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

Abstract

The invention discloses a composite biological agent based on enterobacter hormaechei-bacillus megaterium, 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 cellulose and starch in cigars are difficult to degrade, and an effective microbiological method for improving the tobacco quality is lacked. According to the present invention, with the composite biological agent of the enterobacter hormaechei XW-01 strain with the preservation number of CCTCC NO: M20242595, the bacillus megaterium XW-02 strain with the preservation number of CCTCC NO: M2025185, and the metabolites thereof, the cellulose content and the starch content in the tobacco leaf can be reduced, the irritation of the tobacco leaf during the burning and smoking process can be improved, and the overall quality and the smoking safety of the cigar can be improved; the two types of microorganisms cooperate to achieve a synergistic effect, and the composite biological agent prepared from the microorganisms has the technical advantages of being simple, convenient and efficient in technological process, low in tobacco leaf cost, short in period, safe, reliable, capable of obviously reducing the content of cellulose and starch in tobacco leaves and improving the comprehensive quality of the tobacco leaves and the like, and has good application and popularization value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation, in particular to a compound biological preparation based on Enterobacter hormaechei-Bacillus megaterium, preparation thereof and application thereof in improving tobacco quality. BACKGROUND

[0002] Cigar, as an important branch of Nicotiana genus, Nicotiana has attracted much attention in recent years. With many tobacco companies laying out cigar raw material production, the optimization of its fermentation process has become the core direction of technical research. However, the current cigar fermentation field still faces key bottlenecks: lack of mature fermentation process system, application research of microbial fermentation technology is particularly weak, and systematic research of compound microbial agent in cigar fermentation is almost blank.

[0003] Cigar fermentation is the core link to improve tobacco quality. Through microbial metabolic activity, macromolecular substances in tobacco are transformed, irritant components are reduced, and aroma substances are enriched, ultimately achieving significant improvement in sensory quality. However, the chemical properties of cigar raw materials pose unique challenges - their cellulose and starch content is significantly higher than that of ordinary cigarette raw materials. The pyrolysis products of these components during combustion can severely damage the quality: Cellulose: pyrolysis generates substances such as guaiacol under high-temperature anaerobic conditions, producing woody aroma and grassy flavor, which mask the cigar's natural aroma; at the same time, carbon monoxide is generated, which further reacts to produce harmful substances such as formaldehyde, polycyclic aromatic hydrocarbons (IARC Class 1 carcinogens), etc.

[0004] Starch: anaerobic pyrolysis generates carcinogenic or irritating substances such as polycyclic aromatic hydrocarbons, benzene, aldehydes, ketones, phenols, etc., directly causing bitterness during smoking.

[0005] It is worth noting that although microbial fermentation technology has been widely applied in food, feed, and other fields, and some companies in the tobacco industry have attempted to accelerate cigar aging or regulate aroma components through single-strain fermentation, research on compound microbial agent fermentation for cigar raw materials is still in its infancy. Existing technologies focus on the functional verification of single strains, lack depth analysis of the synergistic action mechanism of microbial flora and substrate metabolic pathways, and it is difficult to achieve efficient directional degradation of cellulose and starch. Therefore, developing compound microbial agents with synergistic degradation ability and constructing a fermentation process system suitable for the characteristics of cigar raw materials has become a key path to improve the quality of domestic cigars and break through technical barriers.

[0006] The information disclosed in this BACKGROUND section is only for the purpose of enhancing the understanding of the background of the present disclosure and should not be taken as admitting that such information is prior art known to those of ordinary skill in the art. SUMMARY

[0007] The inventors have found that Enterobacter hormaechei (CCTCC NO: M 20242595) and Bacillus megaterium (CCTCC NO: M 2025185) can secrete cellulase and amylase, and the two have a synergistic effect, and the compound biological agent can stably degrade cellulose and starch in cigar tobacco leaves, effectively improving the quality of tobacco leaves. Enterobacter hormaechei Bacillus megaterium The inventors have found that Enterobacter hormaechei (CCTCC NO: M 20242595) and Bacillus megaterium (CCTCC NO: M 2025185) can secrete cellulase and amylase, and the two have a synergistic effect, and the compound biological agent can stably degrade cellulose and starch in cigar tobacco leaves, effectively improving the quality of tobacco leaves.

[0008] According to one aspect of the present disclosure, a compound biological agent is provided, comprising: Enterobacter hormaechei (CCTCC NO: M 20242595) and / or its metabolites, Bacillus megaterium (CCTCC NO: M 2025185) and / or its metabolites.

[0009] According to another aspect of the present disclosure, the compound biological agent is used in at least one of the following (1) to (7): (1) degrading cellulose in tobacco or preparing a preparation for degrading cellulose in tobacco; (2) degrading starch in tobacco or preparing a preparation for degrading starch in tobacco; (3) degrading total sugar in tobacco or preparing a preparation for degrading total sugar in tobacco; (4) degrading reducing sugar in tobacco or preparing a preparation for degrading reducing sugar in tobacco; (5) increasing the tensile value of tobacco or preparing a preparation for increasing the tensile value of tobacco; (6) improving the sensory quality of tobacco or preparing a preparation for improving the sensory quality of tobacco, the sensory quality including at least one of aroma quality, aroma amount, odor, irritation, aftertaste, strength, concentration, and softness; (7) improving the quality of cigarettes or cigar tobacco or preparing a preparation for improving the quality of cigarettes or cigar tobacco.

[0010] According to still another aspect of the present disclosure, a preparation method of the compound biological agent is provided, comprising the following steps: (1) activating the strains: inoculating Enterobacter hormaechei (CCTCC NO: M 20242595) and Bacillus megaterium (CCTCC NO: M 2025185) on NA solid medium, and culturing at 25-35°C for 24-48h; (2) preparing a seed liquid: scraping the bacterial bodies cultured in step (1), inoculating in NA liquid medium, and culturing at 25-35°C with 160-200 r / min shaking for 10-14h to obtain a seed liquid; ​(3) Enlargement culture: inoculate the seed liquid of Enterobacter hornaechei XW-01 obtained in step (2) into the corresponding cellulase production liquid medium at 1-4% of the weight of the medium, and culture at 25-35°C and 160-200 r / min for 46-50 h; Synchronously inoculate the seed liquid of Bacillus megaterium XW-02 obtained in step (2) into the corresponding amylase production liquid medium at 1-4% of the weight of the medium, and culture at 25-35°C and 160-200 r / min for 46-50 h; (4) Preparation of a composite biological agent: take the supernatant of the culture medium after culture in step (3) to obtain a crude enzyme preparation; mix the enzyme preparation of Enterobacter hornaechei XW-01 with the enzyme preparation of Bacillus megaterium XW-02 to obtain a composite biological agent.

[0011] In some embodiments of the present disclosure, in the step (3), the cellulase production liquid medium for Enterobacter hornaechei XW-01 is composed of fructose 9 g / L, tryptone 15 g / L, and MnSO4 7 g / L.

[0012] In some embodiments of the present disclosure, in the step (3), the amylase production liquid medium for Bacillus megaterium XW-02 is composed of glucose 1 g / L, wheat bran powder 10 g / L, and KCL 11 g / L.

[0013] According to another aspect of the present disclosure, a method for processing cigar leaves is provided, comprising the following steps: (1) Spray the composite biological agent to the leaves at 20-30% of the mass of the leaves to be processed, and adjust the water content of the leaves to 30-40% and then seal, and ferment at 33-37°C and 70-80% humidity for 3-5 days; (2) After fermentation, dry and balance the water content, and the product is obtained.

[0014] The one or more technical solutions provided in the embodiments of the present disclosure have at least the following technical effects or advantages: 1. Enterobacter hornaechei XW-01 and Bacillus megaterium XW-02 can produce specific enzymes during fermentation, which can effectively decompose cellulose in cigar leaves. The reduction of cellulose content can reduce the generation of guaiacol, propylene aldehyde and other pungent and irritating substances by more than 30% when cigar is burned, and can avoid decarboxylation and dehydration reactions caused by high burning cone temperature, reduce the content of IARC confirmed carcinogens such as formaldehyde and catechol, and has a positive effect on improving the taste and safety of tobacco leaves.

[0015] 2. The microorganisms in the complex microbial agent can secrete starch-degrading enzymes. High starch content can significantly reduce the quality of cigar tobacco, specifically in the form of abnormal combustion, reduced smoke quality, increased harmful substances, and deteriorated smoking experience. The degradation of starch helps to increase combustion durability, reduce irritation, enhance smoking comfort, and avoid the neutralization of acidic substances in the smoke by starch cleavage products (such as ammonia), resulting in excessive alkalinity of the smoke, causing "pressure" phenomenon, masking the original cigar aroma, and providing favorable conditions for the formation of tobacco aroma.

[0016] 3. The complex biological agent is far superior to single microbial agent or conventional fermentation in reducing total sugar and reducing sugar in tobacco and improving draw value. Studies have shown that the metabolic enzymes of Enterobacter hormaechei XW-01 and Bacillus megaterium XW-02 can be compounded and synergized, and the biological agent prepared by compounding them has a simple and efficient process, low cost, short processing period, and is safe and reliable, thus having good popularization and application value in the field of cigarette manufacturing. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The figure is the determination result of cellulase enzyme activity of XW-01 and XW-02 in an embodiment of the present application.

[0018] Figure 2 The figure is the determination result of amylase enzyme activity of XW-01 and XW-02 in an embodiment of the present application.

[0019] Figure 3 The figure is the determination result of cellulase enzyme activity of the complex microbial agent in an embodiment of the present application.

[0020] Figure 4 The figure is the determination result of amylase enzyme activity of the complex microbial agent in an embodiment of the present application.

[0021] Figure 5 The figure is the determination result of cellulose content of tobacco leaves treated with enzyme preparation in an embodiment of the present application.

[0022] Figure 6 The figure is the determination result of starch content of tobacco leaves treated with enzyme preparation in an embodiment of the present application.

[0023] Figure 7 The figure is the determination result of total sugar content of tobacco leaves treated with enzyme preparation in an embodiment of the present application.

[0024] Figure 8 The figure is the determination result of reducing sugar content of tobacco leaves treated with enzyme preparation in an embodiment of the present application.

[0025] Figure 9 The figure is the determination result of draw value of tobacco leaves treated with enzyme preparation in an embodiment of the present application. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] The following examples involve Enterobacter cholerae (C. cholerae). Enterobacter hormaechei The XW-01 strain was deposited on November 20, 2024, 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 20242595. The screening and identification process is detailed in patent application CN202510853944.1. The involved *Bacillus megaterium* (…) Bacillus megaterium The XW-02 strain was deposited at the China Center for Type Culture Collection on January 20, 2025, with accession number CCTCC NO: M 2025185. The screening and identification process can be found in patent application CN202510787799.1.

[0029] Example 1: Preparation of Compound Biological Agents To use *Enterobacter holmieae* XW-01 and *Bacillus megaterium* XW-02 strains to degrade cellulose and starch in tobacco leaves, the two strains must first be prepared into easy-to-use biological agents. The specific preparation process is as follows: (1) Activation of strains: The preserved Enterobacter holmie XW-01 strain and Bacillus megaterium XW-02 strain 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 12h to obtain the seed culture. (3) Expanded culture: The Enterobacter holmium XW-01 seed culture from step (2) was inoculated at 3% into cellulase selective induction liquid medium (medium composition: fructose 9g / L, tryptone 15g / L, MnSO4 7g / L) and cultured at 30℃ and 180r / min for 48h. Synchronously inoculate the Bacillus megaterium XW-02 seed liquid in step (2) into the amylase-producing selective induction liquid medium (medium composition: glucose 1 g / L, wheat bran powder 10 g / L, KCl 11 g / L) at 3% of the medium weight, and culture at 30°C and 180 r / min for 48 h.

[0030] (4) Preparation of the composite biological agent: centrifuge the fermentation liquid obtained in step (3) at 4°C and 8000 r / min for 10 min, and then collect the supernatant to obtain two corresponding crude enzyme agents. Take the two crude enzymes, solvent and additives, etc. in proportion, mix uniformly, and obtain the composite biological agent.

[0031] The cellulase enzyme activity of the two crude enzyme agents was detected by using an enzyme-linked kit and an enzyme marker, and the results are shown in Table 1. Figure 1 As can be seen, both strains have high cellulase enzyme activity, in which the cellulase enzyme activity of XW-01 is as high as 16.11 U / ml, and the cellulase enzyme activity of XW-02 is as high as 14.17 U / ml. It is shown that both strains can be applied to the fermentation of cigar to degrade the cellulose content in tobacco leaves, so as to improve the aroma quality of cigar tobacco leaves.

[0032] The amylase enzyme activity of the two crude enzyme agents was detected by using an enzyme-linked kit and an enzyme marker, and the results are shown in Table 2. Figure 2 As can be seen, both strains have high amylase enzyme activity, in which the amylase enzyme activity of XW-01 is as high as 12.43 U / ml, and the amylase enzyme activity of XW-02 is as high as 16.932 U / ml. It is shown that both strains can be applied to the fermentation of cigar to degrade the starch content in cigar tobacco leaves, so as to improve the overall quality of cigar tobacco leaves.

[0033] Example Two, verification test of the composition ratio optimization of the composite enzyme agent 1. Preparation of the fermentation agent The two crude enzyme agents obtained in Example One and distilled water were used to prepare the following fermentation agents (in volume percentage): CK: distilled water; T1: 5% XW-01 crude enzyme agent, 15% XW-02 crude enzyme agent, 80% distilled water; T2: 10% XW-01 crude enzyme agent, 10% XW-02 crude enzyme agent, 80% distilled water; T3: 15% XW-01 crude enzyme agent, 5% XW-02 crude enzyme agent, 80% distilled water; T4: 20% XW-01 crude enzyme agent, 80% distilled water; T5: 20% XW-02 crude enzyme agent, 80% distilled water.

[0034] The cellulose-degrading enzyme activity and starch-degrading enzyme activity in the fermentation liquor of the five compound microbial agents were detected according to the tobacco industry standard by using an enzyme-labeled instrument method, and were marked as T1, T2, T3, T4 and T5, respectively. The results are shown in Figure 3 、 Figure 4 It can be seen from Figure 3 that the cellulose-degrading enzyme activity of the T3 compound enzyme preparation is the highest (22.73 U / ml), which is increased by about 39.9% and 62.47% compared with the crude enzyme preparations of XW-01 or XW-02 alone, respectively. Figure 4 It can be seen from that the starch-degrading enzyme activity of the T1 and T3 compound microbial agents is relatively high, which is 22.85 U / ml and 22.36 U / ml, respectively. Compared with the starch-degrading enzyme activity of the XW-01 and XW-02 microbial agents, it is increased by 40.7% and 58.46%, respectively.

[0035] 2. Test treatment The cigar leaves to be treated were uniformly sprayed (in the form of spray) with distilled water and the above compound enzyme preparations at 35% of the dry weight of the cigar leaves, sealed with double-layer sealed bags, and fermented at 35°C and 70% RH for 4 days to obtain the fermented cigar leaves.

[0036] The tensile force of the fermented cigar leaves was measured after the fermented cigar leaves were balanced at 26°C and 80% RH for 24 h. The cellulose content, starch content, total sugar content and reducing sugar content in the cigar leaves treated with CK, T1, T2, T3, T4 and T5 were measured, respectively.

[0037] 3. Test results Cellulose content: The cellulose content of the cigar leaves under each treatment was measured (cellulose content kit of Enzyme-linked Biological Technology Co., Ltd.), and the results are shown in Figure 5 From the graph, it can be seen that the cellulose content under the T3 (15% XW-01 crude enzyme preparation, 5% XW-02 crude enzyme preparation and 80% distilled water) treatment is the lowest, which is degraded from 124.92 mg / g before fermentation to 71.236 mg / g, with a degradation rate of 42.97%. It is shown that the compound preparation has a more significant effect on degrading the cellulose content in cigar fermentation than the single microbial agent and the conventional fermentation.

[0038] Starch content: The starch content of the cigar leaves under each treatment was measured (starch content kit of Enzyme-linked Biological Technology Co., Ltd.), and the results are shown in Figure 6As can be seen from the figure, the starch content of the tobacco leaves treated by the composite microbial agent (T1 and T3) fermentation is more obviously decreased, from 19.25 mg / g before fermentation to 11.18 mg / g and 11.05 mg / g respectively, and the degradation rates are 41.55% and 42.59% respectively. It is shown that the starch content of the tobacco leaves treated by the composite microbial agent fermentation is more obviously decreased than that of the tobacco leaves treated by the single strain and the conventional fermentation, which has a wide and far-reaching influence on improving the physical properties and aroma quality of the 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 Sugar in Tobacco and Tobacco Products by Continuous Flow Method”, and the results are shown in Table 2. Figure 7

[0040] Reducing sugar content determination: The reducing sugar content was determined according to the Tobacco Industry Standard YC / T 159-2019 “Determination of Water-soluble Sugar in Tobacco and Tobacco Products by Continuous Flow Method”, and the results are shown in Table 3. Figure 8

[0041] It can be seen that the total sugar and reducing sugar contents of the cigar tobacco leaves treated by different fermentation treatments are decreased, and the total sugar and reducing sugar of the tobacco leaves treated by the composite microbial agent fermentation (T1, T2, T3) are more obviously decreased, which is due to the fact that the cellulose and starch are degraded into sugar and then a series of chemical reactions generate aroma substances or compounds affecting color change.

[0042] Tension value detection: The tension values of the cigar tobacco leaves treated by different fermentation treatments were determined after the tobacco leaves were balanced at 26°C and 90% RH for 48 h (determined by a Baosheng technology texture analyzer), and the results are shown in Table 4. Figure 9 As can be seen from the results, the composite microbial agent of T3 scheme has the best effect on improving the tension, which improves the overall quality of the cigar to a certain extent. Compared with before fermentation, the composite microbial agent fermentation (T1, T2, T3) and the single strain microbial agent fermentation (T4, T5), the tension value of the tobacco leaves treated by the composite microbial agent fermentation is higher, which is about 28.87% higher. The tension value is one of the important indicators for evaluating the industrial usability of the cigar tobacco leaves, and the increase of the tension value has a significant influence on the usability of the cigar during rolling.

[0043] Example Three, Fermentation Cellulose Degradation Effect and Sensory Evaluation of Cigar Tobacco Leaves The cigar tobacco leaves were treated by fermentation according to the method in Example Two, wherein the tobacco leaves sprayed with equal amount of sterile water were marked as CK, and the cigar tobacco leaves treated by different fermentation preparations (CK, T1, T2, T3, T4, T5) in Example Two were marked as CK, T1, T2, T3, T4 and T5 in turn.

[0044] ​​Sensory evaluation method: the unfermented and fermented cigar tobacco leaves treated by each fermentation preparation were rolled into cigarettes, and then the moisture content was balanced at 80% RH and 26℃ for 48h. The sensory quality of the cigarettes was evaluated by experts in terms of aroma quality, aroma amount, offensive odor, irritation, aftertaste, strength, density and softness. The evaluation standard or specification referred to GB 15269.4-2011 "Cigar Tobacco" and YC / T 138-1998 "Sensory Evaluation Method of Tobacco and Tobacco Products". The evaluation results are shown in Table 1.

[0045] Table 1 Sensory evaluation results of fermented cigar tobacco leaves .

[0046] As shown in Table 1, the irritation of the cigar treated by T1 was reduced, the aroma quality was increased, the aftertaste was clean and comfortable, and the comprehensive score was the highest among all treatment schemes. It is indicated that the T1 treatment process has a significant advantage in improving the quality of cigar, can optimize the sensory quality of cigar to the greatest extent, and is one of the effective means to improve the quality of cigar.

[0047] In summary, the specific enzymes produced by the synergistic fermentation of Enterobacter hormaechei XW-01 and Bacillus megaterium XW-02 can effectively degrade cellulose and starch in cigar tobacco leaves. The mechanism is that cellulose decomposition reduces the generation of pungent substances (guaiacol, propylene aldehyde, etc.) and carcinogens (formaldehyde, catechol) in the burning process. Overall, the sensory quality is systematically improved, which is reflected in the effective improvement of the core indicators: aroma quality↑ (5.0→5.5); offensive odor↓ / aftertaste↑ (offensive odor score 6.0→obvious optimization; aftertaste is cleaner); irritation↓ (burning pungency is reduced).

[0048] Although some preferred embodiments of the present application have been described, those skilled in the art who have the basic inventive concept can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0049] 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 inventive concept. 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 is also intended to include these modifications and variations.

Claims

1. A composite biological preparation, characterized in that, comprising Enterobacter hormaechei (CCTCC NO: M 20242595) Enterobacter hormaechei ) XW-01 or / and its metabolites, Bacillus megaterium (CCTCC NO: M 2025185) Bacillus megaterium ) XW-02 or / and its metabolites.

2. The use of the complex biological preparation of claim 1 in at least one of the following (1)~(7): (1) degrading cellulose in tobacco or preparing a preparation for degrading cellulose in tobacco; (2) degrading starch in tobacco or preparing a preparation for degrading starch in tobacco; (3) degrading total sugar in tobacco or preparing a preparation for degrading total sugar in tobacco; (4) degrading reducing sugar in tobacco or preparing a preparation for degrading reducing sugar in tobacco; (5) improving the draw value of tobacco or preparing a preparation for improving the draw value of tobacco; (6) improving the sensory quality of tobacco or preparing a preparation for improving the sensory quality of tobacco, the sensory quality including at least one of aroma quality, aroma amount, odor, irritation, aftertaste, strength, concentration, and softness; (7) improving the quality of cigarettes or cigars or preparing a preparation for improving the quality of cigarettes or cigars.

3. A process for the preparation of the complex biological preparation as claimed in claim 1, characterized in that, comprising the following steps: (1) activating the strains: Enterobacter hormaechei XW-01 with the preservation number CCTCC NO: M 20242595 and Bacillus megaterium XW-02 with the preservation number CCTCC NO: M 2025185 are inoculated on NA solid culture medium and cultured at 25~35℃ for 24~48h; (2) preparing the seed liquid: the bacteria obtained by activating the culture in step (1) are scraped and inoculated in NA liquid culture medium, and cultured at 25~35℃ with 160~200 r / min shaking for 10~14h to obtain the seed liquid; (3) expanding the culture: the seed liquid of Enterobacter hormaechei XW-01 obtained in step (2) is inoculated in cellulase-producing liquid culture medium at 1~4% of the weight of the culture medium, and cultured at a temperature of 25~35℃ with a shaking speed of 160~200 r / min for 46~50h; at the same time, the seed liquid of Bacillus megaterium XW-02 obtained in step (2) is inoculated in amylase-producing liquid culture medium at 1~4% of the weight of the culture medium, and cultured at a temperature of 25~35℃ with a shaking speed of 160~200 r / min for 46~50h; (4) preparing the complex biological preparation: the supernatant of the culture medium after the culture in step (3) is taken to obtain the crude enzyme preparation; and the enzyme preparation of Enterobacter hormaechei XW-01 and the enzyme preparation of Bacillus megaterium XW-02 are mixed to obtain the complex biological preparation.

4. The production method according to claim 3, characterized by, In the step (3), the cellulase-producing liquid culture medium for Enterobacter hormaechei XW-01 comprises: fructose 9 g / L, tryptone 15 g / L, and MnSO4 7 g / L.

5. The preparation method according to claim 3, characterized in that, In the step (3), the amylase-producing liquid culture medium for Bacillus megaterium XW-02 comprises: glucose 1 g / L, wheat bran powder 10 g / L, and KCL 11 g / L.

6. A method of treating cigar leaf, the method comprising: comprising the following steps: (1) according to 20~30% of the mass of the tobacco to be treated, the complex biological preparation of claim 3 is sprayed on the tobacco, the moisture content of the tobacco is adjusted to 30~40%, and then the tobacco is closed and fermented at 33~37℃ and 70~80% humidity for 3~5d; (2) After the fermentation is completed, it is dried and balanced in moisture, and then it is finished.

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