Composite biological agent based on bacillus megaterium-bacillus subtilis, preparation of composite biological agent and application of composite biological agent in improvement of tobacco quality

Abstract

The invention relates to a composite biological agent based on bacillus megaterium-bacillus subtilis, preparation of the composite biological agent and application of the composite biological agent in tobacco quality improvement, and aims to solve the technical problems that pectin and protein in cigars are difficult to degrade, and an effective microbiological method for improving the tobacco quality is lacked. According to the present invention, the biological agent compounded from the bacillus megaterium XW-02 strain with the preservation number of CCTCC NO: M 2025185, the bacillus subtilis MY-01 strain with the preservation number of CCTCC NO: M 20242596 and the metabolites thereof can reduce the pectin content and the protein content in the tobacco leaves, improve the irritation of the tobacco leaves during the burning and smoking process, and improve the overall quality and the smoking safety of cigars; the two kinds of microorganisms cooperate to achieve a synergistic effect, the prepared composite biological agent has the technical advantages that the technological process is simple, convenient and efficient, the tobacco leaf cost is low, the period is short, safety and reliability are achieved, the content of pectin and protein in the tobacco leaves can be obviously reduced, the comprehensive quality of the tobacco leaves can be improved, and good application and popularization value is achieved.

Description

Technical Field

[0001] This invention application relates to the field of tobacco processing technology, specifically to a compound biological agent based on Bacillus megaterium and Bacillus subtilis, its preparation, and its application in improving tobacco quality. Background Technology

[0002] As an important economic crop, tobacco, particularly cigars, has seen significant growth in the domestic market in recent years, propelling the domestic cigar industry into a phase of rapid development. However, improving the quality of raw materials for domestic cigars still faces key technological bottlenecks: the high content of naturally occurring pectin and protein in the raw materials directly affects the sensory quality and safety of the product.

[0003] Pectin is a key component of the cell wall structure of cigar tobacco leaves, mainly found between the cell wall and cell layers. Together with cellulose and hemicellulose, it forms the cytoskeleton, providing structural support and maintaining the shape and firmness of the tobacco leaves. Furthermore, as a hydrophilic colloid, pectin significantly affects the hygroscopicity and elasticity of tobacco leaves. During fermentation, pectin can be degraded by microorganisms or enzymes, releasing substances such as methanol, which can further transform into irritating components like formaldehyde and formic acid, directly impacting the taste and safety of the smoke.

[0004] Protein is a core substance in the physiological metabolism of tobacco leaves, participating in various biochemical reactions. Its degradation products (such as amino acids) are important precursors of aroma substances, and an appropriate amount of protein can enhance the aroma quality and quantity of tobacco smoke. However, when the protein content is too high, combustion will produce harmful substances such as feather odor, quinoline, and hydrogen cyanide, which not only affect the smoking experience but also threaten health. In addition, protein content is negatively correlated with tobacco leaf grade; excessive protein will lead to bitter smoke and reduce quality.

[0005] To improve tobacco quality and enhance the quality of cigarette raw materials, it is necessary to effectively degrade macromolecules such as pectin and protein in tobacco, maintaining their content within a reasonable range. However, pectin and protein have relatively stable structures and are difficult to degrade during the natural aging process of tobacco leaves. With the widespread application of cigar tobacco and the increasing awareness of its importance, researching and developing new degradation technologies has broad and practical significance for improving cigarette quality and increasing the economic benefits for enterprises.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] The inventors discovered through research that *Bacillus megaterium* with accession number CCTCC NO: M 2025185 ( Bacillus megateriumBacillus subtilis strain XW-02, with preservation number CCTCC NO: M 20242596 ( Bacillus subtilis The MY-01 strain can secrete pectinase and protease, and the two have a synergistic effect. Its compound biological agent can stably degrade proteins and pectin in cigar tobacco leaves, effectively improving the quality of tobacco leaves.

[0008] According to one aspect of this disclosure, a compound biological agent is provided, comprising: Bacillus megaterium XW-02 with accession number CCTCC NO: M2025185 and / or its metabolites, and Bacillus subtilis MY-01 with accession number CCTCC NO: M 20242596 and / or its metabolites.

[0009] According to another aspect of this disclosure, the compound biological agent is used in at least one of the following (1) to (8): (1) Degrading pectin in tobacco or preparing preparations that degrade pectin in tobacco; (2) Degrading proteins in tobacco or preparing preparations that degrade proteins in tobacco; (3) Degrading total sugar in tobacco or preparing preparations for degrading total sugar in tobacco; (4) Degrading reducing sugars in tobacco or preparing preparations that degrade reducing sugars in tobacco; (5) Degrading nicotine in tobacco or preparing preparations that degrade nicotine in tobacco; (6) To increase the tensile strength of tobacco or to prepare formulations that increase the tensile strength of tobacco; (7) Improving the sensory quality of tobacco or preparing preparations for improving the sensory quality of tobacco, wherein the sensory quality includes at least one of aroma quality, off-flavors, aroma quantity, smoothness, irritation, aftertaste, strength, and concentration; (8) To improve the quality of cigarettes or cigars or to prepare preparations that improve the quality of cigarettes or cigars.

[0010] According to another aspect of this disclosure, the method for preparing the above-mentioned compound biological agent includes the following steps: (1) Activation of bacterial strains: Bacillus megaterium XW-02 with preservation number CCTCC NO: M 2025185 and Bacillus subtilis MY-01 with preservation number CCTCC NO: M 20242596 were inoculated onto NA solid medium and cultured at 25-35℃ for 24-48h. (2) Preparation of seed culture: scrape off the bacterial cells cultured in step (1) and inoculate them into NA liquid culture medium. Culture at 25-35℃ and 160-200 r / min for 10-14 h with shaking to obtain seed culture. (3) Expanded culture: The Bacillus megaterium XW-02 seed culture obtained in step (2) was inoculated into pectinase selective induction liquid medium at 1-4% of the culture medium weight, and cultured at 25℃-35℃ and 160-200r / min for 46-50h. Simultaneously, the Bacillus subtilis MY-01 seed culture obtained in step (2) was inoculated into protease-selective induction liquid medium at 1-4% of the culture medium weight, and cultured at 25℃-35℃ and 160-200 r / min for 46-50 hours. (4) Preparation of compound biological agents: Take the supernatant of the culture medium after culturing in step (3) to obtain crude enzyme preparation; mix the obtained Bacillus megaterium XW-02 enzyme preparation and Bacillus subtilis MY-01 enzyme preparation in proportion to obtain the final product.

[0011] In some embodiments of this disclosure, in step (3), the liquid culture medium for Bacillus megaterium XW-02 to produce pectinase consists of: 1 g / L glucose, 10 g / L wheat bran powder, and 11 g / L KCl.

[0012] In some embodiments of this disclosure, in step (3), the liquid culture medium for Bacillus subtilis MY-01 protease production consists of: sodium carboxymethyl cellulose 20 g / L, beef extract 3 g / L, peptone 10 g / L, and NaCl 5 g / L.

[0013] According to another aspect of this disclosure, a method for processing cigar tobacco leaves is provided, comprising the following steps: (1) Spray the above compound biological agent onto the tobacco leaves to be treated at 20-30% of the weight of the tobacco leaves to be treated and adjust the moisture content of the tobacco leaves to 30-40% and then seal it and ferment it at 33-37℃ and 70-80% humidity for 3-5 days. (2) After fermentation, the product is dried and the moisture content is balanced.

[0014] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Bacillus megaterium XW-02 and Bacillus subtilis MY-01 produce specific enzymes during fermentation, which effectively break down pectin in cigar tobacco leaves. As a major component of cell wall structure, reducing pectin content helps improve the physical properties and combustion performance of tobacco leaves. Through microbial fermentation, pectin is degraded into smaller molecules, such as galacturonic acid, which positively impacts the taste and safety of tobacco leaves.

[0015] 2. The microorganisms in the compound inoculant can secrete proteases, which degrade the proteins in cigar tobacco leaves into smaller molecules such as amino acids. Proteins produce unpleasant odors and harmful substances during combustion; reducing their content helps improve the combustion quality and safety of the tobacco leaves. Protein degradation also helps release more aroma precursors, providing favorable conditions for the formation of tobacco aroma.

[0016] 3. The compound biological agents are far more effective than single-agent agents or conventional fermentation in reducing total sugar, reducing sugar, and nicotine in tobacco leaves. This indicates that the metabolic enzymes of Bacillus megaterium XW-02 and Bacillus subtilis MY-01 can be combined and enhanced. The biological agents prepared by combining them for tobacco processing are simple, efficient, low-cost, have a short processing cycle, and are safe and reliable. Therefore, they have good potential for widespread application in the cigarette manufacturing industry. Attached Figure Description

[0017] Figure 1 This is a graph showing the enzyme activity assay results of neutral proteases XW-02 and MY-01 in one embodiment of this application.

[0018] Figure 2 This is a graph showing the pectinase activity assay results of XW-02 and MY-01 in one embodiment of this application.

[0019] Figure 3 This is a graph showing the results of neutral protease activity assay of a compound bacterial agent in one embodiment of this application.

[0020] Figure 4 This is a graph showing the results of pectinase activity assay of a compound microbial agent in one embodiment of this application.

[0021] Figure 5 This is a graph showing the protein content determination results of tobacco leaves after enzyme treatment in one embodiment of this application.

[0022] Figure 6 This is a graph showing the results of pectin content determination in tobacco leaves after enzyme treatment in one embodiment of this application.

[0023] Figure 7 This is a graph showing the results of total sugar content determination of tobacco leaves after enzyme treatment in one embodiment of this application.

[0024] Figure 8 This is a graph showing the results of reducing sugar content determination of tobacco leaves after enzyme treatment in one embodiment of this application.

[0025] Figure 9 This is a graph showing the nicotine content determination results of tobacco leaves after enzyme treatment in one embodiment of this application.

[0026] Figure 10 This is a graph showing the tensile strength test results of tobacco leaves after enzyme treatment in one embodiment of this application. Detailed Implementation

[0027] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, 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.

[0029] The following examples involve Bacillus megaterium (Beta) Bacillus megaterium The XW-02 strain was deposited on January 20, 2025, at the China Center for Type Culture Collection (address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), with accession number CCTCC NO: M 2025185. The screening and identification process is detailed in patent application CN202510787799.1. The involved Bacillus subtilis strain (…) Bacillus subtilis The MY-01 strain was deposited at the China Center for Type Culture Collection on November 20, 2024, with accession number CCTCC NO: M20242596. The screening and identification process can be found in patent application CN202510853864.6.

[0030] Example 1: Preparation of Compound Biological Agents To use Bacillus megaterium XW-02 to degrade pectin in tobacco leaves and Bacillus subtilis MY-01 to degrade proteins in tobacco leaves, both strains must first be prepared into easy-to-use biological agents. The specific preparation process is as follows: (1) Activation of strains: The preserved Bacillus megaterium strain XW-02 and Bacillus subtilis strain MY-01 were inoculated onto NA solid medium by spot inoculation and cultured at 30℃ for 48h to obtain activated strains; (2) Preparation of seed culture: scrape the bacterial cells cultured in step (1), inoculate them into NA liquid culture medium, and culture them in a shaker at 30℃ and 180 r / min for 12 h to obtain the seed culture. (3) Expanded culture: The Bacillus megaterium XW-02 seed culture from step (2) was inoculated at 3% in a selective induction liquid medium for pectinase production (medium composition: 1 g / L glucose, 10 g / L wheat bran powder, 11 g / L KCl) and cultured at 30℃ and 180 r / min for 48 h. Simultaneously, the Bacillus subtilis MY-01 seed culture from step (2) was inoculated into a protease-selective induction liquid medium at 3% of the culture medium weight (culture medium composition: sodium carboxymethyl cellulose 20 g / L, beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L), and cultured at 30℃ and 180 r / min for 48 h. (4) Preparation of compound biological agent: After centrifuging the fermentation broth obtained in step (3) at 4℃ and 8000r / min for 10min, collect the supernatant to obtain the corresponding crude enzyme preparation. Take two crude enzymes, solvent and auxiliary agents in proportion, mix them evenly to obtain the compound biological agent.

[0031] The neutral protease activities of the two strains were detected using an enzyme-linked immunosorbent assay (ELISA) kit and an ELISA reader. The results are as follows: Figure 1 As shown, both strains exhibit high neutral protease activity, with XW-02 showing the highest activity at 13.73 U / ml and MY-01 showing the highest activity at 19.15 U / ml. This indicates that both strains can be used in cigar fermentation to degrade the protein content in tobacco leaves, thereby improving the aroma quality of cigar tobacco.

[0032] The pectinase activity of the two strains was detected using an enzyme-linked immunosorbent assay (ELISA) kit and an ELISA reader. The results are as follows: Figure 2 As shown, both strains exhibit high pectinase activity, with XW-02 showing the highest activity at 15.86 U / ml and MY-01 showing the highest activity at 14.94 U / ml. This indicates that both strains can be used in cigar fermentation to degrade the pectin content in cigar tobacco leaves, thereby improving the overall quality of the cigar tobacco leaves.

[0033] Example 2: Optimization and Verification Experiment of Composition Ratio of Compound Enzyme Preparation 1. Preparation of fermentation agents The following compound enzyme preparations (by volume percentage) were prepared using the crude enzyme preparation obtained in Example 1 and distilled water: CK: Distilled water; T1: 5% MY-01 crude enzyme preparation, 15% XW-02 crude enzyme preparation, 80% distilled water; T2: 10% MY-01 crude enzyme preparation, 10% XW-02 crude enzyme preparation, 80% distilled water; T3: 15% MY-01 crude enzyme preparation, 5% XW-02 crude enzyme preparation, 80% distilled water; T4: 20% XW-02 crude enzyme preparation, 80% distilled water; T5: 20% MY-01 crude enzyme preparation, 80% distilled water.

[0034] The neutral protease and pectinase activities of each compound enzyme preparation were measured separately, and the results are as follows: Figure 3 , Figure 4 As shown. By Figure 3 It can be seen that the T3 complex enzyme preparation has the highest neutral protease activity (23.84 U / ml), which is approximately 73.63% and 27.1% higher than the preparations using XW-02 or MY-01 alone, respectively. Figure 4 It can be seen that the T3 compound enzyme preparation has the highest pectinase activity of approximately 20.79 U / ml, which is 23.71% and 39.15% higher than that of XW-02 or MY-01 preparations used alone.

[0035] 2. Test Treatment Take cigar tobacco leaves of the same batch with consistent quality, and spray them evenly with distilled water and the above-mentioned compound enzyme preparations at 35% of the dry weight of the cigar tobacco leaves. Seal them in double-layer sealed bags and ferment them at 35℃ and 70%RH for 4 days to obtain fermented cigar tobacco leaves.

[0036] The tensile strength of fermented cigar tobacco leaves was measured after balancing the moisture content for 24 hours at 26℃ and 80%RH. The protein, pectin, total sugar, reducing sugar, and nicotine contents of cigar tobacco leaves fermented under CK, T1, T2, T3, T4, and T5 treatments were also determined.

[0037] 3. Experimental Results Protein content determination: The protein content of cigar tobacco leaves under different treatments before and after fermentation was determined using a flow analyzer. The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that the T3 (15% MY-01, 5% XW-02, 80% distilled water) fermentation treatment showed the highest protein degradation rate, at 27.8±0.3%. The protein degradation rates of the other treatments (CK, T1, T2, T4, and T5) were 18%, 22.3±0.3%, 24.5±0.3%, 19.6±0.3%, and 21.7±0.3%, respectively. This indicates that the compound preparation has a more significant effect on protein degradation in cigar fermentation compared to single-agent fermentation and conventional fermentation.

[0038] Pectin content determination: The pectin content of cigar tobacco leaves treated with different methods before and after fermentation was detected using an enzyme-linked immunosorbent assay (ELISA) kit and an ELISA reader. The results are as follows: Figure 6 As shown. From Figure 6It can be seen that the pectin content of fermented tobacco leaves (T1, T2, T3) treated with compound microbial agent fermentation broth decreased significantly, with the highest pectin degradation rate reaching 24.6%±0.3%. The pectin degradation rates from CK to T5 were 18.6±0.3%, 24.6±0.3%, 24.3±0.3%, 23.8±0.3%, 21.3±0.3%, and 19.3±0.3%, respectively. The surface compound preparation has a significant synergistic effect on pectin degradation in cigar fermentation compared to single microbial agents and conventional fermentation, and has a substantial synergistic effect on improving the aroma and physical quality of tobacco leaves.

[0039] Total sugar content determination: The total sugar content was determined according to the tobacco industry standard YC / T 159-2019 "Determination of water-soluble sugars in tobacco and tobacco products - Continuous flow method". The results are as follows: Figure 7 As shown.

[0040] Determination of reducing sugar content: The reducing sugar content was determined according to the tobacco industry standard YC / T 159-2019 "Determination of water-soluble sugars in tobacco and tobacco products - Continuous flow method". The results are as follows. Figure 8 As shown.

[0041] Nicotine content determination: Nicotine content was determined according to tobacco industry standard YC / T 246-2008 (GC), and the results are as follows. Figure 9 As shown.

[0042] It can be seen that the total sugar, reducing sugar, and nicotine content of cigar tobacco leaves decreased after different fermentation treatments. The decrease in total sugar and reducing sugar content varied greatly. The decrease in total sugar and reducing sugar content was more obvious in tobacco leaves fermented with compound microbial agents (T1, T2, T3). This is because after pectin and cellulose degrade into sugars, they generate aroma substances or compounds that affect color changes through caramelization, browning, and Maillard reactions.

[0043] Tensile strength testing: After fermentation, the tobacco leaves were placed in a constant temperature and humidity chamber at 90% RH and 26℃ for 48 hours to equilibrate the moisture. The leaves were then sliced ​​and tested. The tensile strength of the tobacco leaves under different fermentation treatments was measured using a texture analyzer from Baosheng Technology. The results are as follows: Figure 10 As shown in the figure, the results indicate that the tensile strength of cigar tobacco leaves fermented with the compound enzyme preparation is higher than that of tobacco leaves treated with the control (CK) and those fermented with enzyme solutions alone. Tensile strength is a crucial indicator in the evaluation of the usability of cigar tobacco leaves in the cigar industry, and an increase in tensile strength significantly impacts the usability during cigar rolling.

[0044] Example 3: Protein Degradation Effect and Sensory Evaluation of Cigar Tobacco Leaf Fermentation Cigar tobacco leaves were fermented according to the method in Example 2. The tobacco leaves sprayed with an equal amount of sterile water were denoted as CK. The tobacco leaves that were fermented with different compound enzyme preparations (T1, T2, T3, T4, T5) in Example 2 were denoted as CK, T1, T2, T3, T4 and T5 in sequence.

[0045] Sensory evaluation method: Cigars treated with compound enzyme preparations were rolled into tobacco and equilibrated at 80% RH and 26℃ for 48 hours. Then, sensory evaluation experts were organized to assess the quality of the tobacco in eight aspects: aroma, aroma quantity, off-flavors, irritation, aftertaste, strength, concentration, and smoothness. The evaluation standards or specifications referred to GB 15269.4-2011 "Cigars" and YC / T 138-1998 "Sensory Evaluation Methods for Tobacco and Tobacco Products". The evaluation results are shown in Table 1.

[0046] Table 1 Sensory evaluation results of cigar tobacco leaves after fermentation .

[0047] As shown in Table 1, the T3 treatment resulted in cigars with reduced harshness, enhanced aroma, and a cleaner, more pleasant aftertaste, achieving the highest overall score among all treatment options. This indicates that the T3 treatment process has significant advantages in improving cigar quality, maximizing sensory optimization and serving as an effective means of enhancing cigar quality.

[0048] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0049] Obviously, those skilled in the art can make various modifications and variations to this disclosure without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations to this disclosure fall within the scope of the claims of this application and their equivalents, this invention also intends to include such modifications and variations.

Claims

1. A compound biological agent, characterized in that, Contains: Bacillus megaterium XW-02 with accession number CCTCC NO: M 2025185 and / or its metabolites, and Bacillus subtilis MY-01 with accession number CCTCC NO: M 20242596 and / or its metabolites.

2. The use of the compound biological agent according to claim 1 in at least one of the following (1) to (8): (1) Degrading pectin in tobacco or preparing preparations that degrade pectin in tobacco; (2) Degrading proteins in tobacco or preparing preparations that degrade proteins in tobacco; (3) Degrading total sugar in tobacco or preparing preparations for degrading total sugar in tobacco; (4) Degrading reducing sugars in tobacco or preparing preparations that degrade reducing sugars in tobacco; (5) Degrading nicotine in tobacco or preparing preparations that degrade nicotine in tobacco; (6) To increase the tensile strength of tobacco or to prepare formulations that increase the tensile strength of tobacco; (7) Improving the sensory quality of tobacco or preparing preparations for improving the sensory quality of tobacco, wherein the sensory quality includes at least one of aroma quality, off-flavors, aroma quantity, smoothness, irritation, aftertaste, strength, and concentration; (8) Improve the quality of cigarettes or cigars or prepare preparations that improve the quality of cigarettes or cigars.

3. The method for preparing the compound biological agent according to claim 1, characterized in that, Includes the following steps: (1) Activation of bacterial strains: Bacillus megaterium XW-02 with preservation number CCTCC NO: M 2025185 and Bacillus subtilis MY-01 with preservation number CCTCC NO: M 20242596 were inoculated onto NA solid medium and cultured at 25-35℃ for 24-48h. (2) Preparation of seed culture: scrape off the bacterial cells cultured in step (1) and inoculate them into NA liquid culture medium. Culture at 25-35℃ and 160-200 r / min for 10-14 h with shaking to obtain seed culture. (3) Expanded culture: The Bacillus megaterium XW-02 seed culture obtained in step (2) was inoculated into pectinase-producing liquid culture medium at 1-4% of the culture medium weight, and cultured at 25℃-35℃ and 160-200 r / min for 46-50 hours. Simultaneously, the Bacillus subtilis MY-01 seed culture obtained in step (2) was inoculated into the protease-producing liquid culture medium at 1-4% of the culture medium weight, and cultured at 25℃-35℃ and 160-200r / min for 46-50h. (4) Preparation of compound biological agents: Take the supernatant of the culture medium after culturing in step (3) to obtain crude enzyme preparation; mix the obtained Bacillus megaterium XW-02 enzyme preparation and Bacillus subtilis MY-01 enzyme preparation in proportion to obtain the final product.

4. The preparation method according to claim 3, characterized in that, In step (3), the liquid culture medium for producing pectinase from Bacillus megaterium XW-02 consists of: 1 g / L glucose, 10 g / L wheat bran powder, and 11 g / L KCl.

5. The preparation method according to claim 3, characterized in that, In step (3), the liquid culture medium for Bacillus subtilis MY-01 protease production consists of: sodium carboxymethyl cellulose 20 g / L, beef extract 3 g / L, peptone 10 g / L, and NaCl 5 g / L.

6. A method for processing cigar tobacco leaves, characterized in that, Includes the following steps: (1) Spray the compound biological agent described in claim 3 onto the tobacco leaves to be treated at 20-30% of the mass of the tobacco leaves to be treated, and adjust the moisture content of the tobacco leaves to 30-40% and then seal it and ferment it at 33-37°C and 70-80% humidity for 3-5 days. (2) After fermentation, the product is dried and the moisture content is balanced.

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