Application of bacillus megatherium XW-02 in tobacco quality improvement
By fermenting cigar tobacco leaves with Bacillus megaterium XW-02, the problem of excessive pectin content is solved, the effective degradation of pectin is achieved, and the appearance and sensory quality of cigars are improved, which has both environmental and economic advantages.
Patent Information
- Application Number
- CN202510787800.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-13
- Publication Date
- 2025-10-10
AI Technical Summary
Excessively high pectin content in cigar tobacco leaves leads to processing difficulties, reduced appearance quality and health risks, and existing technologies are difficult to effectively degrade pectin.
Bacillus megaterium XW-02 and its fermentation products were used to degrade pectin in tobacco leaves through microbial fermentation. Wheat bran was used as a nitrogen source to prepare a pectin-degrading preparation, which was sprayed on the tobacco leaves and then fermented under specific conditions.
Significantly reduce pectin content, improve the appearance and sensory quality of cigars, reduce unpleasant odors, enhance aroma quality and taste, while achieving environmental and economic benefits and simplifying operating procedures.
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Figure CN120753431A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of bioengineering technology, and in particular to application of Bacillus megaterium XW-02 in improving tobacco quality. Background Art
[0002] Pectin, a key component of tobacco cell wall structure, is a hydrophilic colloidal substance widely found in the cell walls and intercellular layers of tobacco leaves. Together with cellulose and hemicellulose, it forms a complex network of cell walls, providing essential structural support for tobacco leaves and maintaining their shape and firmness. The pectin content and properties profoundly influence the softness, viscosity, processing properties, and final quality of cigar tobacco leaves. Cigar tobacco contains significantly higher pectin content than flue-cured tobacco, typically reaching 12% to 17%, compared to only 4% to 6%. This difference presents unique challenges during cigar processing. High pectin content can lead to sticking of tobacco leaves during rolling, resulting in uneven dark spots, which severely impacts the appearance and industrial viability of cigars. Furthermore, high pectin content produces a pungent odor and increases tar content during combustion. Pectin is also a precursor to harmful volatile carbonyl compounds such as formaldehyde and acetaldehyde in cigar smoke, posing a potential threat to human health.
[0003] Pectin is highly stable in tobacco leaves and difficult to effectively break down during natural fermentation. Therefore, during the fermentation process of cigar tobacco stacks, special measures are required to reduce the pectin content to improve the quality and smoking safety of cigars. Controlling pectin content not only helps address the problem of excessive pectin content in cigar tobacco leaves but is also crucial to improving the industrial viability and smoking safety of cigars.
[0004] 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
[0005] The inventors have discovered that Bacillus megaterium XW-02, deposited with CCTCC NO: M2025185, and its fermentation products can effectively degrade pectin in tobacco leaves. Using them to process and produce cigar tobacco leaves can significantly improve the quality of cigars, reduce costs, and increase the economic benefits of cigars.
[0006] In one aspect of the present disclosure, Bacillus megaterium XW-02 with a deposit number of CCTCC NO: M2025185 or its metabolite enzyme product is used in at least one of the following (1) to (6): (1) Degrading pectin in tobacco leaves or preparing a preparation for degrading pectin in tobacco leaves; (2) Degrading total sugar in tobacco leaves or preparing a preparation for degrading total sugar in tobacco leaves; (3) Improving the tensile strength of tobacco leaves or preparing reagents for improving the tensile strength of tobacco leaves; (4) Improving the sensory qualities of tobacco leaves or preparing preparations for improving the sensory qualities of tobacco leaves, wherein the sensory qualities include at least one of aroma quality, miscellaneous odors, aroma quantity, mellowness, pungency, aftertaste, strength, and concentration; (5) Improving the quality of cigarettes or preparing preparations that improve the quality of cigarettes; (6) Preparation of pectinase.
[0007] According to another aspect of the present disclosure, a method for preparing a microbial preparation is provided, comprising the following steps: (1) Activation of strains: Bacillus megaterium XW-02 strain with the deposit number CCTCC NO: M2025185 was inoculated onto NA solid medium by the spot inoculation method and cultured at 28-32°C for 24-48 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 28-32°C and 160-200 rpm for 10-15 h to obtain the seed solution; (3) Expanding the culture: inoculating the seed solution into a fermentation medium at an inoculum rate of 2-4%, and culturing the culture medium in a shaker at a temperature of 28-32°C and a rotation speed of 160-200 r / min for 46-50 h; the fermentation medium contains 0.8-1.2 g / L glucose, 8-12 g / L wheat bran powder, and 10-12 g / L KCl; (4) Preparation of bacterial agent: centrifuge the fermentation liquid from the previous step and take the supernatant.
[0008] According to another aspect of the present disclosure, a method for processing cigar tobacco leaves is provided, comprising the following steps: (1) Adjust the moisture content of the tobacco leaves to be treated to 28-32%; (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 33-37°C and 70-80% humidity for 3-5 days; (3) After fermentation is completed, it is dried and the moisture is balanced.
[0009] One or more technical solutions provided in the embodiments of this application have at least any of the following technical effects or advantages: 1. Effectively reduce pectin content: The pectin content of cigar tobacco fermented with the optimized extracellular crude enzyme solution of Bacillus megaterium XW-02 is significantly reduced compared to before fermentation. After a certain period of treatment, the pectin content of cigar tobacco is reduced by 19.5%. This helps to reduce the sticking of tobacco leaves during the rolling process, avoid the formation of uneven dark spots, and improve the appearance quality of cigars.
[0010] 2. Improved Cigar Quality: Reducing the pectin content in cigars effectively reduces the unpleasant odor and irritation that can occur with excessive pectin content, significantly improving the aroma and taste of the cigars. Professional smoking evaluation teams have shown that the treated cigars have reduced irritation, bitterness, and other unpleasant odors, while improving their aroma and taste, significantly enhancing their overall quality.
[0011] 3. Environmentally friendly and low-cost: The optimized induction conditions of this invention utilize wheat bran as a nitrogen source. Wheat bran is a common agricultural waste that is widely available and inexpensive. This not only reduces production costs but also realizes the resource utilization of agricultural waste, with good economic and environmental benefits. Furthermore, the entire treatment process, based on microbial fermentation, is more environmentally friendly than traditional chemical treatment methods, without introducing harmful chemicals, thus meeting current requirements for environmental protection and sustainable development.
[0012] 4. Simple operation and easy promotion: The preparation process of the fermentation microbial preparation of Bacillus megaterium XW-02 is simple and efficient, the fermentation cost is low, and compared with the stacking fermentation process, the processing cycle is shorter and the safety is higher. It can effectively reduce the pectin content in cigar tobacco leaves and effectively improve the industrial availability of cigar tobacco leaves. Therefore, this technology has good promotion and application value in the field of cigar fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] 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.
[0014] Figure 2 This is a diagram of the XW-02 strain in one embodiment of the application.
[0015] Figure 3 This is a graph showing the results of measuring the pectin content in tobacco leaves after being treated with a bacterial agent in an embodiment of the application.
[0016] 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.
[0017] Figure 5 This is a graph showing the tensile strength measurement results of tobacco leaves after being treated with a bacterial agent in an embodiment of the application.
[0018] Figure 6 This is a graph showing the results of measuring the pectinase activity of the XW-02 strain after induction treatment in an embodiment of the application. DETAILED DESCRIPTION
[0019] 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.
[0020] 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.
[0021] The Bacillus megaterium XW-02 strain involved in the following examples belongs to Bacillus Megaterium, with a taxonomic name of Bacillus Megaterium XW-02, and is 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.
[0022] Example 1. Screening and identification of Bacillus megaterium XW-02 strain (1) Strain screening: 10 g of tobacco leaves (produced in Guangxi) were added to 90 mL of sterile water under sterile conditions, and then placed in a constant temperature shaker at 180 rpm for 1 day. After the end, 10 mL of the culture solution was transferred to the sterile operating table and diluted with sterile water to 10 -4 , 10 -5 and 10 -6 Three gradients of dilutions were prepared. 100 μL of each dilution was spread onto CMC selective medium plates (pectin 10.0 g / L, K₂HPO₄ 1.0 g / L, NH₄NO₃ 1.0 g / L, MgSO₄•7H₂O 0.2 g / L, CaCl₂ 0.02 g / L, agar 15.0 g / L, natural pH). Each gradient was replicated three times. The plates were incubated at 30°C in an electric incubator for 24 h. Single colonies were picked from the culture medium and streaked onto NA medium plates for isolation and purification.
[0023] (2) Identification of bacterial species: The selected strains were inoculated into NA solid culture medium and cultured at 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 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; followed by extension at 72°C for 5 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).
[0024] Finally, XW-02 was identified as Bacillus Megaterium (strain morphology as Figure 2 It is currently deposited in China Center for Type Culture Collection, Wuhan University, with the deposit number CCTCC NO: M2025185.
[0025] Example 2: Preparation of Bacillus megaterium XW-02 bacterial agent (1) Strain activation: Bacillus megaterium XW-02 was inoculated onto NA solid culture medium by the dot-graft method and cultured at 30°C for 24-48 hours to obtain the activated strain.
[0026] (2) Seed liquid preparation: Scrape the XW-02 strain from the NA solid culture medium and inoculate it into the NA liquid culture medium. Shake and culture it in a shaker at 180 r / min and 30°C for 12 h to obtain the seed liquid.
[0027] (3) Preparation of liquid culture medium for pectinase production T1: NA liquid medium (3 g / L beef extract, 5 g / L NaCl, 10 g / L peptone) T2: 1g / L glucose, 10g / L wheat bran powder, 11g / L KCl (4) Preparation of microbial fermentation broth: The seed liquid obtained in step (2) was inoculated into the liquid culture medium in step (3) at an inoculum rate of 3%, and cultured at a temperature of 30 °C and a shaking speed of 180 r / min for 48 h. The OD 600 =2.0.
[0028] (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.
[0029] (6) Fermentation treatment: T1: Distilled water fermentation T2: Fermentation of extracellular crude enzyme in NA liquid medium T3: Fermentation of extracellular crude enzyme solution in T2 culture medium in step (3) 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 extracellular enzyme solution obtained in step (5) was evenly sprayed in the form of a spray according to 30% of the dry weight of the cigar leaves, and then sealed with a double-layer self-sealing bag. The cigar leaves were then fermented at 35°C and 80% RH for 3-5 days to obtain cigar leaves treated with the crude extracellular enzyme solution. After the fermentation, the leaves were placed in a constant temperature and humidity chamber at 26°C and 65% RH for 24-48 hours to balance the moisture content. The tensile strength of the fermented leaves was then tested, and sensory evaluation was performed.
[0030] Pectinase activity determination method: Reference to the study of the determination method of pectinase activity[J]. Journal of Northwest Agriculture, 2004(4): 134-137. Pectin content determination method: Use an enzyme-linked reagent kit and dilute acid to hydrolyze the original pectin, which is converted into galacturonic acid together with the original soluble pectin. The product is then condensed with strong acid and carbazole to form a purple-red compound with a characteristic absorption peak at 530 mm.
[0031] Tensile strength determination method: Cut the tobacco leaves into 2*5cm squares, use the Baosheng Technology texture analyzer to replace the tensile detection module, and test the tensile strength of the tobacco leaves. Each treatment is tested with 5 replicates.
[0032] Method for determination of total sugar content: The total sugar content is determined in accordance with the tobacco industry standard YC / T 159-2002 / 2019 "Continuous flow method for determination of water-soluble sugars in tobacco and tobacco products".
[0033] Depend on Figure 3 It can be seen that compared with the control group, the pectin content of tobacco leaves treated with the extracellular crude enzyme solution of Bacillus megaterium XW-02 was reduced, among which the degradation effect of T3 treatment was the best, decreasing by 19.5%, indicating that the pectinase produced by Bacillus megaterium XW-02 can effectively reduce the pectin content in cigar tobacco; in addition, Figure 4 It can be seen that the total sugar content has also decreased slightly; and after fermentation with the extracellular crude enzyme solution of Bacillus megaterium XW-02, with the proper degradation of pectin, the tensile strength value of the cigar tobacco leaves has increased to a certain extent compared with before fermentation, from 1.1413N to 1.454N, which greatly improves the usability in the cigar rolling process.
[0034] Example 4: Optimization of induction conditions for pectinase production by Bacillus megaterium XW-02 Glucose (1 g / L) was used as the carbon source, KCl (11 g / L) was used as the inorganic salt, and wheat bran powder (10 g / L) was added as the nitrogen source. The results of the pectinase activity test were as follows: Figure 6 shown As can be seen from the figure, the pectinase activity of Bacillus megaterium XW-02 increased by about 67.54% after the optimization of the culture medium compared with NA culture medium, and the highest pectinase activity after optimization could reach 15.86U / ml.
[0035] Example 5: Protein 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 the same amount of sterile water were recorded as CK, and the tobacco leaves sprayed with the extracellular crude enzyme solution produced by induction culture in different liquid fermentation media (T1, T2, T3) in Example 2 were recorded as T1, T2, and T3, respectively.
[0036] 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)%. Experts then conducted a sensory evaluation of the cigarettes based on eight aspects: aroma quality, off-flavors, aroma quantity, mellowness, pungency, aftertaste, strength, and strength. (Experts evaluated cigars according to current cigar evaluation standards: aroma quality refers to the quality of the aroma, aroma quantity refers to the amount of aroma in the smoke, off-flavors refer to unpleasant odors in the smoke, pungency refers to the irritation of the smoke in the mouth and throat, aftertaste refers to the quality of the residual taste in the mouth after exhalation, strength refers to the evaluator's satisfaction with the physiological intensity of the smoke, strength refers to the concentration of the smoke after exhalation, and mellowness refers to the overall feeling of the smoke upon inhalation.) The evaluation results are shown in Table 1.
[0037] Table 1 Sensory evaluation score table of cigars after fermentation
[0038] As shown in Table 1, the treatment of cigar tobacco leaves with crude extracellular enzymes of Bacillus megaterium XW-02 induced by optimized culture media (T1-T3) has achieved the following significant technical effects: 1. Significantly reduce pectin content: The pectin content of the treated cigar tobacco leaves is effectively reduced (reduced by 19.5% in the example), which improves the processing performance of the tobacco leaves and reduces the sticking phenomenon during the rolling process.
[0039] 2. Comprehensively improve the sensory quality of cigars: Taking the optimal T3 treatment plan as an example: the total sensory evaluation score was the highest (49.5 points, a significant increase from 42 points before treatment).
[0040] Key improvements: Pungency significantly reduced (score: 7.0 vs. 4.5 before treatment), off-flavors significantly reduced (score: 6.0 vs. 4.5 before treatment), a cleaner, more pleasant finish with improved mellowness (score: 7.0 vs. 6.0 before treatment), and a moderate increase in strength (score: 6.5 vs. 5.5 before treatment). Aroma quality, volume, and concentration remained stable. The T3 treatment maximizes the aroma quality and taste of the cigar, minimizing unpleasant sensations.
[0041] 3. Embody environmental protection and economic advantages: The optimized culture medium (especially that used in the T3 program) uses wheat bran as the nitrogen source, which is low-cost and realizes the resource utilization of agricultural waste; the entire treatment process is based on microbial fermentation, which is green and environmentally friendly, and no harmful chemicals are introduced.
[0042] 4. Verify the practicality and superiority of the technical solution: The T3 treatment solution has the best effect, which fully proves that the Bacillus megaterium XW-02 fermentation preparation and its application method of the present invention have significant advantages in effectively degrading pectin and improving the quality of cigars. At the same time, it has the characteristics of simple operation, low cost, short cycle, safety and environmental protection, and has high promotion and application value.
[0043] In summary, the above treatments, especially the T3 treatment, successfully achieved the technical effects of effectively degrading cigar tobacco leaf pectin (reducing it by 19.5%), significantly improving sensory quality (the total score increased to 49.5 points, and key adverse indicators were significantly improved), and achieving both environmental and economic benefits. These results fully verify the superiority and industrial application potential of the technical solution of the present invention.
[0044] Cigars treated with T3 showed reduced pungency, increased strength, a clean and comfortable aftertaste, and improved mellowness. The overall score was the highest among all treatments. This demonstrates that T3 treatment offers significant advantages in enhancing cigar quality, maximizing its sensory qualities and making it an effective means of improving cigar quality.
[0045] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0046] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications of the present invention fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.
Claims
1. Use of Bacillus megaterium XW-02 with a deposit number of CCTCC NO: M2025185 or its metabolite enzyme products in at least one of the following (1) to (6): (1) Degrading pectin in tobacco leaves or preparing a preparation for degrading pectin in tobacco leaves; (2) Degrading total sugar in tobacco leaves or preparing a preparation for degrading total sugar in tobacco leaves; (3) Improving the tensile strength of tobacco leaves or preparing preparations for improving the tensile strength of tobacco leaves; (4) Improving the sensory qualities of tobacco leaves or preparing preparations for improving the sensory qualities of tobacco leaves, wherein the sensory qualities include at least one of aroma quality, miscellaneous odors, aroma quantity, mellowness, pungency, aftertaste, strength, and concentration; (5) Improving the quality of cigarettes or preparing preparations that improve the quality of cigarettes; (6) Preparation of pectinase.
2. A method for preparing a microbial preparation, characterized in that: The steps include: (1) Activation of bacterial strains: Bacillus megaterium XW-02 with the deposit number CCTCC NO: M2025185 was inoculated onto NA solid medium by the spot inoculation method and cultured at 28-32°C for 24-48 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 28-32°C and 160-200 rpm for 10-15 h to obtain the seed solution; (3) Expanding the culture: inoculating the seed solution into a fermentation medium at an inoculum rate of 2-4%, and culturing the culture in a shaker at a temperature of 28-32° C. and a rotation speed of 160-200 r / min for 46-50 h; the fermentation medium contains 0.8-1.2 g / L glucose, 8-12 g / L wheat bran powder, and 10-12 g / L KCl; (4) Preparation of bacterial agent: centrifuge the fermentation liquid from the previous step and take the supernatant.
3. 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 28-32%; (2) Spray the microbial preparation described in claim 2 onto the tobacco leaves in an amount of 20-30% by mass of the tobacco leaves to be treated, seal the leaves, and ferment them at 33-37°C and 70-80% humidity for 3-5 days; (3) After fermentation is completed, it is dried and the moisture is balanced.