Bacillus ginsengisoli, fermentation method of tobacco leaves, fermented tobacco leaves, and cigarette product

Abstract

The application provides a ginseng soil novel bacillus, a fermentation method of tobacco leaves, fermented tobacco leaves and a cigarette product. The ginseng soil novel bacillus (Neobacillus ginsengisoli) SCT-B5 has been preserved in the China General Microbiological Culture Collection Center on October 10, 2025, and the address is No. 1, Xibaheyi, Chaoyang District, Beijing, and the preservation number is CGMCC No. 36126. The ginseng soil novel bacillus SCT-B5 has high yields of protease, amylase and cellulase, the protease produced in the fermentation process can reduce the content of protein in tobacco leaves, thereby reducing the irritability and offensive odor of the tobacco leaves; the cellulase can reduce the cellulose content in the tobacco leaves, decompose the cellulose into small molecule sugars, and improve the sweetness and alcohol degree of the smoke.

Description

Technical Field

[0001] This application relates to the field of tobacco fermentation technology, and in particular to a method for fermenting *Bacillus ginseng*, tobacco leaves, fermented tobacco leaves, and cigarette products. Background Technology

[0002] Fermentation can promote changes in the chemical composition and physical properties of tobacco leaves. Specifically, macromolecules such as cellulose, protein and starch are degraded during fermentation to produce substances such as glucose and amino acids. Glucose and amino acids can enhance the sweetness of tobacco leaves while reducing off-flavors and irritation. Amino acids can also undergo Maillard reactions with sugars to produce aroma substances such as pyrazines, aldehydes and ketones, which can enhance the roasted and nutty aromas of tobacco leaves.

[0003] The natural fermentation and aging process of tobacco leaves is lengthy and difficult to control. Therefore, traditional techniques involve inoculating tobacco leaves with microorganisms to enhance fermentation, thereby shortening the fermentation time and imparting a natural, mellow aroma while removing bitter and grassy odors. However, the effect of traditional microorganisms on enhancing tobacco fermentation is limited. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide a novel Bacillus subtilis that significantly enhances the sensory properties of tobacco leaves, as well as a method for fermenting tobacco leaves, fermented tobacco leaves, and cigarette products.

[0005] According to a first aspect of the embodiments of this application, a strain of Neobacillus ginsengisoli SCT-B5 is provided. The Neobacillus ginsengisoli was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36126.

[0006] According to a second aspect of the embodiments of this application, a method for fermenting tobacco leaves is provided, comprising the following steps:

[0007] The above-mentioned novel Bacillus subtilis was used to ferment tobacco leaves.

[0008] In some embodiments, the tobacco leaves include cigar tobacco leaves.

[0009] In some embodiments, the fermentation method described above includes the following steps:

[0010] The *Bacillus ginseng* was cultured to prepare a bacterial solution; and,

[0011] The bacterial solution is mixed with the tobacco leaves and fermented.

[0012] In some embodiments, culturing the *Bacillus subtilis* from ginseng soil includes the following steps:

[0013] The novel Bacillus ginseng was subjected to activation culture and proliferation culture in sequence.

[0014] In some embodiments, the amount of bacterial solution added is 0.5wt% to 5wt% of the mass of the tobacco leaves.

[0015] In some embodiments, the concentration of the bacterial solution is 8 × 10⁻⁶. 4 CFU / mL ~12×10 4 CFU / mL.

[0016] In some of these embodiments, the fermentation process is carried out at a temperature of 30°C to 45°C for 15 to 45 days, with a relative humidity of 70% to 80%.

[0017] According to a third aspect of the embodiments of this application, a fermented tobacco leaf is provided, which is obtained by fermenting the tobacco leaf using the above-described tobacco leaf fermentation method.

[0018] According to a fourth aspect of the embodiments of this application, a cigarette article is provided, comprising the fermented tobacco leaves described above.

[0019] Compared with traditional technologies, this application has the following advantages:

[0020] Through extensive research, the applicant of this application screened and obtained a novel Bacillus subtilis strain SCT-B5 with high yields of protease, amylase, and cellulase. The protease produced by this strain during fermentation can reduce the protein content in tobacco leaves, thereby reducing the irritation and off-flavors of tobacco leaves. The cellulase can reduce the cellulose content in tobacco leaves, breaking it down into small molecule sugars, thereby improving the sweetness and mellowness of the smoke. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the specific embodiments of this application, the drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings.

[0022] Figure 1 This is a colony morphology diagram of the novel Bacillus ginseng SCT-B5 strain used in this application.

[0023] Figure 2 Gram-stained microscopic image of the novel Bacillus ginseng SCT-B5 strain of this application;

[0024] Figure 3The growth curve of *Bacillus subtilis* SCT-B5 is shown.

[0025] Figure 4 The growth curve of Bacillus endophyta is shown.

[0026] The Neobacillus ginsengisoli SCT-B5 strain provided in this application was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36126. This strain was received and registered by the collection center on October 10, 2025, and was confirmed to be a viable strain by the collection center on the same day. Detailed Implementation

[0027] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0029] In this application, terms such as "multiple", "various", "multiple times", and "multi-dimensional" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0030] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0031] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0032] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0033] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0034] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0035] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0036] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0037] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0038] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0039] In this application, "room temperature" or "normal temperature" generally refers to 4℃~35℃, for example 20℃±5℃.

[0040] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0041] Some embodiments of this application provide a strain of Neobacillus ginsengisoli SCT-B5, which was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36126.

[0042] The above-mentioned strains have a high capacity for producing protease, amylase and cellulase. The protease produced during fermentation can reduce the protein content in tobacco leaves, thereby reducing the irritation and off-flavors of tobacco leaves; the cellulase can reduce the cellulose content in tobacco leaves, breaking it down into small molecule sugars, thereby improving the sweetness and mellowness of the smoke.

[0043] In some of these embodiments, the novel Bacillus ginseng is derived from tobacco leaves.

[0044] Understandably, the novel Bacillus subtilis strain selected from tobacco leaves is more likely to colonize tobacco leaves and fine-tune the evolution of the tobacco community in a direction conducive to product fermentation.

[0045] Some embodiments of this application also provide a method for fermenting tobacco leaves, including the following steps:

[0046] The above-mentioned novel Bacillus subtilis was used to ferment tobacco leaves.

[0047] In some embodiments, the tobacco leaves include cigar tobacco leaves.

[0048] In some embodiments, the above-mentioned tobacco leaf fermentation method includes the following steps:

[0049] Culture of *Bacillus subtilis* from ginseng soil to prepare bacterial culture; and,

[0050] The bacterial solution is mixed with tobacco leaves and fermented.

[0051] In some embodiments, culturing *Bacillus subtilis* from ginseng includes the following steps:

[0052] The novel Bacillus subtilis of ginseng was subjected to activation culture and proliferation culture in sequence.

[0053] In some specific examples, the activation culture step includes: inoculating the strain into LB liquid medium at a volume ratio of 1% to 2%, and culturing it at 35℃ to 37℃ and 150 rpm to 200 rpm for 1 to 2 days to obtain the seed culture.

[0054] In some specific examples, the proliferation culture steps include: inoculating the seed culture into the tobacco extract culture medium at a volume ratio of 1% to 2%, and culturing it at 35℃ to 37℃ and 150 rpm to 200 rpm for 1 to 2 days.

[0055] In some specific examples, the tobacco extract culture medium, by weight percentage, comprises 3%–7% cigar tobacco dust and 93%–97% water.

[0056] As an example, the mass percentage of cigar tobacco dust in the tobacco extract culture medium can be 3%, 4%, 5%, 6%, 7%, or any value within the range of any two of the above points.

[0057] As an example, the water mass percentage in the tobacco extract culture medium can be 93%, 94%, 95%, 96%, 97%, or any value within the range of any two of the above points.

[0058] Furthermore, the tobacco extract culture medium comprises 5% cigar tobacco dust and 95% water by weight percentage.

[0059] In some embodiments, the amount of bacterial solution added is 0.5wt% to 5wt% of the mass of the tobacco leaves.

[0060] As an example, the amount of bacterial solution added can be 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, or 5wt% of the mass of the tobacco leaves, or any value within the range formed by any two of the above points.

[0061] Furthermore, the amount of bacterial solution added is 0.5wt% to 2wt% of the mass of the tobacco leaves.

[0062] In some of these embodiments, the concentration of the bacterial solution is 8 × 10⁻⁶. 4 CFU / mL ~12×10 4 CFU / mL.

[0063] As an example, the concentration of the bacterial solution can be 8 × 10⁻⁶. 4 CFU / mL, 9×10 4 CFU / mL, 10×10 4 CFU / mL, 11×10 4CFU / mL, 12×10 4 CFU / mL, or any value within the range formed by any two of the above point values.

[0064] Furthermore, the concentration of the bacterial solution was 9 × 10⁻⁶. 4 CFU / mL ~11×10 4 CFU / mL.

[0065] Furthermore, the concentration of the bacterial solution was 10 × 10⁻⁶. 4 CFU / mL.

[0066] In some of these embodiments, the fermentation process is carried out at a temperature of 30°C to 45°C for 15 to 45 days, with a relative humidity of 70% to 80%.

[0067] As an example, the fermentation temperature can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, or any value within the range formed by any two of the above points.

[0068] As an example, the fermentation time can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, or 45 days, or any value within the range formed by any two of the above point values.

[0069] As an example, the humidity of the fermentation process can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or any value within the range of any two of the above points.

[0070] The above-mentioned tobacco fermentation method has the advantages of simple process and convenient operation; and the use of the above-mentioned Bacillus subtilis to ferment tobacco leaves can significantly increase the aroma of tobacco leaves and improve the sensory quality of tobacco leaves.

[0071] Some embodiments of this application also provide a fermented tobacco leaf, which is obtained by fermenting the tobacco leaf using the above-described tobacco leaf fermentation method.

[0072] Some embodiments of this application also provide a cigarette product comprising the aforementioned fermented tobacco leaves.

[0073] The present application will be further described below with reference to specific embodiments and comparative examples, but these should not be construed as limiting the scope of protection of the present application. Unless otherwise specified, the raw materials involved in the following specific embodiments are all commercially available, the instruments used are all commercially available, and the processes involved are conventionally selected by those skilled in the art unless otherwise specified.

[0074] Example 1

[0075] (1) Strains Isolation

[0076] Cigar tobacco leaves were shredded using sterile scissors and added to LB liquid medium. The mixture was incubated at 37°C for 48 hours to obtain the culture. The culture was then diluted 10 times. 5 The sample was then spread onto LB agar plates containing 5% cigar tobacco dust and incubated at 37°C for 48 hours to obtain a single colony, namely Bacillus subtilis SCT-B5.

[0077] (2) Strain identification

[0078] (2.1) The colony characteristics of the above strains on LB agar medium are as follows: the colonies are round, small in diameter, milky white in color, with neat and smooth edges, moist texture, low elevation, and opaque; Gram staining microscopic examination shows that they are Gram positive bacilli, with straight rod-shaped and blunt ends, and are scattered or arranged in pairs / short chains; the strains grow well at 30℃~37℃, with the optimal growth temperature being 35℃, and growth is inhibited at temperatures below 28℃ or above 40℃.

[0079] Figure 1 The above strains are shown as colony morphology diagrams on LB agar medium. Figure 2 These are Gram-stained electron microscope images of the above-mentioned strains. Figure 1 and Figure 2 It can be seen that the overall colony morphology and Gram staining microscopic examination results of the novel Bacillus ginseng SCT-B5 on LB agar medium are consistent with the characteristics of the Bacillus genus.

[0080] (2.2) The molecular fragment of the 16S rRNA gene region of the screened Bacillus subtilis SCT-B5 was amplified by PCR (primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2). After passing the agarose gel electrophoresis test, the 16S rRNA sequence of the strain was obtained, as shown in SEQ ID NO:3.

[0081] SEQ ID NO:1

[0082] AGAGTTTGATCCTGGCTCAG.

[0083] SEQ ID NO:2

[0084] GGTTACCTTGTTACGACTT。

[0085] SEQ ID NO:3

[0086]

[0087] The above gene sequence was compared with the gene sequences of some strains already registered in GenBank. Combined with the morphological identification results, it was shown that strain SCT-B5 belongs to Neobacillus ginsengisoli.

[0088] This strain was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36126.

[0089] (3) The strain's utilization of nitrogen and carbon sources

[0090] The above-mentioned strains were cultured for two generations in activation medium and the OD was adjusted. 600 After reaching level 1, the inoculum was inoculated into different culture media at a rate of 2%, and the absorbance was measured after incubation at 37℃ and 200rpm for 48h.

[0091] The formula for the carbon source liquid culture medium is as follows: each 1L of liquid culture medium contains 2g of ammonium sulfate, 0.2g of magnesium sulfate, 0.5g of sodium dihydrogen phosphate, 0.1g of calcium chloride, 5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, and 10g of carbon source (selected from glucose, lactose, maltose, fructose, sucrose, cellobiose, trehalose, starch, or xylan); sterilize at 115℃ for 30min.

[0092] The formula for the nitrogen source liquid culture medium is as follows: Each 1L of liquid culture medium contains 1.36g of potassium dihydrogen phosphate, 0.1g of calcium chloride, 2.13g of disodium hydrogen phosphate, 10g of glucose, 0.2g of magnesium sulfate, 5g of sodium chloride, 0.02g of ferrous sulfate, and 10g of nitrogen source (selected from soybean meal powder, soybean peptone, tryptone or yeast powder respectively); sterilize at 115℃ for 30min.

[0093] The same treatment method and culture medium were used to culture Bacillus endophyticus screened from tobacco leaves, and the utilization of different carbon and nitrogen sources by different strains was compared; the results are shown in Table 1.

[0094] Table 1

[0095]

[0096] Note: The data in Table 1 refer to absorbance (OD) before and after inoculation (excluding blank culture medium). 600 (difference)

[0097] As shown in Table 1, the *Bacillus ginseng* strain SCT-B5 screened in this application can utilize carbon sources such as glucose, lactose, maltose, fructose, sucrose, cellobiose, trehalose, and starch, and nitrogen sources such as soybean meal, soybean peptone, tryptone, and yeast extract. In Table 1, "-" indicates that the strain does not grow.

[0098] (4) Enzyme production characteristics of the strain

[0099] The aforementioned *Bacillus ginseng* SCT-B5 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 37℃ and 200 rpm for 2 days. The fermentation broth was centrifuged to obtain the supernatant. 10 μL of the supernatant was transferred to a protease-producing identification medium and cultured at 37℃ for 24 h. The diameter of the hydrolysis zone or clear zone was measured using calipers to determine the enzyme activity. *Bacillus endophyticus* sieving from tobacco leaves was used as a control. The results are shown in Table 2.

[0100] The formula for 1L protease production identification medium is as follows: KH2PO4 0.36g, MgSO4 0.5g, ZnCl2 0.014g, Na2HPO4·7H2O 1.07g, NaCl 0.16g, CaCl2 0.002g, FeSO4 0.002g, casein 4g, tryptone 0.05g, agar 20g, adjust the pH to 6.5~7.0.

[0101] Table 2

[0102]

[0103] Based on the above results, compared with Bacillus endophytes, the novel Bacillus ginseng SCT-B5 screened in this application formed a larger transparent zone on the culture medium, indicating that it has a stronger protease production capacity.

[0104] To further investigate the amylase production activity of *Bacillus subtilis* SCT-B5, the OD of the activated bacterial culture after two generations was measured. 600 After adjusting to 1, total amylase activity and α-amylase activity were determined using an α-amylase activity kit and a total amylase activity kit (α-amylase test kit (micro method), catalog number JN24263X, Shanghai Jining Industrial Co., Ltd.), respectively. β-amylase activity was then calculated based on the total and α-amylase activities; specifically, β-amylase activity = total amylase activity - α-amylase activity. One unit of amylase activity is defined as per milliliter of culture medium (OD). 600 =1) 1 mg of reducing sugar is produced per minute by catalysis.

[0105] One mL of activated bacterial culture after two generations was centrifuged at 6000 g and 4℃ for 2 min, the supernatant was discarded, and 1 mL of extract was added to resuspend the bacterial cells. The cells were then disrupted by sonication on ice (50 W power, 3 seconds sonication, 7-second interval, total time 3 min). The cells were then centrifuged at 10000 g and 4℃ for 10 min, and the supernatant was placed on ice for testing. The exo-β-1,4-glucanase activity kit (Exo-β-1,4-glucanase (C1) / Cellbiose hydrolase test kit (micro-method / ), catalog number JN24605X, Shanghai Jining Industrial Co., Ltd.) was used for testing. One unit of exo-β-1,4-glucanase (cellulase) activity is defined as the production of 1 μg of glucose per minute by 10,000 bacteria or cells. *Bacillus endophyte* was used as a control. The results are shown in Table 3.

[0106] Table 3

[0107]

[0108] As shown in Table 3, the amylase and exo-β-1,4-glucanase activities of the novel Bacillus ginseng SCT-B5 strain screened in this application were significantly higher than those of Bacillus endophyta.

[0109] (5) Growth curve of the strain

[0110] Two generations of activated *Bacillus subtilis* SCT-B5 and *Bacillus plantarum* were inoculated into LB medium and tobacco broth medium at an inoculation rate of 2%, respectively. The growth curves of the strains were determined using an online cell OD value detection bioreactor (ZWYF-290, Shanghai Zhicheng).

[0111] The LB medium formula is as follows: 10g tryptone, 5g yeast extract, 10g NaCl, 1000mL water, adjust pH to 7.0, and sterilize at 121℃ for 20min.

[0112] The formula for tobacco dust culture medium is as follows: Add 60g of tobacco dust to 1200mL of 100℃ hot water, soak for 2h, filter with 4 layers of gauze, take the supernatant and centrifuge again (10000rpm, 10min), take 1000mL of supernatant, add 10g of glucose, after the glucose is fully dissolved, dispense into 100mL / bottle, sterilize at 115℃ for 30min.

[0113] Figure 3 The growth curve of *Bacillus subtilis* SCT-B5 is shown. Figure 4 This is the growth curve of *Bacillus endophyte*. (From...) Figure 3It can be seen that *Bacillus subtilis* SCT-B5, grown in LB medium (nutrient-rich), exhibits faster growth rates, higher peak values, longer stationary phases, and delayed mortality at all stages. While it can also grow well in tobacco-based medium (nutrient-limited), its growth rate slows, peak values ​​decrease, stationary phase shortens, and mortality occurs earlier. Figure 4 It is known that *Bacillus endophyticus* exhibits an extremely long lag phase in tobacco dust culture medium (due to difficulty in bacterial adaptation), but its growth potential explodes in the later stages (logarithmic growth rate surpasses that of *Bacillus endophyticus*, with a higher peak value), yet its stationary phase is extremely short or nonexistent, and it declines very rapidly. From Figure 3 and Figure 4 It can be seen that *Bacillus ginseng* SCT-B5 is more adaptable to tobacco culture medium than *Bacillus endophyte*, meaning it is more likely to colonize tobacco leaves.

[0114] (6) Effects of strains on tobacco fermentation

[0115] Bacillus subtilis SCT-B5 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 37℃ and 200 rpm for 2 days to obtain the seed culture. 1% (v / v) of the seed culture was inoculated into tobacco extract medium (containing 5 wt% cigar tobacco powder and 95 wt% water) and cultured in a constant temperature shaker at 37℃ and 200 rpm for 2 days to obtain the fermentation broth. 10 mL of the fermentation broth was diluted with sterile water to a final concentration of 10 mL. 5 The bacterial solution was sprayed at 1% of the tobacco leaf weight onto Hainan No. 2 cigar tobacco leaves from Hainan Jianheng and Dexue No. 1 cigar tobacco leaves from Shifang, Sichuan, with a moisture content of 20%. Fermentation was carried out at 30℃ and 80% relative humidity for 30 days. The volatile components in the tobacco leaves were determined by GC-MS (Hu Wanrong, Cai Wen, Zheng Zhaojun, et al. Effects of fermentation medium and fermentation process on the quality of cigar tobacco leaves [J]. Tobacco Science and Technology, 2023, 56(2): 41-52.).

[0116] The total content of volatile components in tobacco leaf samples before and after fermentation was determined, and the results are shown in Table 4. In Table 4, "-" indicates that the corresponding volatile component was not detected.

[0117] Table 4

[0118]

[0119]

[0120] As shown in Table 4, the total content of volatile components in fermented tobacco leaves increased significantly. The volatile component content in Hainan No. 2 cigar tobacco leaves and Dexue No. 1 cigar tobacco leaves increased from the original 9338.62 μg / g and 5533.05 μg / g to 9777.51 μg / g and 6591.9 μg / g, respectively. The content of 19 volatile components increased in fermented tobacco leaves, including nicotine and its degradation products (nornicotine, cotinine, neonicotine), as well as 3-hydroxy-β-dihydrodamascone, 2-butyl-1-octanol, phytol, etc. The levels of six volatile components decreased in the fermented tobacco leaves, including tetrahydro-2,5-dimethyl-2H-pyranol, phytone, megastigmatrienone, 4-(3-hydroxy-1-butenyl)-3,5,5-trimethyl-2-cyclohexen-1-one, 5-ethyl-5-methyl-decane, and dihydroactinol.

[0121] (7) Effects of strains on the sensory quality of tobacco leaves

[0122] The above-mentioned *Bacillus subtilis* SCT-B5 was inoculated into LB liquid medium for tobacco extract and cultured in a constant temperature shaker at 37°C and 200 rpm for 2 days to obtain the seed culture. The seed culture was then inoculated into tobacco extract medium (containing 5 wt% cigar tobacco dust and 95 wt% water) at a 1% (v / v) inoculation ratio and cultured in a constant temperature shaker at 37°C and 200 rpm for 2 days to obtain the fermentation broth. 10 mL of the fermentation broth was diluted with sterile water to a final concentration of 10 mL. 5 The bacterial solution was sprayed at 1 wt% of the tobacco leaf weight onto Chuxue No. 14 cigar tobacco leaves from Hubei Province with a moisture content of 22%. Fermentation was carried out for 15 days at 35℃ and 75% relative humidity. After professional tasting, the aroma and sweetness of the tobacco leaves were significantly increased. The sensory evaluation results are shown in Table 5.

[0123] Table 5

[0124]

[0125] The mass percentage of conventional chemical components in tobacco leaves was determined using a flow analyzer, and the results are shown in Table 6. As can be seen from Table 6, after fermentation treatment of tobacco leaves with the aforementioned Bacillus subtilis SCT-B5, the total sugar and reducing sugar content in the tobacco leaves was significantly increased. The increase in total sugar and reducing sugar content is also one of the reasons for the increased sweetness of the smoke.

[0126] Table 6

[0127]

[0128] The results above indicate that fermentation of tobacco leaves using the novel Bacillus subtilis SCT-B5 obtained through the above screening can effectively improve and enhance the sensory quality of tobacco leaves.

[0129] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0130] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A strain of Neobacillus ginsengisoli SCT-B5, characterized in that, The aforementioned *Bacillus ginseng* was deposited on October 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36126.

2. A method for fermenting tobacco leaves, characterized in that, Includes the following steps: The novel Bacillus subtilis described in claim 1 is used to ferment tobacco leaves.

3. The method for fermenting tobacco leaves according to claim 2, characterized in that, The tobacco leaves include cigar tobacco leaves.

4. The method for fermenting tobacco leaves according to claim 2, characterized in that, Includes the following steps: The *Bacillus ginseng* was cultured to prepare a bacterial culture; and, The bacterial solution is mixed with the tobacco leaves and fermented.

5. The method for fermenting tobacco leaves according to claim 4, characterized in that, The cultivation of the novel Bacillus subtilis from ginseng soil includes the following steps: The novel Bacillus ginseng was subjected to activation culture and proliferation culture in sequence.

6. The method for fermenting tobacco leaves according to claim 4, characterized in that, The amount of bacterial solution added is 0.5wt% to 5wt% of the mass of the tobacco leaves.

7. The method for fermenting tobacco leaves according to claim 4, characterized in that, The concentration of the bacterial solution is 8 × 10⁻⁶. 4 CFU / mL ~12×10 4 CFU / mL.

8. The method for fermenting tobacco leaves according to any one of claims 2 to 7, characterized in that, The fermentation process is carried out at a temperature of 30℃ to 45℃ for 15 to 45 days, with a relative humidity of 70% to 80%.

9. A fermented tobacco leaf, characterized in that, The tobacco leaves are fermented using the fermentation method described in any one of claims 2 to 8.

10. A cigarette product, characterized in that, Includes the fermented tobacco leaves as described in claim 9.

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