vinyl-2-methoxyphenol, and 4-ethyl-2-methoxyphenol.

[0047] In some specific embodiments of the present invention, the rice wine prepared by the above application method has an increased content of various flavor substances, specifically including at least one of 2-ethylhexanol, benzyl alcohol, isoamyl acetate, phenethyl acetate, acetic acid, octanone, and 4-vinylphenol.

[0048] In one embodiment of the present invention, the use of *Fibrian sacchariformis* LZLJ1-6 or a microbial agent containing therein in fermented foods is provided. The fermented food is at least one of sourdough, enzymes, soy sauce, or fermented soybean paste.

[0049] In one embodiment of the present invention, the application of *Fibrian sacchariformis* LZLJ1-6 or a microbial agent containing therein in a brewing starter is provided. The brewing starter is at least one of bran starter, small starter, medium-temperature starter, or high-temperature starter.

[0050] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0051] The culture medium and detection method involved in the embodiments of the present invention are as follows: I. Culture medium formulation Bengal Red Solid Medium: Peptone 5 g / L, Glucose 10 g / L, Potassium dihydrogen phosphate 1 g / L, Magnesium sulfate 0.5 g / L, Bengal Red 0.03 g / L, Chloramphenicol 0.1 g / L, Agar 15 g / L, pH 7.0, autoclaved at 115°C for 20 minutes.

[0052] YPD liquid culture medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, pH 7.0, autoclaved at 115℃ for 20 minutes.

[0053] 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.

[0054] Glutinous rice juice fermentation medium: Mix glutinous rice flour and water in a beaker at a ratio of 1:50 (g / mL) (mix with a small amount of cold water first, then add hot water), heat on an electric stove to boiling, keep for 2-3 minutes until the liquid is slightly transparent, filter the glutinous rice particles with four layers of gauze, and sterilize the filtrate at 121℃ for 20 minutes.

[0055] Malt extract liquid culture medium: 130 g / L malt extract powder, 0.1 g / L chloramphenicol, pH 7.0, autoclaved at 115°C for 20 minutes.

[0056] II. Methods for detecting volatile substances The specific method for determining volatile products using headspace solid-phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS) is as follows: Take the sample and add it to the headspace vial, add 2g NaCl, 4mL pure water, and 10μL internal standard (0.1644mg / mL 2-octanol). After equilibration at 55℃ for 5min, extract with a 50 / 30μm DVB / CAR / PDMS extraction head at 55℃ for 45min. After extraction, desorb at 250℃ for 5min at the GC inlet.

[0057] GC-MS detection chromatographic conditions Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: initial temperature 40℃, hold for 1 min, increase to 75℃ at 2.5℃ / min, increase to 162℃ at 3℃ / min, increase to 230℃ at 6℃ / min, hold for 5 min; carrier gas is high-purity helium (0.8mL / min); injection port temperature 250℃, splitless.

[0058] 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.

[0059] Using 0.1644 mg / mL 2-octanol as an internal standard, the compound search results were matched with the NIST standard spectral library, and the similarity was more than 80%, confirming it as the target compound.

[0060] Example 1: Isolation and screening of *Nardostachys ferruginea* LZLJ1-6 (1) Take 10 g of traditional rice wine starter sample and put it into a 250 mL Erlenmeyer flask containing glass beads and 90 mL of sterile water. Shake on a shaker at room temperature and 150 r / min for 30 min to prepare a sample bacterial suspension for later use. Take 1 mL of the supernatant of the starter culture suspension and 9 mL of sterile water into a 15 mL centrifuge tube to prepare 10 g of traditional rice wine starter sample and put it into a 15 mL centrifuge tube to prepare 10 mL of traditional rice wine starter sample and put it into a 15 mL centrifuge tube to prepare 10 mL of traditional rice wine starter sample and put it into a 15 mL centrifuge tube to prepare 15 ... -2 The diluted solution was serially diluted using this method to prepare 10 solutions. -2 10 -3 10 -4 10 -5 10 -6 The dilution was prepared by spreading 100 μL of each dilution onto Bengal Red solid agar plates and incubating at 30°C for 2–3 days. Vigorous single colonies were then picked and streaked multiple times until all single colonies were purified. The isolated single colonies were inoculated into glutinous rice juice fermentation medium and incubated at 30°C with a shaking speed of 120 rpm for 4–6 days. Based on colony morphology and sensory evaluation of the fermented juice flavor, a strain LZLJ1-6 was selected. This strain exhibited milky white, round colonies with neat edges, a smooth and moist surface, and was easily picked. Its colony morphology is shown in the figure below. Figure 1 As shown. The fermented glutinous rice juice exhibits a rich, complex floral and fruity aroma, as shown in the GCMS chromatogram. Figure 2 As shown in Table 1, the flavor compounds were not detected ("—" indicates not detected). The bacteria were stored at -80°C in 20% glycerol for further analysis.

[0061] Table 1. Results of flavor compound detection (mg / L)

[0062] Table 1 shows that, compared with the blank glutinous rice juice, strain LZLJ1-6 was able to metabolize and produce a variety of flavor compounds in the glutinous rice juice fermentation medium, including n-hexanol, phenethyl alcohol, isoamyl alcohol, 2-ethylhexanol, isoamyl acetate, phenethyl acetate, nonyl lactone, and 2-octanone. Among them, the content of 2-octanone was particularly prominent, at 0.5688 mg / L, accounting for 50.07% of the total flavor compounds. The glutinous rice juice fermented by this strain exhibited a rich, complex floral and fruity aroma and had excellent aroma-producing characteristics.

[0063] (2) Take 1 mL of the bacterial culture of strain LZLJ1-6 selected in Example 1, centrifuge at 10000 rpm for 5 min, remove the supernatant, and 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 and take the supernatant. Add an equal volume of chloroform-isoamyl alcohol (24:1), mix well, centrifuge at 12000 rpm for 5 min and take the supernatant. Wash twice with ethanol solution, centrifuge at 12000 rpm for 5 min and remove the supernatant. Dry the precipitate and resuspend it in 50 μL of sterile water to obtain the strain DNA template. The obtained DNA template is used for species identification. Using the universal fungal primers ITS1 / ITS4, the extracted strain genomic DNA is amplified by PCR. Sequencing yields the ITS fragment as shown in SEQ ID NO:1. The sequencing results are compared with the NCBI database using BLAST to determine the species.

[0064] Among them, the primers used for PCR are: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 2) ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 3) Based on morphological characteristics and molecular identification, strain LZLJ1-6 was finally identified as *Saccharomyces ferrugins*, and deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 2025590. The deposit date is March 27, 2025. The address of the depository is: Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. Classification name: Cyberlindnera fabianii .

[0065] Example 2: Metabolic capacity of *Neptunia ferruginea* LZLJ1-6 After the glycerol preservation tubes of the bacterial strain were dissolved, they were inoculated into 5 mL of YPD liquid medium and cultured at 30°C for 48 h. After three generations of activation, the inoculum was added to 400 mL of YPD liquid medium and malt extract liquid medium at a 1% (v / v) inoculation rate, respectively. The culture temperature was 30°C, the shaking speed was 120 rpm, and the culture time was 72 h. After fermentation, the fermentation broth was obtained. After centrifugation at 10000 rpm for 5 min, the supernatant was collected for volatile flavor compound detection. The uninoculated medium was used as a blank control. The GCMS detection chromatogram of fermentation in YPD liquid medium is shown below. Figure 3 As shown, the GCMS detection chromatogram of malt extract fermentation in liquid culture medium is as follows: Figure 4 As shown in Table 2, the results of the analysis of volatile flavor compounds are shown in Table 2 ("——" indicates not detected).

[0066] Table 2. Results of volatile flavor compounds (mg / L)

[0067] Table 2 shows that strain LZLJ1-6 can metabolize and produce abundant flavor compounds in both YPD liquid medium and wort liquid medium, including ethanol, isoamyl alcohol, phenethyl alcohol, ethyl acetate, isoamyl acetate, phenethyl acetate, and 2-octanone. The content of 2-octanone in YPD liquid medium is as high as 1.6031 mg / L, while in wort liquid medium it is 0.7823 mg / L, further confirming that this strain has the inherent metabolic ability to synthesize 2-octanone. In addition, acetic acid, isobutyric acid, phenylacetaldehyde, ethyl butyrate, and 3-ethyl-2,5-methylpyrazine were also detected in wort liquid medium, indicating that it has good metabolic adaptability to different brewing substrates.

[0068] Example 3: Preparation of *Nardella hyacinthiae* LZLJ1-6 inoculum from *Fibriania seppuku*. (1) Take the glycerol preservation tube of Febian Seberlindnerella vaginalis LZLJ1-6 and inoculate it into 1 mL of YPD liquid medium at an inoculation rate of 10% (v / v). Incubate it in a shaker at 120 r / min to activate the strain and obtain activated bacterial solution. Transfer the activated bacterial solution to 10 mL of YPD liquid medium at an inoculation rate of 5% (v / v) and incubate it in a shaker at 120 r / min to obtain seed liquid.

[0069] (2) Weigh out wheat bran, with the weight of wheat bran in each bottle being 8%~10% of the volume of the Erlenmeyer flask (i.e., 40~50g of material in a 500mL Erlenmeyer flask). Add 70%~80% water (based on the dry weight of the wheat bran), stir evenly, and sterilize at 121℃ for 30 min. Remove the flask and break up any lumps of wheat bran while it is still hot. After cooling, inoculate the seed liquid at 2%~5% (v / m) of the dry weight of the wheat bran, stir evenly, and incubate at 30℃ for 2 days. Dry and pulverize at 40℃ to obtain the inoculum.

[0070] Example 4: Application of *Saccharomyces cerevisiae* LZLJ1-6 inoculum from the Fiberon Silberlind strain. Take fresh, mold-free glutinous rice, wash it, soak it for 4-8 hours, drain it, and steam it for 30-40 minutes. Add about 30% (by dry weight of glutinous rice) of cooled boiled water to loosen the steamed glutinous rice and spread it out to cool to 28-35℃. Add commercial rice wine yeast at 0.2% (v / m) of the dry weight of glutinous rice. The yeast strains are Rhizopus and brewer's yeast. Ferment at 28℃ for 2 days.

[0071] After fermentation, the fermentation mash was centrifuged, and the supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization. The absence of microbial residue in the filtrate was verified on YPD solid culture plates. In the sterilized supernatant, the experimental group added *Saccharomyces cerevisiae* LZLJ1-6 inoculum (concentration of 10⁻⁶) at 0.2% (v / m) of the dry weight of the glutinous rice. 5 (CFU / g), while no addition was made to the control group. After stirring evenly, fermentation continued at 28℃ for 2 days. After fermentation, samples were taken for volatile flavor compound analysis, and the GCMS chromatogram is shown below. Figure 5 As shown in Table 3, some flavor compounds were detected ("——" indicates not detected).

[0072] Table 3. Detection results of some volatile compounds in rice wine (mg / L)

[0073] Table 3 shows that, compared with the control group, the rice wine in the experimental group with added *Saccharomyces cerevisiae* LZLJ1-6 significantly increased in both the types and contents of flavor compounds. Specifically, the content of octanone increased from 0.0849 mg / L to 0.1333 mg / L, an increase of 57%. Simultaneously, various flavor compounds were newly added, including 2-undecen-1-ol, ethyl 3-phenylpropionate, ethyl 3-hydroxyhexanoate, ethyl 3-hydroxyoctanoate, ethyl acetate, 3-methylvaleric acid, acetophenone, 4-vinyl-2-methoxyphenol, and 4-ethyl-2-methoxyphenol. Furthermore, the contents of 2-ethylhexanol, benzyl alcohol, isoamyl acetate, phenylethyl acetate, and acetic acid were also further increased. These results indicate that this strain can utilize precursors produced by *Rhizopus* and *Saccharomyces cerevisiae* (such as ethyl palmitate, ethyl linoleate, and 4-ethylphenol) to further ferment and synthesize various flavor compounds, thereby significantly enhancing the flavor richness and aroma complexity of the rice wine.

Claims

1. Fabian Seberlindnerella vaginalis ( Cyberlindnera fabianii LZLJ1-6, characterized in that, The accession number is: CCTCC NO: M 2025590.

2. A microbial inoculant, characterized in that: Live or dry cells containing the *Fibrians cerevisiae* LZLJ1-6 as described in claim 1.

3. The application of the *Fibriania cerevisiae* LZLJ1-6 as described in claim 1 or the microbial inoculum as described in claim 2 in the production of flavor substances, characterized in that... The flavoring substances include at least one of the following: ethanol, n-hexanol, 3-nonen-1-ol, benzyl alcohol, phenethyl alcohol, n-heptanol, n-octanol, nonanol, isobutanol, isoamyl alcohol, linalool, 2-ethylhexanol, dodecanol, ethyl acetate, isoamyl acetate, phenethyl acetate, isoamyl propionate, ethyl butyrate, ethyl hexanoate, propyloctyl lactone, propylnonyl lactone, diisobutyl phthalate, acetic acid, butyric acid, isobutyric acid, 3-methylvaleric acid, nonanal, phenylacetaldehyde, 2,3-dihydro-2,2,6-trimethylbenzaldehyde, 2-octanone, methylheptenone, acetophenone, geranylacetone, 4-vinyl-2-methoxyphenol, 1,3-di-tert-butylbenzene, and 3-ethyl-2,5-methylpyrazine.

4. The application of the *Fibrian sacchariformis* LZLJ1-6 as described in claim 1 or the microbial agent as described in claim 2 in fermented wine brewing, fermented food, or yeast starter.

5. The application according to claim 4, characterized in that: The fermented wine is at least one of rice wine, yellow wine, sake, beer, wine, or fruit wine; the fermented food is at least one of sourdough, enzyme, soy sauce, or broad bean paste; and the yeast is at least one of bran yeast, small yeast, medium-temperature large yeast, or high-temperature large yeast.

6. The use of the *Fibrian sacchariformis* LZLJ1-6 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of rice wine.

7. The application according to claim 6, characterized in that: The application is to increase the richness of flavor compounds in rice wine; The flavoring substances include at least one of 2-undecen-1-ol, trans-2-octen-1-ol, 3-nonen-1-ol, nonanol, ethyl 3-phenylpropionate, ethyl 3-hydroxyhexanoate, ethyl 3-hydroxyoctanoate, ethyl acetate, phenethyl butyrate, 3-methylvaleric acid, acetophenone, geranylacetone, 4-vinyl-2-methoxyphenol, and 4-ethyl-2-methoxyphenol.

8. The application according to claim 6, characterized in that: The application is to increase the content of flavor substances in rice wine; The flavoring substances include at least one of 2-ethylhexanol, benzyl alcohol, isoamyl acetate, phenethyl acetate, acetic acid, octanone, and 4-vinylphenol.

9. The application according to any one of claims 6 to 8, characterized in that, The method of application includes the following steps: S1. Wash, soak, steam, and cool the glutinous rice, then inoculate it with commercial rice wine yeast for the first fermentation. S2. After the initial fermentation is complete, centrifuge to collect the supernatant, and then filter to remove bacteria; S3. Inoculate the supernatant after step S2 with microbial inoculants for further fermentation to obtain rice wine.

10. The application according to claim 9, characterized in that: In step S1, the inoculation amount of the commercial rice wine starter is 0.2%~0.8% (v / m) of the dry weight of glutinous rice. In step S1, the temperature of the initial fermentation is 25~30℃, and the fermentation time is 2~3 days; In step S2, a 0.22μm filter membrane is used for filtration and sterilization. In step S3, the inoculation amount of the microbial agent is 0.2%~0.5% (v / m) of the dry weight of glutinous rice. In step S3, the concentration of the microbial agent is 10. 8 ~10 9 CFU / g; In step S3, the temperature for the second fermentation is 25~30℃, and the fermentation time is 2~3 days.

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