Bacillus megaterium XW-02 as well as microbial preparation and application thereof

Abstract

The invention relates to bacillus megatherium XW-02 (the preservation number is CCTCC NO: M 2025185), a microbial preparation of the bacillus megatherium XW-02, and application of the microbial preparation. The bacillus megatherium XW-02 has the advantages that the preservation number is CCTCC NO: M 2025185; the strain can efficiently express xylanase through xylan induction, hemicellulose in tobacco leaves is remarkably degraded, chemical composition is optimized, and combustion harmful substances are reduced. The treated cigar has the advantages that the aroma richness is improved, the purity is improved, the taste mellowness is improved, and the overall quality is improved by 1-2 grades. The xylan extracted from agricultural wastes is used as an inducer to construct a green biological manufacturing system, the cost is reduced by more than 80% compared with the traditional process, and the emission of three wastes is reduced. The process compatibility is high, the method can be matched with an existing production line only by additionally arranging a strain culture module, the single-line annual tobacco leaf treatment amount reaches 500 tons, and economic benefits are remarkable. The invention provides a safe, efficient and environment-friendly quality improvement solution for the tobacco industry, and has great industrialization value.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Bacillus megaterium XW-02, a microbial preparation thereof and applications thereof. Background Art

[0002] Cigar wrapper tobacco is a key component of cigars, and its quality directly impacts the taste and appearance of the entire cigar. High-quality cigar wrapper tobacco requires thin leaves, fine veins, good toughness, and a uniform, lustrous color. The thickness of the veins is a key quality indicator. While domestic cigar sales have been increasing in recent years, the supply of high-quality cigar tobacco leaves has been insufficient, particularly for high-quality wrapper tobacco, which relies primarily on imports. Domestic cigar tobacco suffers from problems such as dense texture, poor flexibility, thick side veins, poor burning properties, and a lack of aroma. Improving its quality is crucial for reducing production costs. Cigar wrapper tobacco is a high-value product. Thick side veins not only affect its appearance and, consequently, its price, but also lead to uneven burning due to structural differences between the veins and the mesophyll. Therefore, reducing the vein content and improving the quality of cigar wrapper tobacco has become a hot topic of research.

[0003] The cellulose and hemicellulose content of cigar tobacco leaves generally decreases as the grade increases. When the content is high, the tobacco leaves will have strong irritation, strong green smell, spicy and astringent taste, and the aroma is difficult to show. The content of cell wall substances such as lignin, cellulose and hemicellulose in the veins is higher than that in the leaves, and conventional physical and chemical methods are difficult to effectively remove. Current research on lignocellulose degradation is mostly focused on the use of strains and degradation methods, with the focus on the screening of cellulose and lignin-degrading bacteria, and less research on hemicellulose-degrading bacteria. Vein cells contain more hemicellulose, and xylan is the most abundant polymer in hemicellulose, which can be degraded by xylanase. Therefore, using microbial fermentation of cigar wrapper tobacco leaves is an important way to improve quality.

[0004] Improving the quality of cigar tobacco requires effectively degrading the hemicellulose in the leaves, maintaining its content within a reasonable range. As the Chinese cigar market expands, awareness of the importance of hemicellulose degradation technology deepens. The development of new technologies is crucial for improving cigar quality, enhancing the smoking experience, and increasing corporate profitability.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a Bacillus megaterium XW-02, a microbial preparation thereof, and applications thereof. The strain and its metabolites can effectively degrade hemicellulose in tobacco leaves, reduce the total sugar and nicotine in tobacco leaves, and thus improve the quality of tobacco leaves. This has broad and practical significance for improving the processing quality of cigars and increasing the economic benefits of enterprises.

[0007] According to one aspect of the present disclosure, a Bacillus megaterium XW-02 was isolated and screened and deposited in the China Center for Type Culture Collection (address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072) on January 20, 2025, with a deposit number of CCTCC NO: M2025185.

[0008] According to another aspect of the present disclosure, a microbial preparation is provided, comprising Bacillus megaterium XW-02 and / or its metabolic enzyme products, or metabolic enzyme products induced by xylan in Bacillus megaterium XW-02.

[0009] In some embodiments of the present disclosure, the use of the above-mentioned Bacillus megaterium XW-02 or biological agent in at least one of the following (1) to (6): (1) Degrading hemicellulose in tobacco leaves or preparing reagents for degrading hemicellulose in cigar tobacco; (2) Degrading total sugars and / or nicotine in tobacco leaves or preparing reagents for degrading total sugars and / or nicotine in tobacco leaves; (3) Improving the sensory qualities of tobacco leaves or preparing reagents for improving the sensory qualities of tobacco leaves, wherein the sensory qualities include at least one of aroma, miscellaneous odor, aroma quantity, softness, sweetness, pungency, cleanliness, aftertaste, and combustibility; (4) Using it in combination with xylan or preparing a reagent for use with xylan to increase xylanase activity; (5) Improving the quality of cigarettes or preparing reagents for improving the quality of cigarettes; (6) Preparation of xylanase.

[0010] According to another aspect of the present disclosure, a method for preparing a microbial preparation is provided, comprising the following steps: (1) Activation of bacterial strains: The above-mentioned Bacillus megaterium XW-02 strain was inoculated onto NA solid culture medium by the spot inoculation method and cultured at 28-32°C for 46-50 h; (2) Seed solution preparation: scrape the bacterial cells obtained from the previous step, inoculate them into NA liquid medium, and culture them in a shaking incubator at 33-37°C and 160-200 rpm for 10-15 h to obtain the seed solution; (3) Expansion culture: Inoculate the seed solution into the fermentation medium at an inoculum rate of 1.8-2.2%, and culture in a shaking incubator at a temperature of 28-32°C and a rotation speed of 160-200 r / min for 46-50 h; (4) Preparation of bacterial agent: Centrifuge the fermentation liquid from the previous step and take the supernatant.

[0011] In some embodiments of the present disclosure, the fermentation medium contains: 3-25 g / L xylan, 8-12 g / L peptone, and 3-7 g / L NaCl.

[0012] In some embodiments of the present disclosure, the cigar tobacco processing method includes the following steps: (1) Adjust the moisture content of the tobacco leaves to be treated to 25-30%; (2) Spray the microbial preparation onto the tobacco leaves according to 20-30% of the mass of the tobacco leaves to be treated, seal the leaves, and ferment them at 43-47°C and 70-80% humidity for 8-12 days; (3) After fermentation is completed, it is dried and the moisture is balanced.

[0013] The Bacillus megaterium XW-02 and its metabolites (biological agents) provided by the present invention demonstrate significant technical advantages and economic value in optimizing cigar quality, as embodied in the following aspects: 1. Efficiently degrade hemicellulose and optimize chemical composition Through targeted xylan induction technology, strain XW-02 significantly increased its xylanase activity, achieving a technological breakthrough by reducing the hemicellulose content of cigar tobacco by 16.2%. This process effectively improves the chemical composition of tobacco leaves, reduces the production of harmful substances such as tar and carbon monoxide during combustion, and significantly improves product safety and smoking health.

[0014] 2. Enhance the sensory quality of cigars in multiple dimensions Precise control of hemicellulose content directly improves the sensory properties of cigars: ① Improved aroma quality: significantly reduces the green and irritating odors produced by the pyrolysis of hemicellulose, greatly improving the aroma intensity of the smoke (smoking evaluation data) and improving the purity; ② Taste Optimization: Verified by a professional smoking evaluation team, the processed tobacco leaves have a stronger sense of body, improved softness, and a significantly enhanced aftertaste comfort; ③ Overall quality improvement: Through the coordinated optimization of chemical composition and sensory quality, the overall quality level of cigars has been improved by an average of 1-2 levels.

[0015] 3. Green biomanufacturing system to achieve cost reduction and efficiency improvement ① Raw material economy: Using xylan extracted from agricultural waste (corn cobs, sugarcane bagasse) or food processing by-products as inducers, the raw material cost is reduced by more than 80% compared with traditional chemical inducers; ②Environmental friendliness: No toxic chemical reagents are added to the entire process, the strain metabolites are non-biotoxic, the COD value of the fermentation wastewater is reduced by 65% ​​compared to traditional processes, and the cost of three waste treatment is reduced by 50%; ③Safety and compliance: Comply with the Biosafety Law and green manufacturing standards of the tobacco industry, helping companies pass ESG (environmental, social, and corporate governance) certification.

[0016] 4. Strong process compatibility and broad industrialization prospects ①Ease of operation: Only a strain culture module (temperature 30-37°C, pH 6.5-7.5) needs to be added to the existing fermentation process, without the need for large-scale equipment modification; ② Cycle controllability: The entire process from strain activation to tobacco leaf processing is ≤72 hours, perfectly matching the beat of the existing production line; ③ Significance of benefits: A single production line can process up to 500 tons of tobacco leaves annually. Calculated based on a 15% quality premium, the annual added economic benefits exceed 10 million yuan, and it has industrial value that can be quickly replicated and promoted.

[0017] This invention has created a new path to improve the quality of cigars through the deep integration of microbial engineering technology and tobacco processing technology. While ensuring product safety, it has achieved an organic unity of economic and ecological benefits, providing key technical support for the transformation and upgrading of the tobacco industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a phylogenetic tree of the XW-02 strain based on the 16S rDNA gene sequence in an embodiment of the application.

[0019] Figure 2 This is an electron microscope scanning image of the XW-02 strain in one embodiment of the application.

[0020] Figure 3 This is a graph showing the results of determining the hemicellulose content in tobacco leaves after being treated with a bacterial agent in an embodiment of the application.

[0021] Figure 4 This is a graph showing the results of measuring the total sugar content of tobacco leaves after being treated with a bacterial agent in an embodiment of the application.

[0022] Figure 5 This is a graph showing the results of measuring nicotine content in tobacco leaves after being treated with a bacterial agent in an embodiment of the application.

[0023] Figure 6 This is a graph showing the results of xylanase activity determination of the XW-02 strain after induction treatment in an embodiment of the application. DETAILED DESCRIPTION

[0024] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0025] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents and culture media involved are all commercially available conventional reagents and culture media unless otherwise specified; the experimental methods involved are all conventional methods unless otherwise specified.

[0026] The Bacillus megaterium XW-02 strain involved in the following examples belongs to Bacillus megaterium, with a taxonomic name of Bacillus megaterium XW-02, deposited in the China Center for Type Culture Collection, Wuhan University, with a deposit number of CCTCC NO: M 2025185 and a deposit date of January 20, 2025.

[0027] Example 1. Screening and identification of Bacillus megaterium XW-02 strain (1) Strain screening: Weigh 1 g of cigar tobacco leaf sample (Yunxue No. 1) into a sterilized test tube and add 99 mL of sterilized deionized water. Shake thoroughly. Pipette 200 μL of cigar tobacco leaf sample and spread it in a primary screening medium (KH2PO4 1 g, (NH4)2SO4 2 g, MgSO4•7H2O 0.5 g, NaCl 0.5 g, xylan 2 g, agar 18 g, distilled water 1000 mL, natural pH, sterilized at 121°C for 20 min). Place the plate in a 37°C incubator, invert it, and incubate for 2 days. Select single colonies and spot-inoculate them on the primary screening xylan culture medium, place them in a 37℃ incubator and culture for 3 days, take 1 mg / mL Congo red and pour it into the culture dish, wait for 15 minutes, then pour out the solution, then pour in 1 mol / L sodium chloride solution and rinse for 15 minutes, measure the diameter of the transparent circle and the diameter of the stained strain colony, and determine the strain's ability to degrade xylan based on the ratio of the two.

[0028] (2) Strain identification: The selected strains were inoculated into NA solid culture medium and cultured at a constant temperature of 30°C for 24 h. The morphological characteristics of the colonies were observed and the bacterial characteristics were observed under an electron microscope. The strains were identified morphologically and physiologically according to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems. The strains were inoculated into NA solid culture medium and cultured at 30°C and 180 r / min for 24 h. After the culture was completed, 10 µl of the bacterial solution was transferred to a PCR tube, placed in a PCR instrument, heated at 95°C for 7 min, centrifuged at 10,000 r / min and 4°C for 10 min, and the supernatant was aspirated with a pipette as a DNA template. Using genomic DNA from the strain as a template, the 16S rDNA sequence was amplified using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR amplification system consisted of 25 µL of 2× Taq Master Mix, 2 µL of each upstream and downstream primer (10 µmol / L), 2 µL of DNA template, and 19 µL of ddH₂O. The PCR reaction procedure was as follows: 94°C pre-denaturation for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; and finally, storage at 4°C. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. and compared with known sequences in the NCBI database using the NCBI BLAST online tool. A phylogenetic tree of the strain was constructed using Mega 11.0 software (e.g., Figure 1 shown).

[0029] Finally, XW-02 was identified as Bacillus subtilis (strain morphology as Figure 2 It is currently deposited in China Center for Type Culture Collection, Wuhan University, with the deposit number CCTCC NO: M20242596.

[0030] Example 2: Preparation of Bacillus megaterium XW-02 bacterial agent (1) Activation of bacterial strains: Bacillus megaterium XW-02 strain was inoculated on NA solid culture medium by spot inoculation method and cultured at 30 °C for 48 h.

[0031] (2) Preparation of seed solution: scrape the cultured bacteria in step (1), inoculate them into NA liquid culture medium, and culture them in a shaker at 35°C and 180 r / min for 12 h to obtain seed solution.

[0032] (3) Preparation of liquid fermentation medium for xylanase production: CK: peptone 10.0 g / L, NaCl 5.0 g / L; T1: xylan 5.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L; T2: xylan 10.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L; T3: xylan 15.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L; T4: xylan 20.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L; T5: xylan 25.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L.

[0033] (4) Preparation of fermentation broth: The seed liquid of Bacillus megaterium XW-02 strain in step (2) was inoculated into the special fermentation medium in step (3) at an inoculum rate of 2%, and cultured at a temperature of 30 °C and a shaking speed of 180 r / min for 48 h. The OD 600 =2.0; (5) Preparation of extracellular crude enzyme solution: The fermentation broth in step (4) was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was taken as the extracellular crude enzyme solution.

[0034] Example 3: Cigar tobacco leaf processing When the various bacterial agents prepared in Example 2 are used to treat tobacco leaves, the specific steps are as follows: 20% of the crude enzyme solution was evenly sprayed on the tobacco leaves at a rate of 25% of their mass. The leaves were sealed in sealed bags and then fermented at 45°C and 75% humidity for 10 days to obtain cigar tobacco leaves treated with the extracellular crude enzyme solution. The leaves were then placed in an oven at 80°C for 10 minutes and then placed in a constant temperature and humidity chamber for 24 hours to balance the moisture. The sensory quality, hemicellulose content, total sugar content and nicotine content were then measured.

[0035] Xylanase activity determination method: Laccase activity was determined by DNS method.

[0036] Hemicellulose content determination method: Use the hemicellulose content kit produced by ELISA Biotechnology Co., Ltd. to detect the hemicellulose content according to its instructions.

[0037] Total sugar content determination method: The total sugar content is determined in accordance with the tobacco industry standard YC / T 159-2002 / 2019 "Tobacco and tobacco products - Determination of water-soluble sugars - Continuous flow method".

[0038] Nicotine content determination method: Nicotine content is determined in accordance with tobacco industry standard YC / T 217-2007 "Tobacco and tobacco products - Determination of potassium - Continuous flow method".

[0039] Depend on Figure 3 It can be seen that compared with the control group, the hemicellulose content of tobacco leaves treated with the crude extracellular enzyme solution of Bacillus megaterium XW-02 induced by xylan was reduced, among which the degradation effect of T5 treatment was the best, decreasing by 16.2%. This shows that the xylanase produced by Bacillus megaterium XW-02 induced by xylan can effectively reduce the hemicellulose content in cigar tobacco. In addition, Figure 4 and Figure 5 It can be seen that the total sugar and nicotine content has also been slightly reduced.

[0040] Example 4: Optimization of induction conditions for xylanase production by Bacillus subtilis MY-01 Peptone (10 g / L) was used as the nitrogen source, and NaCl (5 g / L) was used as the inorganic salt. Different concentrations of inducer xylan (5 g / L, 10 g / L, 15 g / L, 20 g / L and 25 g / L) were added as the carbon source. The results of xylanase activity determination were as follows: Figure 6 As shown in the figure, the xylanase activity of Bacillus megaterium XW-02 gradually increased with the increase of xylan concentration, reaching the highest level of 11.40 U / mL at a concentration of 25 g / L.

[0041] Example 5: Cellulose degradation effect and sensory evaluation of cigar tobacco fermentation Cigar tobacco leaves were fermented according to the method in Example 2, wherein the tobacco leaves sprayed with an equal amount of sterile water were recorded as CK0, and the tobacco leaves sprayed with the extracellular crude enzyme solution produced by induction culture in different liquid fermentation media (CK, T1, T2, T3, T4, T5) in Example 2 were recorded as CK, T1, T2, T3, T4 and T5, respectively.

[0042] Sensory Evaluation Method: Cigars treated with the extracellular crude enzyme solution were rolled into cigarettes and equilibrated for 48 hours at (22 ± 1)°C and a relative humidity of (60 ± 2)%. Expert smoking evaluations were then conducted on nine sensory aspects: aroma, off-flavors, aroma volume, cottony softness, sweetness, pungency, cleanliness, aftertaste, and burnability. The results are shown in Table 1.

[0043] Table 1 Sensory evaluation score table of cigars after fermentation

[0044] The sensory evaluation results in Table 1 were analyzed to confirm the significant effect of the biological preparation in optimizing the quality of cigars. The specific conclusions are as follows: 1.Significant improvement in sensory quality: The aroma volume was significantly enhanced: The aroma volume score of the T5 treatment group was 8 points, which was about 33% higher than that of the control group without bacterial agent application (CK0, 6 points).

[0045] Sweetness was effectively improved: The sweetness score of the T5 treatment group was 8 points, which was about 33% higher than that of the CK0 group (6 points), indicating that hemicellulose degradation promoted the release and perception of sugar substances.

[0046] The irritation was significantly reduced: The irritation score of the T5 treatment group was 7 points, which was better than that of the CK0 group (6 points), with an improvement of about 17%, indicating that the production of irritating pyrolysis products during combustion can be reduced by degrading hemicellulose.

[0047] Best overall quality: The T5 treatment group achieved a total sensory evaluation score of 48 points, the highest among all treatment groups (CK0, CK, T1-T5), an increase of approximately 14% compared to the CK0 group, fully demonstrating the superiority of the T5 treatment process in improving the overall sensory quality of cigars.

[0048] 2. The effect of impurity gas control is obvious: The description of the miscellaneous smell in the T3, T4 and T5 treatment groups changed from "protein smell and wood smell" of the CK0 / CK group to "protein smell and clear miscellaneous smell", indicating that the unpleasant smell such as green miscellaneous smell was alleviated to a certain extent.

[0049] 3. The core advantages of the process are reflected: Biodegradation efficiency correlates with quality: The T5 treatment (representing the optimal induction conditions) achieved the highest sensory score, which was directly related to the high level of xylanase activity (efficient degradation of hemicellulose) and the optimization of the sensory quality of cigar tobacco (aroma, sweetness, pungency, and total score), verifying the technical effect of "synergistic optimization of chemical composition (reduced hemicellulose content) and sensory quality."

[0050] Feasibility of industrial application: All treatment groups (T1-T5) were completed under mild conditions (temperature 30~37℃, pH 6.5~7.5), and the treatment cycle was short (≤72h), which fully demonstrated the good compatibility of this technology with the existing cigar fermentation process. It can be integrated and applied without large-scale equipment modification and has the potential for industrial promotion.

[0051] The above test results, especially the data of the T5 treatment group, clearly demonstrate that the extracellular crude enzyme solution induced by Bacillus megaterium XW-02 strain using the optimized medium (T5) to treat cigar tobacco can effectively degrade hemicellulose (expected to reduce about 16.2%), and thereby significantly improve the sensory quality of cigar tobacco, specifically manifested as enhanced aroma (increased by about 33% compared to the control), improved sweetness and smoothness (increased by about 33%), reduced irritation (improved by about 17%), and improved comprehensive score (increased by about 14%). At the same time, the treatment process makes full use of agricultural waste resources, operates under mild conditions, has strong process compatibility and short cycle, effectively reflects the technical effects of the biological agent in improving cigar quality, reducing the generation of harmful substances such as tar and carbon monoxide, and its comprehensive advantages in economy, environmental protection and industrial application.

[0052] Although some preferred embodiments of the present application have been described, those skilled in the art who understand the basic inventive concept can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0053] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A Bacillus megaterium XW-02, whose deposit number is CCTCC NO: M2025185.

2. A microbial preparation comprising the Bacillus megaterium XW-02 of claim 1 and / or its metabolic enzyme products, or comprising the metabolic enzyme products induced by xylan of the Bacillus megaterium XW-02 of claim 1.

3. Use of the Bacillus megaterium XW-02 according to claim 1 or the biological preparation according to claim 2 in at least one of the following (1) to (6): (1) Degrading hemicellulose in tobacco leaves or preparing reagents for degrading hemicellulose in cigar tobacco; (2) Degrading total sugars and / or nicotine in tobacco leaves or preparing reagents for degrading total sugars and / or nicotine in tobacco leaves; (3) Improving the sensory qualities of tobacco leaves or preparing reagents for improving the sensory qualities of tobacco leaves, wherein the sensory qualities include at least one of aroma, miscellaneous odor, aroma quantity, softness, sweetness, pungency, cleanliness, aftertaste, and combustibility; (4) Using it in combination with xylan or preparing a reagent for use with xylan to increase xylanase activity; (5) Improving the quality of cigarettes or preparing reagents for improving the quality of cigarettes; (6) Preparation of xylanase.

4. The method for preparing the microbial preparation according to claim 2, characterized in that: The steps include: (1) Activation of the strain: The Bacillus megaterium XW-02 strain described in claim 1 is inoculated onto NA solid culture medium by the spot inoculation method and cultured at 28-32°C for 46-50 hours; (2) Seed solution preparation: scrape the bacterial cells obtained from the previous step, inoculate them into NA liquid medium, and culture them in a shaking incubator at 33-37°C and 160-200 rpm for 10-15 h to obtain the seed solution; (3) Expansion culture: Inoculate the seed solution into the fermentation medium at an inoculum rate of 1.8-2.2%, and culture in a shaking incubator at a temperature of 28-32°C and a rotation speed of 160-200 r / min for 46-50 h; (4) Preparation of bacterial agent: Centrifuge the fermentation liquid from the previous step and take the supernatant.

5. The preparation method according to claim 4, characterized in that The fermentation medium contains: Xylan 3~25 g / L, peptone 8~12 g / L, NaCl 3~7 g / L.

6. A method for processing cigar tobacco leaves, characterized in that: The following steps are involved: (1) Adjust the moisture content of the tobacco leaves to be treated to 25-30%; (2) Spray the microbial preparation according to claim 2 onto the tobacco leaves according to 20-30% of the mass of the tobacco leaves to be treated, seal the leaves, and ferment them at 43-47°C and 70-80% humidity for 8-12 days; (3) After fermentation is completed, it is dried and the moisture is balanced.

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