Bacillus subtilis SXZY-03 and application thereof in tobacco leaf fermentation
By screening and activating Bacillus subtilis SXZY-03, a biological agent was prepared for tobacco fermentation, which solved the problem of incomplete polyphenol degradation, improved the sensory quality of tobacco and maintained its flavor, and achieved green and environmentally friendly tobacco processing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHINA TOBACCO SHAANXI IND
- Filing Date
- 2026-02-12
- Publication Date
- 2026-06-23
AI Technical Summary
In existing tobacco fermentation technologies, polyphenols are not completely degraded, resulting in poor sensory quality. Furthermore, traditional physicochemical methods are inefficient and damage flavor.
A strain of Bacillus subtilis SXZY-03 was screened and isolated. Its polyphenol oxidase activity was activated by an activator, and a biological agent was prepared for tobacco fermentation to optimize enzyme activity and degrade polyphenols, thereby improving the sensory quality of tobacco.
It significantly degrades polyphenols in tobacco leaves, enhances the aroma and flavor of tobacco, reduces bitterness in the aftertaste, increases enzyme activity, and maintains the natural flavor of tobacco, which aligns with the direction of green and environmentally friendly development.
Smart Images
Figure CN122256170A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bio-fermentation technology and relates to a strain of Bacillus subtilis SXZY-03 and its application in tobacco fermentation. Background Technology
[0002] Fermentation of tobacco leaves is a crucial process in cigarette processing. Essentially, it involves the synergistic action of microorganisms and enzymes to decompose and transform macromolecules (such as proteins, starches, and polyphenols) in tobacco leaves, aiming to reduce irritation, improve taste, and enhance sensory quality. Polyphenols, as important secondary metabolites of tobacco leaves, significantly influence the sensory quality of the final product. Excessive polyphenol content can lead to a noticeable astringency and irritation in the smoke, and may also cause a rough aroma and poor permeability due to over-oxidation, thus hindering further improvement in the sensory quality of the tobacco leaves. Therefore, appropriately reducing the polyphenol content in tobacco leaves through biotransformation is of great significance for reducing astringency and improving aroma and smoothness. Polyphenol oxidase plays a vital role in this transformation process, efficiently catalyzing the oxidation of polyphenols, thereby driving the degradation and transformation of aroma precursors such as carotenoids, and having a decisive influence on the formation of the unique aroma style of tobacco leaves. Currently, in the field of tobacco fermentation, utilizing exogenous microorganisms or their enzyme preparations to improve quality has become a research hotspot.
[0003] However, existing tobacco fermentation schemes generally have the following limitations: First, many studies directly use commercial enzyme preparations, which are costly and poorly adapted to the complex tobacco system, resulting in unstable enzyme activity efficiency; second, although there have been reports on fermentation using microorganisms such as Bacillus subtilis, most of these studies focus on direct inoculation of the strains, neglecting to maximize the potential of key enzyme systems through pretreatment methods (such as activator induction), leading to limited enzyme activity enhancement, insufficient polyphenol degradation efficiency, and ultimately insignificant quality improvement; third, existing technologies lack a complete process chain for targeted screening of specific strains with high polyphenol oxidase production potential, systematically enhancing their enzyme activity through activator methods, and then applying the highly efficient enzyme solution to tobacco fermentation.
[0004] Therefore, a directional fermentation technology based on polyphenol-degrading bacteria is needed to solve the technical problems mentioned above in existing tobacco processing, such as incomplete polyphenol degradation leading to poor sensory quality and the low efficiency and flavor degradation of traditional physicochemical methods. Summary of the Invention
[0005] The technical solution of this invention is: a strain of Bacillus subtilis ( Bacillus subtilis SXZY-03, with accession number CCTCC NO:M 2025150, was deposited at the China Center for Type Culture Collection on January 15, 2025.
[0006] The present invention also provides the application of the above-mentioned Bacillus subtilis or its fermentation products in tobacco fermentation.
[0007] Preferably, the fermentation product includes polyphenol oxidase.
[0008] This invention also provides a biological agent prepared from Bacillus subtilis or its fermentation products, which is used to: degrade polyphenols in tobacco; improve the sensory quality of tobacco, characterized by at least one of aroma quality, aroma intensity, woody off-flavors, irritation, aftertaste, strength, concentration, and smoothness; enhance aroma concentration and reduce bitterness in the aftertaste; and secrete or prepare polyphenol oxidase; this biological agent is an enzyme preparation for tobacco processing.
[0009] Preferably, the method for preparing the biological agent includes the following steps: Step 1: Activate the bacterial strain: Inoculate Bacillus subtilis SXZY-03 into NA solid medium and incubate at 30-35℃ for 36-72 hours; Step 2, Preparation of seed culture: Take the bacterial cells cultured in Step 1, inoculate them into NA liquid culture medium, and culture them in a shaker at 30-35℃ and 180-200 r / min for 14-18 hours to obtain the seed culture; Step 3, scale-up culture: Inoculate the seed culture obtained in step 2 into NA liquid medium at 1% to 3% of the weight of the medium, and culture at 30℃ to 35℃ and shaking speed of 180 to 200 r / min for 36 to 48 hours to obtain fermentation broth; Step 4: Preparation of crude enzyme solution: Centrifuge the fermentation broth obtained in step 3 at 3-5℃ and 8000-10000rpm for 8-12 minutes, and take the supernatant, which is the biological agent; the fermentation product is the supernatant obtained after centrifugation of the fermentation broth.
[0010] More preferably, in step 1, the composition of the NA solid culture medium includes: 2-4 g / L beef extract, 8-12 g / L peptone, 4-6 g / L sodium chloride, and 18-22 g / L agar powder; in steps 2 and 3, the composition of the NA liquid culture medium includes: 2-4 g / L beef extract, 8-12 g / L peptone, and 4-6 g / L sodium chloride.
[0011] Preferably, the biological agent is used to process tobacco shreds, comprising the following steps: Step A: Spray the biological agent onto the tobacco shreds at 2% to 6% of the weight of the tobacco leaves to be treated, adjust the moisture content of the tobacco shreds to 18% to 22%, seal, and ferment at 40 to 45°C and 70% to 80% humidity for 24 to 36 hours. Step B: Dry the tobacco shreds at 80℃ for 10-15 minutes to balance the moisture content.
[0012] The beneficial effects of this invention are: 1. This invention possesses highly efficient polyphenol degradation capabilities: The Bacillus subtilis SXZY-03 strain of this invention exhibits excellent polyphenol oxidase production capabilities. After activation and concentration optimization with copper sulfate, manganese sulfate, and ferric sulfate, the polyphenol oxidase activities reached 112.59, 69.26, and 51.26 U·mL, respectively. -1 The concentration was significantly higher than the 24.52 U·mL without activation. -1 This strain can effectively promote the targeted degradation of polyphenols in tobacco leaves. Experimental studies have shown that polyphenols in tobacco treated with enzyme solutions activated by activators are degraded to varying degrees. Among them, the polyphenol degradation effect is the best in tobacco treated with enzyme solutions activated by copper sulfate. The contents of chlorogenic acid, rutin, and hyoscyamine are reduced by 3.09, 3.34, and 0.08 mg / g, respectively, compared with the control (CK), and by 1.58, 1.83, and 0.02 mg / g, respectively, compared with tobacco treated with unactivated enzyme solutions.
[0013] 2. This invention significantly improves the sensory quality of tobacco: By precisely controlling the polyphenol degradation process, this invention directs the conversion of polyphenols in tobacco leaves into low-molecular-weight aroma substances (such as phenylacetaldehyde and phenylethanol), thereby significantly improving the sensory quality of tobacco. Sensory evaluation results show that tobacco treated with the strain of this invention exhibits significant improvements in key indicators such as aroma, aftertaste, and smoothness, with a total score of 45.0 points. Tobacco treated with enzyme solution activated by the activator shows further improvement in sensory quality. Specifically, this is manifested in increased aroma volume, enhanced permeability, reduced astringency, increased smoke smoothness, prominent sweetness, and a significant improvement in the bitterness of the aftertaste, with a total score 3.5–4.5 points higher than the CK group and 1–2 points higher than the T1 group. This fully verifies the progressive synergistic effect of strain SXZY-03 in optimizing the sensory quality of tobacco, providing practical technical support for the development of high-quality tobacco products.
[0014] 3. This invention effectively enhances enzyme activity: By adding trace amounts of activators such as sugars, phenols, and metal ions to the enzyme solution, this invention identified copper sulfate, manganese sulfate, and ferric sulfate as the three substances with the best activation effect on the polyphenol oxidase activity of strain SXZY-03, thereby maximizing the polyphenol oxidase activity and releasing the enzyme's catalytic potential to the greatest extent. This technical solution not only provides a practical and feasible optimization path for the efficient application of polyphenol oxidase, but also provides an important reference for the activity regulation research of other enzyme preparations, and has broad application prospects in multiple fields such as food processing, biotransformation, and environmental remediation.
[0015] 4. This invention provides a new green and environmentally friendly path for tobacco flavor: by using a microbial enzymatic hydrolysis method, polyphenols are degraded in a targeted manner under mild conditions, avoiding the environmental shortcomings and flavor destruction problems of traditional physical or chemical methods. While efficiently improving the quality of tobacco, the natural flavor components of tobacco are fully preserved, which is in line with the high-quality development direction of the tobacco industry of "reducing harm and tar, improving quality and enhancing aroma".
[0016] 5. This invention achieves precise regulation of polyphenols in tobacco by screening the highly efficient polyphenol oxidase-producing strain SXZY-03 and optimizing its cultivation process. It effectively solves the problems of high polyphenol content, strong bitterness, and low polyphenol oxidase activity in existing technologies, providing an innovative technical path for the green improvement of tobacco quality. Attached Figure Description
[0017] Figure 1 A streak plate diagram of Bacillus subtilis SXZY-03 in an embodiment of the present invention and its application in tobacco fermentation; Figure 2 This is a phylogenetic tree diagram constructed based on the 16S rDNA gene sequence of Bacillus subtilis strain SXZY-03 in an embodiment of the present invention; Figure 3 This is a line graph showing the optimized concentration of polyphenol oxidase activator in Bacillus subtilis strain SXZY-03 in this invention. Figure 4 The columnar sample is a result of the degradation of tobacco polyphenols by the Bacillus subtilis SXZY-03 strain in this embodiment of the invention. Detailed Implementation
[0018] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0019] like Figures 1-4 As shown, this invention addresses the technical problems of incomplete polyphenol degradation leading to poor sensory quality in existing tobacco processing, and the low efficiency and flavor degradation of traditional physicochemical methods. It employs a strain of Bacillus subtilis with high polyphenol oxidase content isolated from the surface of tobacco. Bacillus subtilis SXZY-03 was developed, and by exploring the optimal activator to activate enzyme activity, it was applied to tobacco fermentation treatment, achieving outstanding technical effects such as efficient degradation of tobacco polyphenol content, significant improvement of tobacco sensory quality, and reduction of aftertaste bitterness.
[0020] According to one aspect of this disclosure, a strain of Bacillus subtilis ( ) was isolated through screening. Bacillus subtilis SXZY-03 was deposited on January 15, 2025 at the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072, China) with accession number CCTCC NO:M 2025150.
[0021] According to one aspect of this disclosure, a biological agent is provided containing the Bacillus subtilis SXZY-03 and / or its fermentation products, the fermentation products including polyphenol oxidase.
[0022] According to one aspect of this disclosure, the use of Bacillus subtilis SXZY-03 or the biological agent in at least one of the following or in the preparation of a product having at least one of the following functions: (1) Degrading polyphenols in tobacco; (2) Improve the sensory quality of tobacco, wherein the sensory quality is characterized by at least one of aroma quality, aroma quantity, woody off-flavor, irritation, aftertaste, strength, concentration, and softness; (3) Enhance aroma concentration and reduce aftertaste bitterness; (4) Secretion or preparation of polyphenol oxidase.
[0023] According to another aspect of this disclosure, a method for preparing the biological agent is provided, characterized by comprising the following steps: (1) Activation of bacterial strain: The Bacillus subtilis SXZY-03 was inoculated into NA solid medium and cultured at 30-35℃ for 36-72h; (2) Preparation of seed culture: Pick the bacterial cells cultured in step (1), inoculate them into NA liquid culture medium, and culture them in a shaker at 30-35℃ and 180-200 r / min for 14-18h to obtain seed culture; (3) Expanded culture: The seed culture obtained in step (2) is inoculated into NA liquid culture medium at 1% to 3% of the weight of the culture medium, and cultured at 30℃ to 35℃ and shaking speed of 180 to 200 r / min for 36 to 48 hours to obtain fermentation broth; (4) Preparation of crude enzyme solution: Centrifuge the obtained fermentation broth at 3-5℃ and 8000-10000rpm for 8-12 minutes, and take the supernatant, which is the biological agent.
[0024] In some of the disclosed embodiments, the NA solid culture medium composition is as follows: beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, and agar powder 20.0 g / L.
[0025] In some of the disclosed embodiments, the NA liquid culture medium is composed of: 3.0 g / L beef extract, 10.0 g / L peptone, and 5.0 g / L sodium chloride.
[0026] According to another aspect of this disclosure, a method for processing tobacco shreds is provided, characterized by comprising the following steps: (A) Spray the biological agent onto the tobacco shreds at 2% to 6% of the mass of the tobacco leaves to be treated, adjust the moisture content of the tobacco shreds to 18% to 22%, seal, and ferment at 40 to 45°C and 70% to 80% humidity for 24 to 36 hours; (B) Dry the tobacco shreds at 80°C for 10-15 minutes to balance the moisture content.
[0027] Example Example 1: Screening and identification of Bacillus subtilis SXZY-03.
[0028] Bacillus subtilis was isolated from the surface of tobacco leaves in Hanzhong, Shaanxi Province in 2020 and named SXZY-03.
[0029] The specific screening and isolation process was as follows: 2g of Shaanxi Hanzhong tobacco shreds were placed in 40 mL of sterile water and cultured on a shaker at 30℃ and 180 r / min for 48 h. Guaiacin was used as the sole nutrient source to screen for polyphenol-degrading bacteria; strains capable of growing in this medium possessed the ability to degrade polyphenols. Finally, strain SXZY-03 was purified by streaking on NA medium. Figure 1 ).
[0030] The strain sequence was amplified using universal 16S rDNA primers, and the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained sequences were compared with the NCBI database, showing 98.83% homology with Bacillus subtilis, indicating a close phylogenetic relationship between the two (e.g., ...). Figure 2 (As shown).
[0031] Therefore, SXZY-03 was identified as Bacillus subtilis (… Bacillus subtilis It is currently deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 2025150.
[0032] Example 2: Preparation of biological agents.
[0033] (1) Activation of bacterial strain: Bacillus subtilis SXZY-03 was inoculated on NA solid medium (beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar powder 20.0 g / L) and cultured at 30℃ for 36 h; (2) Seed culture preparation: Pick the bacterial cells cultured in step (1) and inoculate them into NA liquid medium (beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L). Culture at 30℃ and shaking at 180-200r / min for 14-18h to obtain seed culture. (3) Expanded culture: The Bacillus subtilis SXZY-03 seed culture obtained in step (2) was inoculated into NA liquid medium at 2% to 4% of the weight of the medium. The culture was carried out at 30°C and shaken at 180 to 200 r / min for 36 to 48 hours to obtain the fermentation broth. (4) Preparation of crude enzyme solution: Centrifuge the obtained fermentation broth at 3-5℃ and 8000-10000rpm for 8-12min, and take the supernatant, which is the biological agent.
[0034] Example 3: Polyphenol oxidase activation test and concentration optimization of Bacillus subtilis strain SXZY-03.
[0035] The crude enzyme solution prepared in Example 2 was supplemented with 20 compounds, including salicylic acid, gallic acid, vanillic acid, ferulic acid, hydroquinone, rutin, chlorogenic acid, copper sulfate, manganese sulfate, ferric sulfate, magnesium sulfate, ammonium sulfate, fructose, glucose, oligosaccharides, sucrose, lactose, chitosan, L-ascorbic acid, and farnesol, to a final concentration of 0.01%. The enzyme solution in NA medium without these compounds served as a control. The solution was incubated in a 30°C water bath for 2 hours, and the polyphenol oxidase activity was measured using a Shanghai Enzyme-Linked Immunosorbent Assay Kit (sample number: ml076354).
[0036]
[0037] The enzyme activity results are shown in Table 1 (Polyphenol oxidase activity results). All substances showed an activating effect on the polyphenol oxidase activity of strain SXZY-03. Among them, the three substances with the most significant activation effects were copper sulfate, manganese sulfate, and ferric sulfate, with enzyme activities reaching 73.20, 55.16, and 41.00 U·mL, respectively. -1 .
[0038] Three substances with the best activation effect were selected, and concentration gradients of 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, and 0.11% were set. The activity of polyphenol oxidase under different concentrations of activator was measured. The results are as follows: Figure 3 When the concentrations of copper sulfate, manganese sulfate, and ferric sulfate were 0.09%, the polyphenol oxidase activity of strain SXZY-03 reached its maximum values of 112.59, 69.26, and 51.26 U·mL, respectively. -1 .
[0039] Example 4: Degradation effect and sensory evaluation results of polyphenols in tobacco fermentation.
[0040] The biological agent from Example 3 was sprayed onto the tobacco shreds at 4% of the weight of the tobacco leaves to be treated. After adjusting the moisture content of the tobacco shreds to 18%–22%, the mixture was sealed and fermented at 40°C and 75% humidity for 24 hours. The tobacco shreds were then dried at 80°C for 15 minutes to balance the moisture content, and the product was obtained.
[0041] Tobacco shreds sprayed with an equal amount of sterile water during the tobacco processing were denoted as CK; tobacco shreds sprayed with an equal amount of crude enzyme solution obtained in NA medium were denoted as T1; tobacco shreds sprayed with an equal amount of enzyme solution activated by 0.09% copper sulfate activator were denoted as T2; tobacco shreds sprayed with an equal amount of enzyme solution activated by 0.07% manganese sulfate activator were denoted as T3; and tobacco shreds sprayed with an equal amount of enzyme solution activated by 0.05% ferric sulfate activator were denoted as T4.
[0042] The dried tobacco shreds were ground into powder, passed through a 60-mesh sieve, and the contents of rutin, chlorogenic acid and hyoscyamine in the tobacco leaves were determined by LC-2010C high performance liquid chromatography according to the method of YC / T 202-2006. The sensory quality of the tobacco shreds was evaluated according to the sensory evaluation method of tobacco in YC / T 138-1998.
[0043] The results are as follows: Figure 4 It was found that the polyphenols in tobacco treated with enzyme solutions activated by the activator were degraded to varying degrees. The enzyme solution activated by copper sulfate showed the best polyphenol degradation effect, with the contents of chlorogenic acid, rutin, and hyoscyamine decreasing by 3.09, 3.34, and 0.08 mg / g compared to the control (CK), and by 1.58, 1.83, and 0.02 mg / g compared to the unactivated enzyme solution. This indicates that the Bacillus subtilis SXZY-03 enzyme solution activated by the activator can effectively increase the degradation of polyphenols in tobacco.
[0044] Table 2 (Sensory Quality Evaluation Table for Treated Tobacco) shows that the T1 treatment group, sprayed with microbial agents, exhibited a significant improvement in sensory quality compared to the CK group after fermentation. Specifically, the aroma was improved, the aroma intensity increased, and the bitterness in the aftertaste was significantly reduced. The enzyme-treated groups T2, T3, and T4, activated by the activator, showed a significant improvement in sensory quality of the tobacco after fermentation compared to the T1 treatment group. The T2 treatment group, in particular, showed improved aroma intensity, reduced astringency, the most significant improvement in aftertaste, and a certain degree of improvement in smoothness. This indicates that treatment with Bacillus subtilis strain SXZY-03 can promote the degradation of polyphenols in tobacco leaves, thereby reducing the bitterness in the aftertaste and releasing aroma substances, resulting in a significant improvement in the overall sensory quality of the tobacco leaves and effectively increasing the utilization value of low-grade tobacco leaves in industrial production.
[0045]
[0046] In summary, this invention represents a significant breakthrough for the tobacco processing industry by screening for highly efficient polyphenol oxidase-producing Bacillus subtilis SXZY-03, optimizing its cultivation process, and employing suitable activators to enhance enzyme activity. Its application in tobacco leaf fermentation not only technically solves problems such as incomplete polyphenol degradation and flavor degradation in existing tobacco processing methods but also aligns with the industry's development direction of "reducing harm and tar, improving quality and aroma." This achievement allows tobacco companies to improve tobacco quality while reducing environmental impact and resource waste. In terms of cost control, this green and environmentally friendly approach avoids the high costs and risks associated with traditional physical or chemical methods. With increasingly stringent requirements for tobacco quality and stricter environmental regulations, the technical solution provided by this invention has broad market application prospects and is expected to play a vital role in the future tobacco industry, driving the entire tobacco industry towards a greener, more efficient, and higher-quality direction.
[0047] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.
Claims
1. A strain of Bacillus subtilis ( Bacillus subtilis SXZY-03, with accession number CCTCC NO:M2025150, was deposited at the China Center for Type Culture Collection on January 15, 2025.
2. The application of Bacillus subtilis or its fermentation products as described in claim 1 in tobacco fermentation.
3. The application of Bacillus subtilis or its fermentation product according to claim 2 in tobacco fermentation, characterized in that, The fermentation products include polyphenol oxidase.
4. A biological agent prepared from Bacillus subtilis or its fermentation product as described in claim 2, wherein the biological agent is used for: Degrades polyphenols in tobacco; Improving the sensory quality of tobacco, wherein the sensory quality is characterized by at least one of aroma quality, aroma quantity, woody off-flavors, irritation, aftertaste, strength, concentration, and smoothness. Enhance aroma concentration and reduce bitterness in the aftertaste; Secretion or preparation of polyphenol oxidase.
5. The biological agent prepared from Bacillus subtilis or its fermentation product according to claim 4, characterized in that, The preparation method of the biological agent includes the following steps: Step 1, Activation of bacterial strain: The Bacillus subtilis SXZY-03 was inoculated into NA solid medium and cultured at 30-35℃ for 36-72 hours; Step 2, Preparation of seed culture: Take the bacterial cells cultured in Step 1, inoculate them into NA liquid culture medium, and culture them in a shaker at 30-35℃ and 180-200 r / min for 14-18 hours to obtain the seed culture; Step 3, scale-up culture: Inoculate the seed culture obtained in step 2 into NA liquid medium at 1% to 3% of the weight of the medium, and culture at 30℃ to 35℃ and shaking speed of 180 to 200 r / min for 36 to 48 hours to obtain fermentation broth; Step 4: Prepare crude enzyme solution: Centrifuge the fermentation broth obtained in step 3 at 3-5℃ and 8000-10000rpm for 8-12 minutes, and take the supernatant, which is the biological agent.
6. The biological agent prepared from Bacillus subtilis or its fermentation product according to claim 5, characterized in that, In step 1, the composition of the NA solid culture medium includes, in g / L: 2-4 g of beef extract, 8-12 g of peptone, 4-6 g of sodium chloride, and 18-22 g of agar powder. In steps 2 and 3, the NA liquid culture medium consists of: 2-4 g / L beef extract, 8-12 g / L peptone, and 4-6 g / L sodium chloride.
7. The biological agent prepared from Bacillus subtilis or its fermentation product according to claim 4, characterized in that, When the biological agent is used to process tobacco, the following steps are included: Step A: Spray the biological agent onto the tobacco shreds at 2% to 6% of the weight of the tobacco leaves to be treated, adjust the moisture content of the tobacco shreds to 18% to 22%, seal, and ferment at 40 to 45°C and 70% to 80% humidity for 24 to 36 hours. Step B: Dry the tobacco shreds at 80℃ for 10-15 minutes to balance the moisture content.