Bacillus subtilis with high yield of terpenoids, screening method and application of bacillus subtilis

Abstract

The invention discloses bacillus subtilis with high yield of terpenoids as well as a screening method and application of the bacillus subtilis, and belongs to the technical field of brewing. The bacillus subtilis YB4 disclosed by the invention has good ethanol tolerance (20%) and sodium chloride tolerance (18%), and has the characteristic of producing terpene substances by fermentation, and the content of terpene compounds produced by fermentation in a glutinous rice saccharification liquid for 5 days is 277.78 + / -13.78 [mu] g / L; the content of terpene compounds produced by fermenting for 20 days in yellow wine brewing is 234.25 + / -14.89 [mu] g / L, and an effective biological way is provided for increasing the content of the terpene compounds in brewed food.

Description

Technical Field

[0001] This invention relates to a Bacillus subtilis strain that produces high levels of terpenoids, its screening method, and its applications, belonging to the field of brewing technology. Background Technology

[0002] Yellow wine is one of the oldest types of alcoholic beverages in my country. Its unique and complex flavor, along with its rich functional components, makes it a favorite among consumers. The flavor and health benefits of yellow wine are crucial factors determining its quality. To date, over 340 volatile components have been identified in yellow wine, fully demonstrating the complexity and diversity of its volatile components.

[0003] Terpenes are important flavor compounds in alcoholic beverages and a key component of the aroma of Shaoxing wine. Terpenes possess aromatic qualities that impart unique sensory qualities to food. Furthermore, they exhibit significant effects in antioxidation, antibacterial activity, enhancing human immunity, and cancer prevention. For example, β-caryophyllene can effectively inhibit oxidative stress and inflammatory responses, while geranylacetone can improve cellular antioxidant capacity by increasing antioxidant enzyme activity and reducing malondialdehyde content. In addition, related studies have shown that terpenes can improve the flavor of alcoholic beverages, making them more delicate and mellow. The rich aroma and balanced, mellow characteristics of Shaoxing wine are particularly appealing to Chinese sensory preferences. Currently, research on terpenes in alcoholic beverages mainly focuses on wines and some fermented fruit wines. Data indicates that the content of terpenes in alcoholic products ranges from 50 to 800 μg / L. However, due to the low detection limit and complex detection methods for terpenes, coupled with the complexity of Shaoxing wine's composition, research on terpenes in Shaoxing wine remains relatively scarce.

[0004] The application of enhanced microorganisms in brewing is currently a mainstream method for improving the quality of alcoholic beverages, mainly reflected in the following aspects: First, by screening and enhancing specific functional microorganisms, the content of certain flavor compounds in the liquor can be increased. In baijiu brewing, enhancing Bacillus subtilis can increase the yield of pyrazine compounds, thereby enhancing specific flavors such as caramel and nutty aromas. Second, enhanced microorganisms can improve the saccharification, esterification, and fermentation power of the starter culture, thereby improving fermentation efficiency and alcohol yield. In addition, by selecting and enhancing specific functional microorganisms, changes in the microbial community caused by environmental factors can be reduced, thus improving the consistency of liquor quality. For example, inoculating functional yeasts such as *Saccharomyces cerevisiae*, *Wickham's yeast*, and *Pichia pastoris* into Maotai starter culture can improve the stability of the brewing system, reduce the impact of environmental factors on the starter culture microbial community, and improve the consistency of baijiu quality.

[0005] Bacillus subtilis has been rated as a Generally Recognized As Safe (GRAS) strain by the U.S. Food and Drug Administration (FDA). It secretes alkaline protease and amylase, participating in the synthesis of various aroma compounds, and is one of the main microorganisms in Maotai-flavor liquor starter. In the literature "Screening of Bacillus subtilis from Daqu Sources Based on Enzymatic Characteristics for Enhanced Brewing," researchers applied Bacillus licheniformis and Bacillus subtilis, both selected for their amylase and protease production capabilities, to the brewing process of light-aroma baijiu. They found that after enhanced fermentation, differential metabolites appeared in both strains, and the content of some flavor compounds increased significantly. The article "Research Progress on the Influence of Starter Microbial Community on the Flavor of Brewed Wine Products" points out that Bacillus subtilis is one of the main bacteria in Maotai-flavor liquor starter, capable of secreting alkaline protease and amylase, and participating in the synthesis of various aromatic compounds. All of the above evidence indicates that Bacillus subtilis, as a high-potential beneficial microorganism, has significant value for the production of alcoholic beverages.

[0006] However, there are currently no reports on the application of enhanced terpene-producing strains in the production of rice wine. Screening for a Bacillus subtilis strain with a high capacity for producing terpenoids is of great significance for improving the flavor and quality of rice wine. Summary of the Invention

[0007] Based on existing technical and process problems, this invention aims to provide a strain that produces high levels of terpenoid compounds, a screening method, and applications.

[0008] This study screened a strain of Bacillus subtilis with strong terpene-producing ability during the brewing process of rice wine and applied it to rice wine brewing. This not only ensured the microbial safety of the rice wine brewing process, but also improved the flavor and health value of rice wine, providing a new theoretical basis and ideas for the development and utilization of new healthy rice wines.

[0009] This invention provides a high-terpene-producing bacterial strain, namely Bacillus subtilis YB4, which was deposited on March 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.64945, located at No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0010] In one embodiment of the present invention, the Bacillus subtilis is obtained from rice wine mash through separation, purification and screening verification.

[0011] In one embodiment of the present invention, the screening step is as follows:

[0012] Step 1: Pretreatment of Shaoxing wine mash

[0013] Take 100mL of rice wine mash and dry it in an oven at 55℃. Crush the dried sample and set it aside. Perform dry heat treatment followed by wet heat treatment on the dried mash sample. The specific procedure is as follows: Weigh 10g of mash sample into a sterile beaker, seal the beaker and place it in an oven at 100℃ for 10min. Then, put the mash sample into an Erlenmeyer flask containing 90mL of sterile physiological saline and glass beads, heat it in a 50℃ constant temperature water bath for 10min, and then shake and incubate it for 30min for later use.

[0014] Step 2: Isolation and purification of bacterial strains

[0015] Take the pretreated distiller's grains suspension from step one and perform a 10-fold serial dilution four times. Take 10... 4 Diluted bacterial suspensions were spread onto isolation medium and incubated upside down in a 37°C incubator. Single colonies with different morphologies were selected and streaked onto solid medium for further isolation and purification.

[0016] Step 3: Screening of terpene-producing microorganisms and verification through liquid fermentation

[0017] Each isolated strain was subjected to liquid fermentation experiments to produce terpenes. The content of terpenes in the fermentation broth was determined by gas chromatography-mass spectrometry, and the strain with the strongest terpene-producing ability was named YB4.

[0018] The method for preparing the liquid fermentation culture medium is as follows: referring to the brewing ratio of rice wine, using raw glutinous rice as the base, steamed glutinous rice (150%, m / m), water (400%, v / m), wheat koji (15%, m / m), saccharifying enzyme (1‰, v / m) and liquefying enzyme (2‰, v / m) are mixed in proportion, stirred evenly, and saccharified in a 60℃ water bath for 4-6 hours until the sugar content reaches above 13. The glutinous rice lees are filtered out, and the saccharified liquid is dispensed into Erlenmeyer flasks and sterilized at 115℃ for 20 minutes.

[0019] The present invention also provides a microbial preparation containing the aforementioned Bacillus subtilis YB4.

[0020] In one embodiment, the number of Bacillus subtilis YB4 cells in the microbial preparation is at least 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0021] The present invention also provides wheat koji containing Bacillus subtilis YB4, wherein the wheat koji is obtained by fermenting wheat inoculated with Bacillus subtilis YB4.

[0022] In one implementation, fermentation lasts for at least 108 hours.

[0023] The present invention also provides a method for producing terpenoids by adding the Bacillus subtilis YB4 or the microbial preparation to a culture medium for fermentation.

[0024] In one embodiment, the culture medium contains grains, including glutinous rice, rice, or wheat.

[0025] In one embodiment, the culture medium is a reaction solution obtained by reacting grains with saccharifying enzymes and liquefying enzymes.

[0026] In one embodiment, the terpenoids include, but are not limited to, myrcene, β-pinene, 2-carene, linalool, linalyl acetate, solanone, and rutinol-R1.

[0027] The present invention also provides a method for preparing rice wine with high terpene content, wherein the wheat koji is added to the rice wine raw material for fermentation.

[0028] In one embodiment, the raw materials for the rice wine also contain glutinous rice and yeast.

[0029] In one embodiment, the yeast is prepared by inoculating yeast into glutinous rice saccharification liquid and fermenting for 36-48 hours.

[0030] In one embodiment, the yeast is yeast jiangnan1#.

[0031] In one embodiment, the preparation method of the glutinous rice saccharification liquid is as follows: glutinous rice is soaked in a rice soaking tank for 30 minutes, the rice slurry is drained, and the rice is steamed for 30-40 minutes. The steamed rice is cooled to 60°C for later use. 2‰ saccharifying enzyme (initial concentration 4×10⁻⁶) is added. 4 U / g) and 1‰ liquefying enzyme (initial addition concentration of 1×10⁻⁶ ... 5 After mixing thoroughly, place the mixture in a water bath at 60℃ for approximately 5 hours, stirring every hour to ensure uniform saccharification. Filter the saccharified liquid through gauze to obtain a sugar solution, then dilute with water to 13±0.5°Bx (20℃) to obtain the glutinous rice saccharification solution.

[0032] In one embodiment, the fermentation is divided into pre-fermentation and post-fermentation; the pre-fermentation is carried out at 28-30°C for 5-6 days; and the post-fermentation is carried out at 15-18°C for 15-20 days.

[0033] In one embodiment, the terpenoids include, but are not limited to, myrcene, β-pinene, 2-carene, linalool, linalyl acetate, solanone, and rutinol-R1.

[0034] The present invention also provides the application of the Bacillus subtilis YB4, the microbial preparation, or the malt in winemaking.

[0035] In one embodiment, the wine includes, but is not limited to, rice wine.

[0036] The present invention also provides the application of the Bacillus subtilis YB4 or the microbial preparation in the preparation of fermented foods.

[0037] Beneficial effects:

[0038] This invention screened and obtained a high-terpene-producing strain of Bacillus subtilis YB4. Its preservation number is CGMCC No. 1.64945. This Bacillus subtilis YB4 exhibits good ethanol tolerance (20%) and sodium chloride tolerance (18%), and possesses the characteristic of fermenting to produce terpenes. In glutinous rice saccharification liquid, fermentation for 5 days produced terpene compounds with a content of 277.78±13.78 μg / L; in rice wine brewing, fermentation for 20 days produced terpene compounds with a content of 234.25±14.89 μg / L.

[0039] This invention provides an effective biological pathway to increase the content of terpenoid compounds in brewed foods. Attached Figure Description

[0040] Figure 1 Colony morphology diagrams of strains YB-1 to YB-4;

[0041] Figure 2 Phylogenetic tree of strains YB-1 to YB-4;

[0042] Figure 3 : Liquid fermentation terpene production curves of Bacillus strains YB-1 to YB-4;

[0043] Figure 4 Growth curve of Bacillus subtilis YB4;

[0044] Figure 5 NaCl tolerance curve of Bacillus subtilis YB4.

[0045] Preservation of biological materials

[0046] A strain of Bacillus subtilis YB4, taxonomically named Bacillus subtilis, was deposited on March 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.64945, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0048] Technical terms:

[0049] In this invention, "inoculation volume" refers to the ratio of the volume of seed solution added to the volume of culture medium after inoculation.

[0050] In this invention, "seed liquid" refers to a microbial fermentation broth containing highly active target strains.

[0051] Unless otherwise specified, the volume-to-mass ratio v / m involved in this invention refers to mL / g.

[0052] The culture media involved in the following examples are as follows:

[0053] LB medium: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 1L distilled water, adjust pH to 7.0 with NaOH. If it is a solid, add 2% agar and sterilize at 115℃ for 20min.

[0054] Liquid fermentation aroma-producing medium: Based on raw glutinous rice, steamed glutinous rice (150%, m / m), water (400%, v / m), wheat (15%, m / m), saccharifying enzyme (1‰, v / m, enzyme activity added to the medium is 7.1 U / g, purchased from Solarbio, Cat#A8750) and liquefying enzyme (2‰, m / m, enzyme activity added to the medium is 35.4 U / g, purchased from Yuanye Biotechnology, Cat#S10017) were mixed in proportion, stirred evenly, and saccharified in a 60℃ water bath for 4-6 hours until the sugar content reached above 13°Bx. The glutinous rice residue was filtered out, and the mixture was sterilized at 115℃ for 20 minutes.

[0055] The analytical methods involved in the following embodiments are as follows:

[0056] Physicochemical indicators of rice wine: alcohol content, total acidity, and amino acid nitrogen were determined according to the methods in the national standard GB13662-2018, and reducing sugar was determined by the 3,5-dinitrosalicylic acid method.

[0057] Sample pretreatment:

[0058] Fermentation broth sample: Take 3 mL of fermentation broth sample and 3 mL of ultrapure water into a 20 mL headspace vial, add 2.5 g of sodium chloride and 5 μL of internal standard (100 mg / L 2-octanol solution), and then quickly tighten the cap;

[0059] Yellow rice wine sample: Take 2 mL of yellow rice wine sample and 4 mL of ultrapure water into a 20 mL headspace vial, add 2.5 g of sodium chloride and 5 μ L of internal standard (100 mg / L 2-octanol solution), and then quickly tighten the cap.

[0060] GC-MS conditions: Extraction was performed using a 50 / 30 DVB / CAR / PDMS extraction head for 60 min at 70 °C, with an equilibration time of 5 min and a stirring speed of 400 rpm. GC / MS analysis was conducted using a Thermo TRACE1300-ISQ system, with samples injected via a Thermo TriPlus RSH autosampler. All samples were injected in splitless mode for 3 min, followed by a 3 min solvent delay. A splitless liner with a 0.75 mm SPME liner was used, with an injection port temperature of 250 °C, constant flow mode, and a flow rate of 1 mL / min.

[0061] The analyzed compounds were resolved on an HP-5MS (30 m * 0.25 mm * 0.25 μm). Analysis was performed at a programmed temperature starting at 40 °C for 1 minute, then increased to 200 °C at 4 °C / min, and then to 230 °C at 20 °C / min, holding for 10 min. The total analysis time was 52.50 min. The GC / MS transfer line temperature was 250 °C, and the ion source temperature was 230 °C. Spectra were obtained using electron collisional ionization (EI, 70 eV) in full scan mode, with a scan range of m / z 50–400. The NIST database was searched, and the concentrations of the compounds were calculated using the internal standard method.

[0062] The following examples involve *Polysporium roseum* A22, which has been disclosed in patent CN117757584A; *Penicillium oxalate* M1816, which has been disclosed in "Enzyme Production Potential of *Penicillium oxalate* M1816 and Its Application in Ferulic Acid Production"; and *Saccharomyces cerevisiae* jiangnan1#, which has been disclosed in patent CN113621528A.

[0063] Example 1: Physiological and biochemical identification of B. subtilis YB4 strain

[0064] (1) Morphological characteristics of strains

[0065] Samples of rice wine mash, after dry heat and wet heat treatment, were plated and incubated for 24 hours. The culture characteristics were observed according to Bergey's Manual of Bacteriological Identification, and single colonies were selected for purification culture. Ultimately, four strains suspected to be Bacillus were screened from the rice wine mash (see attached). Figure 1 As shown), the colonies are notched and round, dirty white in color, with a dark, wrinkled, rough and opaque surface. The spores are oval-cylindrical in shape and located in the center or slightly off-center of the cell. They are named YB1, YB2, YB3 and YB4 respectively.

[0066] (2) DNA extraction

[0067] Take 1.5 mL of bacterial culture, centrifuge at 12000 rpm for 3 min, and wash the precipitate twice. Add 500 μL of sterile water, freeze at -80℃ for 20 min, then incubate at 80℃ for 10 min, repeating the freeze-thaw cycle three times. Add 20 μL of lysozyme (50 mg / mL) and 20 μL of cartilage lysin, mix well, and incubate at 37℃ for 40 min, shaking well at 10 min intervals. Then add 125 μL of LDS (10%) and 10 μL of LCTAB (20 μg / mL), incubate at 37℃ for 40 min, shaking well at 10 min intervals. Add an equal volume of CTAB (2%), incubate at 65℃ for 1 h, shaking well at 20 min intervals. Centrifuge at 12000 rpm for 10 min, collect the supernatant, add an equal volume of chloroform-isoamyl alcohol (V24:1) pre-cooled at -20℃, shake for 30 s, centrifuge at 12000 rpm for 10 min at 4℃, collect the supernatant, and repeat three times until the intermediate layer disappears. Add 0.6 times the volume of isopropanol pre-chilled at -20°C, mix well, and precipitate at -20°C for 1.5 h. Centrifuge at 12,000 rpm for 10 min at 4°C and collect the precipitate. Add 1 mL of 70% ethanol pre-chilled at -20°C, wash the precipitate, centrifuge at 10,000 rpm for 10 min at 4°C, discard the supernatant, repeat three times, and dry at 37°C for 15 min. Finally, add 30 μL of sterile water to dissolve the DNA and store at -20°C.

[0068] (3) PCR amplification and sequencing analysis

[0069] Polymerase chain reaction (PCR) amplification was performed using 27F (5'-CCTACGGGAGGCAGCAG-3') and 1492R (5'-CCGTCAATTCMTTTRAGT-3') primers. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, followed by 94℃ denaturation for 1 min, 56℃ annealing for 1 min, and 72℃ extension for 1.5 min, for a total of 30 cycles, with a final extension at 72℃ for 9 min. 5 μL of PCR product was used to assess DNA extraction efficiency using a 1% agarose gel. The PCR product was sequenced at Sangon Biotech (Shanghai) Co., Ltd. Homologous sequence searches were performed in the National Center for Biotechnology Information (NCBI) database, and multiple sequence alignment was performed using MEGA11 software. A phylogenetic tree was then constructed using neighbor-joining (NJ) method.

[0070] As attached Figure 2As shown, the results indicate that YB1 is *Bacillus atrophaeus*, YB2 is *Bacillus velezensis*, YB3 is *Bacillus tequilensis*, and YB4 is *Bacillus subtilis*. The *Bacillus subtilis* YB4 was deposited on March 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 1.64945.

[0071] Example 2: Comparison of terpene production by different Bacillus strains in liquid fermentation of rice mash

[0072] The YB1–4 strains were validated for terpene production through liquid fermentation in a liquid aroma-producing medium. The specific operational steps are as follows:

[0073] (1) Seed liquid preparation

[0074] Strains YB1, YB2, YB3, and YB4 were picked from the slant and streaked onto LB solid medium. They were then incubated at 37°C for 24 hours. Colonies were then picked from the plates and inoculated into LB liquid medium. The culture was carried out at 37°C and 150 rpm for 24 hours. Seed culture was obtained by subculturing twice.

[0075] (2) Terpenoid production by liquid fermentation

[0076] The seed culture was inoculated into the liquid fermentation aroma-producing medium at an inoculum volume of 10%, with an initial inoculum concentration of 1×10⁻⁶. 8 CFU / mL. The shaker speed was adjusted to 180 rpm, the fermentation temperature was controlled at 37℃, and the fermentation time was 7 days. Samples were taken at 0, 1, 3, 5, and 7 days, with 10 mL of fermentation supernatant taken each time. After fermentation, the supernatant was used for the determination of terpenoid compounds.

[0077] (3) Determination of terpenoids

[0078] The terpene content of four strains at different time points was determined by GC-MS, and the results are shown in Table 1. Curves showing the changes over time were also plotted (see attached table). Figure 3 It can be seen that, under the same fermentation conditions, the yield and variety of terpenoids from Bacillus subtilis strain YB4 are superior to those from the other three Bacillus strains. After 5 days of fermentation, the yield of terpenoids from Bacillus subtilis strain YB4 is 277.78 ± 13.78 μg / L.

[0079] Table 1. Terpenoid compound production (μg / L) by different Bacillus strains

[0080]

[0081]

[0082] Example 3: Growth characteristics of strain YB4

[0083] Seed culture of Bacillus subtilis YB4 was prepared according to the method in Example 2, and used to test the growth characteristics and environmental tolerance of the strain.

[0084] (1) Growth curve

[0085] Bacillus subtilis seed culture YB4 was inoculated into LB liquid medium at a ratio of 1% and cultured at 37℃ and 180 rpm. OD values ​​were measured at 0, 6, 12, 18, 24, 30, and 36 h, and growth curves were plotted. Results are attached. Figure 4 As shown, strain YB4 enters the logarithmic growth phase after 5-12 hours of liquid fermentation and reaches the stable growth phase after about 30 hours.

[0086] (2) Ethanol tolerance

[0087] Bacillus subtilis YB4 seed culture was serially diluted 10 5 After dilution, it was added to ethanol solutions of different final concentrations (10%, 15%, 20%) at a volume ratio of 1:10 (the initial inoculum size of the strain was 1×10⁻⁶). 8 Samples were taken every 24 hours (CFU / mL) for plating, and the colony count was measured to observe the ethanol tolerance of Bacillus subtilis YB4. As shown in Table 2, after soaking in 20% ethanol solution for 5 days, YB4 still had a high survival rate, indicating that Bacillus subtilis YB4 has strong tolerance to ethanol concentrations up to 20%.

[0088] Table 2. Ethanol tolerance of Bacillus subtilis YB4 (Unit: Bacteria / plate)

[0089]

[0090] Note: +++ indicates >100 colonies / plate, ++ indicates 50-100 colonies / plate.

[0091] (3) Salt tolerance

[0092] Different concentrations of NaCl produce different osmotic pressures, which in turn affect microbial growth differently. LB liquid medium with different NaCl concentrations (0%, 1%, 3%, 6%, 9%, 12%, 15%, 18%, w / v) was prepared, and Bacillus subtilis YB4 seed culture was inoculated into the LB liquid medium at a ratio of 1% (v / v) (the initial inoculum size was 1×10⁻⁶). 8CFU / mL), and OD values ​​were measured after incubation at 37℃ for 12 h (the final OD value is the bacterial culture OD value minus the blank group OD value). The blank group was sterilized LB liquid culture medium. (See attached...) Figure 5 As shown, when the sodium chloride concentration exceeds 6%, the OD value of YB4 bacterial culture begins to decrease rapidly, and when the NaCl concentration is 18%, it is close to 0. This indicates that Bacillus subtilis YB4 has extremely high tolerance to NaCl and can survive under high salt concentrations.

[0093] Example 4: YB4 wheat koji and rice wine production

[0094] (1) YB4 Wheat Music Production

[0095] The seed culture of Bacillus subtilis YB4 was prepared according to the method in Example 2.

[0096] Purchase red or yellow soft wheat from Wuxi Huijutong Grain Trade Co., Ltd. Grind and sieve the wheat, ensuring that each grain is crushed into 3-5 pieces in a plum blossom shape. Then accurately weigh out 5 kg for later use.

[0097] Bacillus subtilis YB4 seed culture was inoculated into crushed wheat at a ratio of 7% (v / m), and 38% deionized water was added to make the total moisture content of the wheat koji 45%. The initial concentration of Bacillus subtilis YB4 was 1×10⁻⁶. 8 After mixing CFU / mL evenly, place the mixture in a constant temperature and humidity incubator (temperature 45℃, humidity 85%) and incubate for 60h to complete the preparation of Bacillus subtilis raw wheat koji. During this period, it is necessary to pay close attention to the state of the wheat koji. If the wheat koji is too dry, spray water with a water bottle and turn the koji appropriately. The moisture content of the wheat koji should be controlled at 30-45%.

[0098] (2) Production of wine mother

[0099] Yeast activation: Streaking of *Saccharomyces cerevisiae* (jiangnan1#) from glycerol tubes onto plates in a sterile environment and incubating at 28°C for 48 hours. Then, inoculate single yeast colonies from the plates into YPD medium in a sterile environment, incubating under sterile conditions at 30°C for 36-48 hours, maintaining a temperature between 28-30°C, not exceeding 30°C. This culture is then ready for use as YPD culture medium.

[0100] Preparation of yeast starter: First, prepare the saccharification solution. Soak glutinous rice in a rice soaking tank for 30 minutes. After draining the rice water, steam the rice for 30-40 minutes. Cool the steamed rice to 60℃ and set aside. Add YB4 wheat koji (15%, m / m) and 2‰ saccharifying enzyme (initial concentration 4×10). 4 U / g) and 1‰ liquefying enzyme (initial addition concentration of 1×10⁻⁶ ... 5After mixing thoroughly, place the mixture in a water bath at 60℃ for approximately 5 hours, stirring every hour to ensure uniform saccharification. Filter the saccharified liquid through gauze to obtain a sugar solution, then dilute with water to 13±0.5°Bx (20℃) to obtain the glutinous rice saccharification solution. Pour the glutinous rice saccharification solution into bottles, seal with film, and sterilize in an autoclave at 115℃ for 20 minutes. Remove and cool before use. Transfer the cultured yeast jiangnan1# broth into the glutinous rice saccharification solution at an inoculation ratio of 3%, with an initial inoculation concentration of 1×10⁻⁶. 7 The concentration of CFU / mL is then increased, and the mixture is incubated at 30℃ for 36-48 hours before it can be used for feeding.

[0101] (3) Fermentation of Shaoxing wine: Add the ingredients according to the formula in Table 3. The total fermentation time is about 20 days. During the fermentation process, pay attention to stirring. The temperature of the pre-fermentation should be controlled below 33℃, and stirring should be done twice a day for a total of 5 days. The temperature of the post-fermentation should be controlled at about 15±2℃ for 15 days.

[0102] Table 3 Ingredients for Yellow Rice Wine Brewing

[0103]

[0104] (4) Determination of physicochemical properties of rice wine

[0105] The physicochemical properties of the fermented rice wine were determined, and the results are shown in Table 4. The rice wine brewed with Bacillus subtilis YB4 wheat koji had an alcohol content of 14.2±0.15 Vol, total acid of 5.88±0.13 g / L, ammonia nitrogen of 0.65±0.02 g / L, and total sugar of 13.33±0.25 g / L upon completion of fermentation, meeting the physicochemical requirements for rice wine in GB / T13662-2018.

[0106] Table 4 Physicochemical Indicators of Shaoxing Wine

[0107]

[0108] (5) Flavor determination of rice wine

[0109] The flavor compounds in rice wine were determined by GC-MS, and the results are shown in Table 5. A total of 65 flavor compounds were detected in YB4 rice wine, including 32 esters, 7 acids, 15 alcohols, 4 terpenes, and 11 other compounds such as aldehydes and phenols. Among the terpenes, citronellol, nerol, geraniol isovalerate, farnesol, and eucalyptol were found, with a total content of 234.25 ± 14.89 μg / L.

[0110] Table 5. Analysis of flavor compounds in rice wine brewed with YB4 wheat koji.

[0111]

[0112]

[0113] Comparative Example 1: Differences in the production of Shaoxing wine by different strains

[0114] The koji made from Bacillus subtilis YB4 in Example 4 was replaced with commonly used laboratory brewing microorganisms (Polyspora roseolae A22 and Penicillium oxalate M1816). Following the method described in Example 4 (where YB4 koji was replaced with the corresponding koji for rice wine brewing during the yeast preparation process), rice wine was brewed under the same conditions. After fermentation, the terpene content was determined by GC-MS, and the results are shown in Table 6. Compared to koji made from Polyspora roseolae A22 and Penicillium oxalate M1816, the rice wine brewed with Bacillus subtilis YB4 koji showed an increased variety of terpene compounds, with a total content reaching 234.25 ± 14.89 μg / L, significantly higher than that brewed with koji made from the other two brewing microorganisms.

[0115] Table 6. Differences in the production of rice wine by different strains

[0116]

[0117]

[0118] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0119] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A strain of Bacillus subtilis YB4, characterized in that, The Bacillus subtilis YB4 was deposited at the China General Microbiological Culture Collection Center on March 1, 2025, with accession number CGMCCNo.1.64945.

2. A microbial preparation containing Bacillus subtilis YB4 as described in claim 1.

3. The microbial preparation as described in claim 2, characterized in that, The number of Bacillus subtilis YB4 cells in the microbial preparation is at least 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

4. A type of wheat koji containing Bacillus subtilis YB4 as described in claim 1, characterized in that, The wheat koji is obtained by inoculating wheat with Bacillus subtilis YB4 and fermenting it.

5. The method as described in claim 4, characterized in that, Fermentation should last at least 108 hours.

6. A method for producing terpenoids, characterized in that, Add the Bacillus subtilis YB4 of claim 1, or the microbial preparation of claim 2 or 3, to the culture medium for fermentation.

7. The method as described in claim 6, characterized in that, The culture medium contains grains, including glutinous rice, rice, or wheat; preferably, the culture medium is a reaction solution obtained by reacting the grains with saccharifying enzymes and liquefying enzymes.

8. The method as described in claim 6 or 7, characterized in that, The terpenoids include, but are not limited to, myrcene, β-pinene, 2-carene, linalool, linalyl acetate, solanone, and rutin-R1.

9. A method for preparing rice wine with high terpene content, characterized in that, The wheat koji described in claim 4 or 5 is added to the raw materials for rice wine for fermentation.

10. The method as described in claim 9, characterized in that, The raw materials for the rice wine also include glutinous rice, water, and yeast.

11. The method as described in claim 10, characterized in that, The yeast starter is obtained by inoculating yeast into glutinous rice saccharification liquid for fermentation; preferably, the yeast includes yeast jiangnan1#.

12. The method as described in claim 10 or 11, characterized in that, The fermentation process is divided into pre-fermentation and post-fermentation; pre-fermentation is carried out at 28-30℃ for 5 days; post-fermentation is carried out at 15-18℃ for 15-20 days.

13. The method as described in claim 12, characterized in that, The terpenoids include, but are not limited to, myrcene, β-pinene, 2-carene, linalool, linalyl acetate, solanone, and rutin-R1.

14. The application of Bacillus subtilis YB4 according to claim 1, or the microbial preparation according to claim 2 or 3, or the wheat koji according to claim 4 or 5 in winemaking, characterized in that, The wines mentioned include, but are not limited to, rice wine.

15. The use of Bacillus subtilis YB4 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in the preparation of fermented foods.

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