Bacillus subtilis and application thereof in preparation of fermented tobacco leaves

Abstract

The invention belongs to the technical field of microorganisms, and relates to bacillus subtilis and application thereof in preparation of fermented tobacco leaves. The classification name of the strain is bacillus subtilis, the strain is preserved in the China Center for Type Culture Collection on March 13, 2025, and the preservation number of the strain is CCTCC NO: M 2025467; the preservation address is No. 299 on eight road in Wuchang District, Wuhan City, Hubei Province. The strain can significantly increase the variety and content of aroma components in the tobacco leaf fermentation process, improves the aroma quality and sensory characteristics of tobacco leaves, and has important application value for improving the quality of tobacco products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Bacillus subtilis and its application in the preparation of fermented tobacco leaves. Background Technology

[0002] Aroma value is a crucial indicator for evaluating tobacco leaf quality, and improving its aroma quality is a hot research topic for tobacco researchers. Currently, the main method for enhancing tobacco aroma is by adding flavorings and fragrances. However, with China's accession to the Framework Convention on Tobacco Control (FTCT), the use of tobacco additives will be subject to increasingly stringent restrictions. Articles 9 and 10 of the FTCT explicitly stipulate "four prohibitions and two restrictions." Canada, the United States, Australia, the European Union, Brazil, and other countries have also successively proposed regulations prohibiting the use of certain additives, providing lists and timelines for prohibited additives. my country's State Tobacco Monopoly Administration is also gradually restricting the use of flavorings and fragrances in cigarettes. Therefore, how to provide consumers with "high aroma, good taste, and low harm" cigarette products while reducing the use of additives is an urgent problem to be solved in the tobacco industry.

[0003] Numerous studies both domestically and internationally have demonstrated that using microbial fermentation technology to improve tobacco quality offers advantages such as being green and safe, producing high-quality aroma compounds, having mild reaction conditions, and a short cycle. Therefore, tobacco industry experts have made bio-flavoring technology a key research focus. However, different microbial strains exhibit significant differences in their metabolic characteristics and flavor-enhancing effects. Therefore, screening for efficient, stable, and suitable functional strains for tobacco fermentation remains crucial for the industrial application of this technology. Summary of the Invention

[0004] To address the above-mentioned technical problems, this invention provides a strain of Bacillus subtilis and its application in the preparation of fermented tobacco leaves. This strain, derived from tobacco leaves themselves, is used in tobacco fermentation treatment and can significantly increase the types and quantities of aroma components in tobacco leaves, thereby improving the overall sensory quality of the tobacco leaves.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of Bacillus subtilis HBZY-TL-B02, whose classification name is Bacillus subtilis (… Bacillus subtilis It was deposited at the China Center for Type Culture Collection on March 13, 2025, with accession number CCTCC NO: M 2025467; the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The nucleotide sequence of the 16S rDNA gene (SEQ ID No. 1) of strain HBZY-TL-B02 is shown below:

[0007] The Bacillus subtilis HBZY-TL-B02 strain provided in this invention, isolated from the surface of aged tobacco leaves, exhibits better tolerance to the dry environment of tobacco leaf surfaces and possesses the function of enhancing tobacco aroma. When used for tobacco leaf fermentation, it effectively increases the variety and content of aroma components in tobacco leaves, with significant increases observed in key tobacco aroma components such as phenylethanol, dihydroactinolone, solanone, geraniol acetone, and megastigmatrienone. Sensory evaluation of cigarettes made from tobacco leaves fermented with this strain shows a richer aroma, better harmony, reduced off-flavors, and improved comfort, sweetness, and aftertaste. This strain has a short fermentation cycle and stable effects, showing promising application prospects in improving the flavor quality of tobacco leaves and enhancing the smoking experience of cigarettes.

[0008] Secondly, the present invention also provides a microbial preparation comprising the above-mentioned Bacillus subtilis HBZY-TL-B02.

[0009] The microbial preparations provided by this invention may contain only Bacillus subtilis HBZY-TL-BO2 or a complex of bacteria including Bacillus subtilis HBZY-TL-BO2; they may also include other substances required for the preparation of the microbial preparations, such as carriers, nutrients, protectants, and fillers. The dosage forms of the microbial preparations include, but are not limited to, suspensions, powders, and granules.

[0010] Preferably, the viable count in the microbial preparation is not less than 1.0 × 10⁻⁶. 7 CFU / mL or 1.0×10 7 CFU / g.

[0011] More preferably, the viable count in the microbial preparation is 1.0 × 10⁻⁶. 7 -1.0×10 9 CFU / mL or 1.0×10 7 -1.0×10 9 CFU / g.

[0012] Thirdly, the present invention also provides a method for preparing a microbial preparation, comprising the following steps: S1: The above-mentioned Bacillus subtilis HBZY-TL-B02 was inoculated into the culture medium for activation culture to obtain live bacterial fermentation broth; S2: Isolate bacterial cells from the live bacteria fermentation broth; S3: The bacterial cells are resuspended in sterile physiological saline to obtain a bacterial suspension.

[0013] Preferably, the culture medium is LB liquid medium.

[0014] Preferably, the activation culture conditions are as follows: at 35-37℃, shake culture for 12-24 h, and shaker speed of 180-200 rpm.

[0015] Preferably, the method for separating and obtaining bacterial cells is to centrifuge at 4-6℃ and 5000-8000 rpm for 5-10 minutes.

[0016] Fourthly, the present invention also provides the application of the above-mentioned Bacillus subtilis HBZY-TL-B02 or the above-mentioned microbial preparation in the preparation of fermented tobacco leaves.

[0017] This invention demonstrates through experiments that, after using Bacillus subtilis HBZY-TL-B02 for tobacco fermentation, the number of aroma compounds in tobacco leaves significantly increased from 60 to 98, with a total increase of 17.3%. The quantities of alcohols, aldehydes, esters, hydrocarbons, acids, phenols, and nitrogen-containing heterocyclic compounds all showed a significant increase. The contents of many key aroma components, such as 3-methyl-2-butanol, farnesol, megastigmatrienone, solanone, and phytone, were also significantly increased.

[0018] Fifthly, the present invention also provides a method for fermenting tobacco leaves, wherein an active bacterial solution containing the above-mentioned Bacillus subtilis HBZY-TL-B02 is evenly applied to the surface of tobacco leaves for fermentation; the moisture content of the tobacco leaves is maintained at 20%-30%.

[0019] The active bacterial solution in this invention contains Bacillus subtilis HBZY-TL-B02, which has metabolic activity, and its physical form is a liquid suitable for spraying, soaking, or other applications, so as to facilitate uniform distribution and colonization on tobacco leaves. Its bacterial concentration, pH value, carrier, etc., can be adjusted according to actual application needs.

[0020] Alternatively, it can be obtained in any of the following ways: Direct source: Microbial preparations in liquid form, which are themselves liquid products containing an effective number of live bacteria.

[0021] Indirect preparation: The solid form of microbial preparations (such as lyophilized powders, granules, etc.) is restored to a liquid suspension system through steps such as rehydration, dissolution, or resuspension.

[0022] Preferably, the active bacterial solution is evenly applied to the surface of the tobacco leaves, so that the final bacterial concentration on the tobacco leaves reaches 3.0 × 10⁻⁶. 5 -5.0×10 7 CFU / g; the fermentation conditions are a temperature of 35-37℃, a humidity of 70%-80%, and a time of 12-24 h.

[0023] More preferably, the final bacterial concentration on the tobacco leaves reaches 5.0 × 10⁻⁶. 7CFU / g; the fermentation conditions were 37°C, 80% humidity, and 12 h.

[0024] In a sixth aspect, the present invention also provides tobacco leaves prepared according to the above-described fermentation method.

[0025] The tobacco leaves provided by this invention can be used as a key raw material or auxiliary component in tobacco products, which helps to improve the smoking taste and enrich the aroma of the final tobacco products. Attached Figure Description

[0026] Figure 1 This is a colony morphology diagram of Bacillus subtilis HBZY-TL-B02 of the present invention; Figure 2 The cell morphology diagram of Bacillus subtilis HBZY-TL-B02 of the present invention is shown. Figure 3 This is an electron microscope image of the morphology of Bacillus subtilis HBZY-TL-B02 of the present invention; Figure 4 The phylogenetic tree of Bacillus subtilis HBZY-TL-B02 of the present invention is constructed based on the 16S rDNA gene sequence. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and verification examples. It should be understood that the specific embodiments and verification examples described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] The strains used in the following embodiments and verification examples of the present invention are from the following sources: Bacillus subtilis HBZY-TL-B02 was deposited at the China Center for Type Culture Collection (CCTCC) on March 13, 2025, with accession number CCTCC NO: M 2025467; the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0029] The culture media and solutions used in the following embodiments of the present invention are as follows: LB medium: 1000 mL distilled water, 10 g trypsin, 5 g yeast extract, 10 g NaCl, adjust pH to 7.0-7.4, autoclave at 121℃ for 20 min. For solid medium, add 20 g agar to the above formula.

[0030] Tobacco leaf extract culture medium: 100 mL tobacco leaf aqueous extract, 900 mL distilled water, 7 g trypsin, 3.5 g yeast extract, 7 g NaCl, natural pH, autoclaved at 121℃ for 20 min. For the solid culture medium, add 20 g agar to the above. The preparation method of tobacco leaf aqueous extract is as follows: 20 g tobacco leaves are added to 300 mL distilled water and boiled for 1 h. After cooling, the extract is filtered through two layers of gauze to obtain the tobacco leaf aqueous extract.

[0031] PBS buffer: 0.24 g KH2PO4, 1.44 g NaH2PO4, 8 g NaCl, 0.2 g KCl, add 800 mL of deionized water and stir thoroughly to dissolve. Then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally bring the volume to 1.0 L.

[0032] Tobacco materials: 2022 Hebei BFE tobacco leaves, 2023 Henan (Yu-Wan) BFSE tobacco leaves and 2020 Yunnan Luoping CFY1 tobacco leaves. All of the above tobacco leaves were provided by Hebei China Tobacco Industry Co., Ltd.

[0033] Unless otherwise specified, the raw materials, reagents and kits used in the following embodiments of the present invention are all commercially available.

[0034] Example 1 This embodiment provides a Bacillus subtilis strain HBZY-TL-B02, which was isolated and screened from the surface of alcoholized tobacco leaves.

[0035] 1. Separation and Screening (1) Sample pretreatment: Take 5 g of alcoholic tobacco leaf sample, cut it into pieces under sterile conditions, soak it in 100 mL of sterile physiological saline, and shake and culture it for 2 h at a temperature of 37℃ and a rotation speed of 180 rpm. Filter it with sterile single-layer gauze, take the filtrate, centrifuge and discard the supernatant, and resuspend it with 5 mL of sterile water to obtain the original bacterial suspension.

[0036] (2) Enrichment culture of the strain: Take 1 mL of the above bacterial suspension and inoculate it into tobacco extract culture medium. Incubate at 37℃ for 24 h. Centrifuge at 5000 rpm for 10 min at 4℃, discard the supernatant and collect the bacterial precipitate. Resuspend the bacterial precipitate with 5 mL of PBS buffer. Take 2 mL of the resuspended solution and inoculate it into tobacco extract culture medium. Repeat the above culture operation 2-3 times to obtain the enriched culture medium.

[0037] (3) Initial screening of strains: Take 1 mL of enrichment culture medium and perform 10-fold serial dilutions to obtain 10 -6Sample dilution at a specific concentration. Spread 100 µL of the dilution onto LB agar plates and incubate at 37°C for 24 h. Collect single colonies for preservation. Add the single colonies to LB liquid medium and incubate for 24–48 h to prepare the seed culture.

[0038] The seed cultures of different bacteria were spread and incubated at 37°C for 12-24 h. The plates were then examined under a microscope. Based on differences in morphology, single colonies of different morphologies were selected and streaked onto LB agar plates and tobacco extract agar plates for purification. Strains that could grow on both agar plates were selected, totaling 18 strains.

[0039] Secondary screening of strains: Eighteen strains were activated to obtain corresponding seed solutions. These seed solutions were inoculated into tobacco extract medium at a 5% (v / v) inoculation rate and fermented at 37℃ and 180 rpm for 24 h. Three replicates were set up for each strain, with the tobacco extract medium serving as a blank control. Sensory evaluation of the fermentation broth after fermentation showed that strain HBZY-TL-B02, while retaining the natural aroma of tobacco, produced a harmonious and prominent pleasant aroma, exhibiting the best overall aroma quality. Therefore, it was selected for further research.

[0040] 2. Strain identification 2.1 Morphological identification of strain HBZY-TL-B02 Colony morphology observation: The HBZY-TL-B02 strain exhibited milky-white, round colonies on LB agar plates. The colony surface was wrinkled and not smooth, with relatively large colonies and irregular edges. See details... Figure 1 .

[0041] Observation of bacterial cell morphology: Under the microscope, the bacterial cells are rod-shaped and Gram-positive. Figure 2 Under scanning electron microscopy, it exhibits a clearly discernible long rod-like morphology. Figure 3 It can be concluded that this strain is a Gram-positive bacillus.

[0042] 2.2 Molecular biological identification of strain HBZY-TL-B02 The 16S rDNA gene fragment of strain HBZY-TL-B02 was amplified using universal primers 27F and 1492R.

[0043] PCR program: 95℃ pre-denaturation for 7 min; 95℃ denaturation for 25 s, 56℃ annealing for 30 s, 72℃ annealing for 20 s to extend primers onto the template, 35 cycles; 72℃ hold for 7 min. Store the obtained PCR product at 4℃.

[0044] The amplified products were sequenced, and the sequencing results are shown in SEQ ID NO.1. The 16S rDNA gene sequence of strain HBZY-TL-B02 was BLAST-aligned in GenBank, and the preliminary taxonomic classification of this bacterium was determined to be within the genus *Bacillus*. Further phylogenetic tree models were constructed. Figure 4 The results showed that this strain was related to Bacillus subtilis (…). Bacillus subtilis The strain is most closely related to the others on the phylogenetic tree, with a similarity of 99.93%. Based on morphological observation, the strain is identified as belonging to Bacillus subtilis.

[0045] This strain was deposited at the China Center for Type Culture Collection on March 13, 2025, with accession number CCTCCNO: M 2025467; the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0046] Example 2 This embodiment provides a Bacillus subtilis HBZY-TL-B02 liquid bacterial agent.

[0047] The preparation method of this bacterium consists of the following steps.

[0048] (1) Pick a single colony of Bacillus subtilis HBZY-TL-B02, inoculate it into 50 mL of LB liquid medium, and culture it at 37℃ and 180 rpm for 24 h to obtain seed liquid.

[0049] (2) Inoculate the seed culture at a rate of 2% (v / v) into 200 mL of LB liquid medium and culture at 37°C and 180 rpm for 24 h to obtain activated bacterial culture.

[0050] (3) Centrifuge the activated bacterial solution at 5000 rpm for 15 min at 4℃, remove the supernatant and collect the bacterial cells.

[0051] (4) The obtained bacterial cells were washed three times with sterile water and then resuspended in 200 mL of sterile water to obtain Bacillus subtilis HBZY-TL-B02 liquid bacterial agent.

[0052] The test results showed that the viable bacteria count of the liquid bacterial agent was 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL.

[0053] Example 3 This embodiment provides a tobacco fermentation method, which consists of the following steps.

[0054] (1) Dilute the Bacillus subtilis HBZY-TL-B02 liquid bacterial agent (provided in Example 2) to adjust the bacterial concentration to 2.5 × 10⁻⁶. 8CFU / mL; 20 mL of diluted bacterial solution is placed in a spray bottle and sprayed evenly onto 100 g of tobacco leaf fragments that have been sterilized at high temperature, so that the moisture content of the tobacco leaves is maintained at 20%-30%.

[0055] (2) Then put the tobacco leaf fragments into a clean sealed bag, make a few small holes, and place it in a constant temperature and humidity incubator at 37℃ and 80% relative humidity for 24 h to ferment.

[0056] (3) After fermentation, the tobacco leaf fragments were placed in an oven at 120°C and baked until the moisture content was 11%-12%. After being cut into strips, they were packaged and stored to obtain HBZY-TL-B02 strain bio-fermented tobacco strips.

[0057] Example 4 This embodiment provides a tobacco fermentation method, which consists of the following steps.

[0058] (1) Take Bacillus subtilis HBZY-TL-B02 liquid bacterial agent (provided in Example 2) and adjust the viable count to 1.5 × 10⁻⁶. 6 Take 20 mL of the diluted bacterial agent (CFU / mL) and put it into a spray bottle. Spray it evenly onto 100 g of tobacco leaf fragments that have been sterilized at high temperature, so that the moisture content of the tobacco leaves is maintained at 20-30%.

[0059] (2) Then put the tobacco leaf fragments into a clean sealed bag, make a few small holes, and place it in a constant temperature and humidity incubator at 37℃ and 70% relative humidity for 12 h to ferment.

[0060] (3) After fermentation, the tobacco leaf fragments were placed in an oven at 120°C and baked until the moisture content was 11%-12%. After being cut into strips, they were packaged and stored to obtain HBZY-TL-B02 strain bio-fermented tobacco strips.

[0061] Test Example 1: Determination of Physicochemical Indicators of Tobacco Leaves Before and After Fermentation Physicochemical properties were determined for the bio-fermented tobacco shreds (sprayed sample) provided in Example 3 and the tobacco shreds prepared by replacing the microbial inoculant with an equal amount of deionized water (control sample). Specifically, the water-soluble sugar content (including total sugar and reducing sugar) was determined using YC / T 159-2002 "Determination of Water-Soluble Sugars in Tobacco and Tobacco Products - Continuous Flow Method"; the total alkaloid (nicotine) content was determined using YC / T 160-2002 "Determination of Total Alkaloids in Tobacco and Tobacco Products - Continuous Flow Method"; the chlorine content was determined using YC / T 162-2002 "Determination of Chlorine in Tobacco and Tobacco Products - Continuous Flow Method"; the total nitrogen content was determined using YC / T 161-2002 "Determination of Total Nitrogen in Tobacco and Tobacco Products - Continuous Flow Method"; and the potassium content was determined using YC / T 173-2003 "Determination of Potassium in Tobacco and Tobacco Products - Flame Photometry". The experimental results are shown in Table 1.

[0062] Table 1 Physicochemical composition analysis of tobacco leaves before and after fermentation

[0063] Note: The experimental tobacco leaves were "2022 Hebei BFE tobacco leaves".

[0064] Table 1 shows that, compared with the control sample, the total sugar and reducing sugar contents in the sprayed sample fermented with Bacillus subtilis HBZY-TL-B02 decreased by 12.56% and 11.87%, respectively; the nicotine content decreased by 9.17%; and the chlorine and potassium contents remained largely unchanged. This indicates that Bacillus subtilis HBZY-TL-B02 has the potential to reduce tobacco irritation, improve combustibility, and enhance aroma quality by regulating the chemical composition of tobacco and promoting material transformation and balance.

[0065] Test Example 2: Detection of aroma components in tobacco leaves before and after fermentation The aroma components of the bio-fermented tobacco shreds (sprayed sample) provided in Example 3 and the tobacco shreds prepared by replacing the microbial agent with an equal amount of deionized water (control sample) were detected.

[0066] Accurately weigh 50 g of tobacco sample and place it in a 1000 mL round-bottom flask, along with 40 g of sodium chloride and 700 mL of distilled water. Connect the other end of the apparatus to a heart-shaped flask, pour in 60 mL of dichloromethane, and place the flask in a 60°C constant-temperature water bath. Connect the inlet and outlet water pipes and turn on the condenser. After the reflux stabilizes, perform simultaneous distillation extraction, ending the extraction after 2.5 h. Transfer the resulting dichloromethane extract to a 150 mL conical flask, add anhydrous sodium sulfate to dry thoroughly, concentrate to 1.5 mL, and analyze using GC / MS.

[0067] GC-MS analysis conditions: Column: DB-5MS (50 m × 0.25 mm × 0.25 µm); Injection temperature: 250℃; Split ratio: 5:1; Delay: 5 min; Carrier gas: He; Carrier gas flow rate: 1 mL / min; Temperature program: 50℃ (1 min) ramped up to 280℃ (5 min) at 4℃ / min; Transfer line temperature: 270℃; Ion source: EI source; Electron energy: 70 eV; Scan range: 35-600 amu. Spectral library: NIST11. Based on the mass spectra, a search and matching process was performed in the standard spectral library. Qualitative analysis was conducted using the peak areas of characteristic mass spectra to determine the aroma components. Phenylacetyl acetate was used as an internal standard for quantitative analysis of the aroma components using the internal standard method. The specific content of each aroma component in the sample was calculated based on the concentrated and diluted volume. The test results are shown in Table 2.

[0068] Table 2. Content of aroma components after tobacco fermentation

[0069] Note: 1. "-" indicates not detected; 2. The experimental tobacco leaf is "2022 Hebei BFE tobacco leaf".

[0070] Table 2 shows that 98 aroma components were detected in the sprayed tobacco leaves, with a total content of 2397.735 μg / g, while 60 aroma components were detected in the control tobacco leaves, with a total content of 2044.928 μg / g. The total amount of aroma components increased by 17.3% after fermentation, and the variety of aroma components also increased. Among them, the number of alcohols increased from 9 to 17, and the content increased from 709.323 µg / g before fermentation to 966.016 µg / g, an increase of 36.2% after fermentation. Among the newly added alcohols, 3-methyl-2-butanol is a commonly used flavoring raw material; farnesol has a mild, fresh, green aroma, similar to lily of the valley, and as an aroma component in tobacco, it can make the aroma fuller and the taste smoother and more pleasant; geraniol is a diterpenoid compound with a faint floral aroma. The contents of important aroma-producing components such as solanone, mesostachitenone, and phytone increased by 9.5%, 29.6%, and 43.8%, respectively, and new compounds such as H-pyran-2-one, methylheptenone, 4-tetaneone, 4-[2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl]-3-buten-2-one, 1-(p-methoxyphenyl)-1,3-butanedione, and (6R,7E,9R)-9-hydroxy-4,7-mesostachiten-3-one were added. This further confirms the positive effect of this strain in improving the aroma quality of tobacco leaves.

[0071] Test Example 3: Sensory Evaluation of Cigarettes Following the method described in Example 3, HBZY-TL-B02 microbial inoculant was used to ferment tobacco shreds from three different origins and grades to obtain the experimental group of fermented tobacco shreds. Meanwhile, samples treated under the same conditions without inoculation served as the control group.

[0072] The fermented tobacco from the experimental group and the tobacco from the control group were hand-rolled into standard cigarettes and placed in an environment of 22℃ and 65% humidity for 48 hours to equilibrate until the moisture content of the cigarettes stabilized. Sensory evaluation was then conducted. The sensory evaluation included nine indicators, each with a maximum score of nine points, for a total of 81 points. The evaluation process was completed by a professionally trained evaluation team in a standard sensory laboratory, with simultaneous recording of the style characteristics and overall quality description (usability evaluation) of each cigarette sample. The evaluation results are shown in Tables 3 and 4.

[0073] Table 3. Sensory Quality Rating Table for Cigarettes

[0074] Note: 1 refers to "2022 Hebei BFE tobacco leaves"; 2 refers to "2023 Henan (Yu-Wan) BFSE tobacco leaves"; 3 refers to "2020 Yunnan Luoping CFY1 tobacco leaves".

[0075] Table 3 shows that, compared with the control group, the average sensory score of the three experimental groups increased by 3.2 points, with reduced off-flavors, increased strength, and improved sweetness and aftertaste. Simultaneously, due to the production of new aroma compounds after fermentation by strain HBZY-TL-B02, the aroma quality and quantity of the cigarettes also significantly increased. This indicates that strain HBZY-TL-B02 can significantly improve the smoking taste and aftertaste of tobacco leaves, enhancing their sensory quality.

[0076] Table 4. Evaluation of Cigarette Usability

[0077] Note: 1 refers to "2022 Hebei BFE tobacco leaves"; 2 refers to "2023 Henan (Yu-Wan) BFSE tobacco leaves"; 3 refers to "2020 Yunnan Luoping CFY1 tobacco leaves".

[0078] As shown in Table 4, the usability evaluation of cigarettes indicates that, overall, after fermentation with strain HBZY-TL-B02, the off-flavors and irritation of the cigarettes decreased, the aroma became richer and more pronounced, and the aroma quantity and harmony were improved.

[0079] In summary, the Bacillus subtilis HBZY-TL-B02 provided by this invention, as a novel microbial resource with a clear aroma-producing function and originating from the tobacco habitat, enriches and expands the microbial germplasm resource bank that can be used for tobacco processing.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis HBZY-TL-B02, characterized in that, Its classification name is Bacillus subtilis ( Bacillus subtilis It was deposited at the China Center for Type Culture Collection on March 13, 2025, with accession number CCTCC NO: M 2025467; the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial preparation, characterized in that, Includes Bacillus subtilis HBZY-TL-B02 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable count in the microbial preparation is not less than 1.0 × 10⁻⁶. 7 CFU / mL or 1.0×10 7 CFU / g.

4. The microbial preparation according to claim 3, characterized in that, The viable count in the microbial preparation is 1.0 × 10⁻⁶. 7 -1.0×10 9 CFU / mL or 1.0×10 7 -1.0×10 9 CFU / g.

5. A method for preparing a microbial preparation, characterized in that, Includes the following steps: S1: The Bacillus subtilis HBZY-TL-B02 of claim 1 is inoculated into a culture medium for activation culture to obtain a live bacterial fermentation broth; S2: Isolate bacterial cells from the live bacteria fermentation broth; S3: The bacterial cells are resuspended in sterile physiological saline to obtain a bacterial suspension.

6. The use of Bacillus subtilis HBZY-TL-B02 as described in claim 1 or the microbial preparations as described in claims 2-4 in the preparation of fermented tobacco leaves.

7. A method for fermenting tobacco leaves, characterized in that, The active bacterial solution containing Bacillus subtilis HBZY-TL-B02 as described in claim 1 is evenly applied to the surface of tobacco leaves for fermentation; the moisture content of the tobacco leaves is maintained at 20%-30%.

8. The fermentation method according to claim 7, characterized in that, The active bacterial solution was evenly applied to the surface of the tobacco leaves, so that the final bacterial concentration on the tobacco leaves reached 3.0 × 10⁻⁶. 5 -5.0×10 7 CFU / g; the fermentation conditions are a temperature of 35-37℃, a humidity of 70%-80%, and a time of 12-24 h.

9. The fermentation method according to claim 8, characterized in that, This resulted in a final bacterial concentration of 5.0 × 10⁻⁶ on the tobacco leaves. 7 CFU / g; the fermentation conditions were 37°C, 80% humidity, and 12 h.

10. Tobacco leaves prepared by the fermentation method according to any one of claims 7-9.

Images