Paenibacillus arborescens and application thereof

Abstract

The invention discloses paenibacillus arborescens and application thereof, and belongs to the technical field of microorganisms. The classification name of the paenibacillus arborescens is paenibacillus arborescens yhs-1, the paenibacillus arborescens yhs-1 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is October 25, 2023, and the preservation number is CCTCC NO: M 20232033. The bacterial strain is screened from tobacco leaf and fruit and vegetable samples, has better degradation capacity on xanthophyll, can degrade xanthophyll in tobacco leaves and generate 18 aromatic substances when being applied to tobacco leaf fermentation, so that the tobacco leaves are sufficient in aroma aftertaste and free of other offensive odor and pungent smell; when the fermented tobacco leaf product is applied to cigarettes, the smoking quality of the cigarettes can be remarkably improved.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology and relates to a dendritic bacillus and its application. Background Art

[0002] Lutein, also known as plant lutein, is an oxygen-containing carotenoid that is widely found in fruits, vegetables, flowers, tobacco and other plants. It is an important flavor precursor. The lutein content in grapes can reach about 1μg / g, while the lutein content in tobacco leaves is even higher, even reaching about 150μg / g. Lutein can be degraded under certain conditions to produce a variety of aroma substances such as β-ionone, β-dihydrodamascone, and megastigmatrienone. These aromatic substances have relatively low thresholds, low irritation, and good aroma quality. They are the source of plant flower and fruit aromas, and are also important components of the flavor of fruit wine and tobacco leaves. However, the degradation mode of lutein will affect the type of degradation products and product quality. Taking tobacco leaves as an example, after adding 0.02% lutein to tobacco, its aroma, miscellaneous odor, and irritation all increase, but the aftertaste becomes worse. The significant increase in its miscellaneous odor and irritation is related to the large amount of benzene and its homologues produced during the thermal decomposition of lutein. Therefore, using appropriate technology to degrade lutein during tobacco processing will help reduce the harmfulness of cigarettes and improve their smoking quality.

[0003] At present, the research on the degradation of lutein is mostly focused on the process of tobacco leaf maturation and baking, and there are few reports in other fields. The degradation methods involved are mostly physical degradation (thermal cracking), chemical degradation and oxidative degradation, while there are few studies on biological degradation. The microorganisms that have been reported to be able to degrade and utilize lutein are mainly Geotrichum sp (candid mold) and Enterobacter hormaechei (hormaechei enterobacter), and their degradation products are β-ionone and 8-methyl-α-ionone, respectively. In addition, two strains of Geotrichum and Bacillus screened from marigold flowers by A. Sanchez-Contreras et al. can also degrade lutein after mixed fermentation, and the products mainly include β-ionone, 7,8-dihydro-β-ionone, 3-hydro-β-ionone and 7,8-dihydro-β-ionol. It is not difficult to find from the above reports that biological degradation of lutein has significant advantages, and the product composition obtained is relatively simple and is recognized as a "natural ingredient". Therefore, the biological degradation of lutein has broad application prospects. Summary of the invention

[0004] In order to solve the above problems, the present application provides a dendritic Bacillus. The applicant screened a strain yhs-1 with good degradation ability for lutein from tobacco leaves and fruit and vegetable samples, and measured its degradation products. The applicant also optimized the culture medium components for the fermentation and degradation of lutein by the strain yhs-1 through single factor and response surface experiments, and finally carried out fermentation application in tobacco leaves, laying the foundation for the development of a new biological flavoring process.

[0005] The specific technical solutions of this application are as follows:

[0006] The present application provides a dendritic Paenibacillus, the classification name of the dendritic Paenibacillus is dendritiformis (Paenibacillus dendritiformis) yhs-1, which has been deposited in the China Center for Type Culture Collection (CCTCC), Wuhan, China, with a deposit date of October 25, 2023, and a deposit number of CCTCC NO: M 20232033.

[0007] The present application also provides a bacterial suspension comprising the dendritic Bacillus described in claim 1.

[0008] Optionally, the bacterial suspension is prepared by the following method:

[0009] Pick a single colony into LB liquid medium and culture it with shaking until the bacterial solution OD 600 The concentration of the culture medium is 0.8-1.2 to obtain seed solution, and the culture is expanded at an inoculation ratio of 1.8-2.2% to obtain bacterial solution. The bacterial solution is centrifuged, and the supernatant is discarded to obtain bacterial precipitation. The bacterial precipitation is resuspended and washed with sterile PBS to obtain bacterial suspension.

[0010] This application also provides the following applications

[0011] Application of the above-mentioned Paenibacillus dendriticus and / or bacterial suspension in degrading lutein.

[0012] Application of the above-mentioned Paenibacillus dendriticus and / or bacterial suspension in tobacco flavor enhancement.

[0013] The application of the above-mentioned Paenibacillus dendriticus and / or bacterial suspension in tobacco leaf fermentation is to spray the bacterial suspension of the Paenibacillus dendriticus on the surface of tobacco shreds for fermentation.

[0014] Optionally, the fermentation conditions are: fermentation at a temperature of 32-40°C and a humidity of 45-65% for 45-60 hours.

[0015] Optionally, the volume mass ratio of the bacterial suspension to the tobacco shreds is 1:45-55.

[0016] Optionally, the concentration of the bacterial suspension is 5×10 7 ~5×1010 CFU / mL.

[0017] The present application also provides the use of the above-mentioned Paenibacillus dendriticus and / or bacterial suspension in improving the quality of tobacco leaves.

[0018] The beneficial effects of this application include but are not limited to:

[0019] 1. The dendritic Bacillus yhs-1 screened from fruits, vegetables and tobacco leaves in the present application can ferment and degrade lutein in tobacco leaves and produce octadecene aldehyde (C 18 H 34 O) and phytol (C 20 H 40 O) and other 18 flavor substances, which are the result of the degradation and transformation of lutein by strain yhs-1. Because fruits, vegetables and tobacco leaves contain a large amount of other nutrients besides lutein, these substances themselves can meet the nutritional needs of strain yhs-1. In this case, the degradation and transformation of lutein has a more complex metabolic pathway.

[0020] These aromatic substances make the tobacco leaves have sufficient aroma aftertaste and the product is free of other impurities and irritating tastes, thereby enhancing the aroma of the tobacco leaves while avoiding the production of harmful gases; the fermented tobacco products used in cigarettes can significantly improve the smoking quality of cigarettes.

[0021] 2. Furthermore, the degradation rate of tobacco leaf lutein by the strain yhs-1 of the present application can reach 23.66% (from 210.5 μg / g before fermentation to 160.7 μg / g); the fermented tobacco leaves have a higher sensory score, with less impurities, less irritating taste, a purer and more comfortable aftertaste, and an increased aroma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0023] Figure 1 This is the standard curve of lutein for this application;

[0024] Figure 2 The degradation rate of lutein by the strain of the present application;

[0025] Figure 3 The effect of fermentation time on the degradation of lutein by yhs-1 strain;

[0026] Figure 4 is the colony morphology of strain yhs-1;

[0027] Figure 5is the phylogenetic tree of strain yhs-1;

[0028] Figure 6 The effect of glucose addition on lutein degradation rate;

[0029] Figure 7 The effect of peptone addition on lutein degradation rate;

[0030] Figure 8 The effect of MgSO4 addition on the degradation rate of lutein;

[0031] Fig. 9 The response surface and contour lines of the interaction between various factors on the degradation rate of lutein;

[0032] Fig.10 Verification of culture medium optimization results. DETAILED DESCRIPTION

[0033] The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples. The present application is further described below in conjunction with the specific implementation methods, but the protection scope of the present application is not limited thereto; unless otherwise specified, the various reagents, instruments, etc. used in the examples are commercially available products.

[0034] Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments and test examples are briefly introduced as follows:

[0035] Materials and methods

[0036] Materials and culture medium

[0037] Materials: Tobacco leaf samples (Inner Mongolia Kunming Cigarette Co., Ltd.), fruit and vegetable samples (Inner Mongolia Agricultural University Supermarket), lutein (Beijing Wanjia Shouhua Biotechnology Co., Ltd.).

[0038] LB medium: peptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, pH = 7.0.

[0039] LB solid medium: peptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, agar 15 g / L, pH = 7.0.

[0040] Basic fermentation medium: glucose 10g / L, peptone 10g / L, yeast extract powder 5g / L, MgSO4 0.2g / L, pH=7.0, sterilize at 121℃ for 20min, then add 1ml / L lutein solution.

[0041] Lutein solution: 0.2 g lutein, 0.2 g Tween 80, 400 ml CH2Cl2. The solvent was evaporated under reduced pressure, dissolved in 10 ml anhydrous ethanol, and filtered through a 0.22 μm sterile filter.

[0042] Instruments and Equipment

[0043] High performance liquid chromatography, Agilent Technologies, USA; gas chromatography-mass spectrometry, Agilent Technologies, USA; clean bench, Shanghai Yiheng Scientific Instrument Co., Ltd.; high pressure sterilizer, Shanghai Xinpu Instrument Equipment Co., Ltd.; high speed centrifuge, Shanghai Chengguan Instrument Co., Ltd.; shaking incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.

[0044] The specific experimental process is as follows:

[0045] 1. Screening of bacterial strains

[0046] Grind tobacco leaves and fruits and vegetables, weigh 4g and put them into a shaking bottle filled with 50mL sterile water, shake thoroughly and filter with gauze, dilute the filtrate to a suitable gradient coated LB solid culture medium plate, culture at 37℃ for 48h, pick a single colony and purify it by streaking, inoculate the purified single colony into LB liquid culture medium, culture at 37℃ and 180r / min for 24h, take 1mL of bacterial solution for glycerol preservation (the applicant screened 198 culturable bacteria from tobacco leaves and fruit and vegetable samples, so all culturable microorganisms were purified and preserved first), and at the same time, inoculate it into the basic fermentation medium at an inoculum of 2% (V / V), culture at 37℃ and 180r / min for 48h to obtain the fermentation liquid, measure the lutein content by high performance liquid chromatography (HPLC), calculate the lutein degradation rate according to the formula, and screen and obtain strains with high lutein degradation rate. Lutein degradation rate = [1-(C 实验 / C 对照 )]×100%, where: C 实验 is the concentration of lutein in the experimental group, C 对照 is the concentration of lutein in the control group, unit: mg / L.

[0047] The lutein content was determined as follows:

[0048] The fermentation broth was centrifuged (4°C, 6500r / min, 10min) to obtain the supernatant, and an equal volume of dichloromethane was added and extracted repeatedly for 3 times to obtain a lutein extract, which was then dried overnight with anhydrous Na2SO4. The solvent was dried with a nitrogen blower, and 2mL of methanol was added. The extract was filtered with a 0.22μm organic filter and used for high performance liquid chromatography (HPLC) analysis. An equal volume of sterile basic fermentation medium was used as a control.

[0049] The chromatographic column selected was a C18 column (4.6 mm × 150 mm, particle size of 4 μm), the column temperature was 30 °C, the mobile phase was methanol: water (97.5:2.5 volume ratio), the flow rate was 1 mL / min, the wavelength was 445 nm, and the injection volume was 20 μL.

[0050] Drawing of lutein standard curve:

[0051] Accurately weigh 10 mg of lutein standard into a 10 mL brown volumetric flask, dissolve it with anhydrous ethanol and dilute to the mark to obtain a standard stock solution with a concentration of 1 mg / mL. Transfer 0.125, 0.25, 0.5, 1.0, 2.0, and 5.0 mL of the standard stock solution to 6 10 mL brown volumetric flasks, dilute to the mark with anhydrous ethanol, and obtain a series of standard working solutions with concentrations of 0.0125, 0.025, 0.05, 0.10, 0.20, and 0.50. After HPLC analysis according to the established chromatographic conditions, a standard curve was prepared with the peak area as the ordinate (Y) and the mass concentration as the abscissa (X). Figure 1 ).

[0052] The degradation rate of lutein was determined by high performance liquid chromatography. Four strains with lutein degradation ability were obtained from 198 culturable bacteria. Among them, the strain named yhs-1 had the highest degradation rate of lutein, which was 21.35% (see Figure 2 ), the highest degradation rate was reached after 48h of fermentation, and there was a downward trend around 60h.

[0053] (See Figure 3 ).

[0054] The colony of strain yhs-1 is round, smooth, light white, and has neat edges (see Figure 4 ).

[0055] 2. Molecular Biological Identification of Strain YHS-1

[0056] Colony PCR was performed using 16S rDNA universal primers 27F and 1492R, and the PCR products were directly sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The measured 16S rDNA gene nucleotide sequence was entered into the GenBank database for BLAST comparison to obtain strain sequences with high 16S rDNA gene sequence similarity. Multiple sequence alignment was performed using MEGA X, and a phylogenetic tree was constructed using Neighbor-joining; Figure 5 As shown, the similarity between strain yhs-1 and Paenibacillus dendritiformis was 98%, and strain yhs-1 was identified as Paenibacillus dendritiformis. Paenibacillus dendritiformis is widely present in plant leaves and is a potential beneficial bacterium.

[0057] The above-mentioned strain yhs-1 is classified and named Paenibacillus dendritiformis yhs-1, and has been deposited in the China Center for Type Culture Collection (CCTCC). The preservation date is October 25, 2023, and the preservation number is CCTCC NO: M 20232033.

[0058] 3. Optimization of culture medium for yhs-1 strain

[0059] The four conditions of yhs-1 inoculation volume of 2% (V / V), rotation speed of 180r / min, temperature of 37℃ and liquid volume of 100mL / 250mL were fixed, and the optimal addition amounts of glucose (5, 10, 15, 20, 25g / L), peptone (5, 10, 15, 20, 25g / L) and MgSO4 (0.1, 0.2, 0.3, 0.4, 0.5g / L) in the basic fermentation medium were determined in turn through single factor experiments, and then a 3-factor 3-level Box-Behnken response surface experiment was designed to further optimize it.

[0060] The results of the single factor experiment are as follows:

[0061] Effect of glucose content on lutein degradation by yhs-1 strain Figure 6 As shown in the figure, with the increase of glucose content, the bacterial concentration increased, and the lutein degradation rate also increased. When the addition amount of glucose was 15g / L, the lutein degradation rate reached the highest level. As the glucose content continued to increase, the lutein degradation rate decreased, which may be due to the excessively high glucose concentration, which inhibited the growth and metabolism of the strain, resulting in a decrease in the ability to degrade lutein.

[0062] Effect of peptone content on lutein degradation by yhs-1 strain Figure 7 As shown in the figure, when the addition amount of peptone was 15g / L, the degradation rate and bacterial concentration of lutein reached the highest level, and then the degradation rate and bacterial concentration decreased with the increase of peptone content.

[0063] Effect of MgSO4 addition on lutein degradation by strain yhs-1 Figure 8 As shown in the figure, the degradation rate of lutein increases first and then decreases with the increase of MgSO4 addition. When the addition amount of MgSO4 is 0.3g / L, the lutein degradation rate and bacterial concentration both reach the highest level. Similar to the effect of glucose and peptone on the degradation of lutein by strain yhs-1, the maximum degradation rate is achieved when the bacterial concentration reaches the highest, indicating that under this fermentation condition, bacterial concentration has the most direct effect on the degradation of lutein.

[0064] Response surface methodology optimization design and results:

[0065] On the basis of the single factor experiment, glucose (A), peptone (B) and MgSO4 (C) were used as independent variables, and the degradation rate of lutein was used as the response value. A 3-factor 3-level design table (Table 1) was made, and the experimental results are shown in Table 2. The experimental results were analyzed by multiple regression fitting using Design-Expert V13 software, and the quadratic regression equation of lutein degradation rate for each factor was obtained: Y = 27.56 + 0.75A + 2.24B + 1.21C + 0.625AB + 0.525AC + 0.1BC-2.21A2-2.83B2-0.98C2, and then variance analysis was performed, and the results are shown in Table 3.

[0066] Table 1 Box-Behnken experimental factors and level design table

[0067] factor -1 0 1 A Glucose 13 15 18 B-peptone 13 15 18 <![CDATA[C MgSO4]]> 0.25 0.3 0.35

[0068] Table 2 Box-Behnken experimental design and results

[0069]

[0070] Table 3 Analysis of variance of regression model

[0071] Sources of variance Average and Degrees of Freedom Mean Square F-number P-value Significance Model 122.86 9 13.65 58.28 <0.0001 ** A 4.50 1 4.5 19.21 0.0032 ** B 40.05 1 40.05 171.00 <0.0001 ** C 11.76 1 11.76 50.22 0.0002 ** AB 1.56 1 1.56 6.67 0.0363 * AC 1.10 1 1.10 4.71 0.0667 BC 0.0400 1 0.0400 0.1708 0.6918 A2 20.47 1 20.47 87.41 <0.0001 ** B2 33.72 1 33.72 143.98 <0.0001 ** C2 4.04 1 4.04 17.27 0.0043 ** Residual 1.64 7 0.2342 Lack of Fit 1.05 3 0.3492 2.36 0.2127 Not significant Error term 0.5920 4 0.1480 sum 124.5 16

[0072] Note: “**” indicates extremely significant (P<0.01), “*” indicates significant (0.01 <P<0.05),P> 0.05 indicates not significant.

[0073] As shown in Table 3, the regression model P < 0.01 indicates that the model is extremely significant, and the lack of fit term P = 0.2127 > 0.05 is not significant, indicating that the model can fit the experimental situation well. 2 =0.9868, indicating that the regression equation has a good fit and can better reflect the relationship between the lutein degradation rate and the glucose, peptone and MgSO4 in the fermentation medium. It can also be seen from Table 3 that the first and second terms have a very significant effect on the results (P<0.01), while the interaction terms AC and BC have no significant effect on the results, and the exchange term AB has a significant effect on the results. From the F value, it can be seen that the degree of influence of each factor on the lutein degradation rate is: B>C>A. That is, peptone in the fermentation medium>MgSO4>glucose.

[0074] The response surface and contour lines of the interaction between glucose, peptone and MgSO4 on the degradation rate of lutein are shown in Figure 2. Fig. 9As shown in the figure, the response surface slope of AB is steeper and the color changes faster, and the contour lines are elliptical and denser, indicating that the interaction effect between AB is significant (P<0.05); on the contrary, the interaction effect between AC and BC is not significant, which is consistent with the results of variance analysis.

[0075] According to the model, the optimal process parameters of the fermentation medium conditions for lutein degradation are: glucose addition 15.958g / L, peptone addition 16.330g / L, MgSO4 addition 0.336g / L. Under this condition, the degradation rate of lutein is 28.62%. For the convenience of actual operation, the fermentation medium conditions are set to glucose addition of 16g / L, peptone addition of 16g / L, and MgSO4 addition of 0.3g / L. Under this setting, three parallel experiments were conducted, and the results are as follows Fig.10 As shown, the degradation rate of lutein was 26.4% after 48 h of fermentation, with a deviation of 7.7% from the theoretical value.

[0076] 4. Determination of lutein degradation products

[0077] The fermentation products of strain yhs-1 were determined by GC-MS, and the different substances with or without lutein addition were obtained (Table 4), including 1 aldehyde, 6 ethers, 5 esters, 4 alkanes and 2 alcohols. 18 H 34 O) and phytol (C 20 H 40 O) are typical flavor substances. The production of these differential substances is the result of the degradation and transformation of lutein by strain yhs-1. Compared with the resting cell method for determining the products of degraded lutein, the determination method of this study is closer to the actual application of the strain, because fruits, vegetables and tobacco leaves contain a large amount of other nutrients besides lutein, which can meet the nutritional needs of strain yhs-1. In this case, the degradation and transformation of lutein has a more complex metabolic pathway.

[0078] Table 4 Different substances produced by fermentation when lutein is added to the culture medium

[0079]

[0080] 5. Application of Bacillus dendrobatidis YHS-1 in tobacco fermentation

[0081] Preparation of bacterial suspension: streak Bacillus dendrobatidis yhs-1 on LB solid medium, culture at 37°C for 24 h, pick a single colony and transfer it to LB liquid medium, culture at 37°C and 180 r / min with shaking until the OD600 of the bacterial solution reaches about 1 to obtain seed solution, expand the culture at a 2% (V / V) inoculation ratio to obtain sufficient bacterial solution, centrifuge the bacterial solution at 4°C and 6000 r / min for 5 min, discard the supernatant to obtain the bacterial precipitate, resuspend and wash the bacterial precipitate with sterile PBS once, and adjust the bacterial concentration to 5×10 8 CFU / mL, and obtain the bacterial suspension for use.

[0082] Tobacco leaf fermentation: Spray the bacterial suspension on the surface of tobacco (cut the tobacco leaves into shreds, width: 2mm) at a mass-to-volume ratio of 50:1 (Kg:L), and ferment at a temperature of 37°C and a humidity of 55% for 48 hours. After the fermentation is completed, the lutein content of the tobacco leaves before and after fermentation is determined, and the smoking is evaluated.

[0083] The sensory quality evaluation standards are as follows: for tobacco leaves before and after fermentation, refer to the standard requirements of YC / T497-2014 in the tobacco industry, and organize smoking evaluation personnel to evaluate the changes in the sensory quality of tobacco leaves before and after fermentation based on 10 indicators, including aroma quality, aroma volume, permeability, concentration, miscellaneous smell, irritation, sweetness, dryness, strength and aftertaste. The full score for each indicator is 10 points. The total score of the sensory quality evaluation is calculated according to the total score of the evaluation indicators. The results are shown in Table 5.

[0084] Table 5 Lutein content in tobacco leaves before and after fermentation and tobacco leaf smoking results

[0085] sample Lutein content (μg / g) Sensory quality evaluation score Tobacco leaves before fermentation 210.5±5.2 80.2±2.7 Tobacco leaves after fermentation 160.7±3.39 86±1.8

[0086] As shown in the table above, after fermentation with strain yhs-1, the lutein content in tobacco leaves dropped from 210.5μg / g before fermentation to 160.7μg / g, and the degradation rate reached 23.66%. The sensory evaluation results showed that compared with unfermented tobacco leaves, fermented tobacco leaves had higher sensory scores, less impurities, less irritating taste, a purer and more comfortable aftertaste, and increased aroma.

[0087] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A dendritic bacillus, characterized in that The classification name of the dendritic Bacillus is Paenibacillus dendritiformis yhs-1, which has been deposited in the China Center for Type Culture Collection (CCTCC) on October 25, 2023, and the deposit number is CCTCC NO: M 20232033.

2. A bacterial suspension, characterized in that: The invention comprises the Paenibacillus dendriticus described in claim 1.

3. The bacterial suspension according to claim 2, characterized in that The bacterial suspension is prepared by the following method: Pick a single colony into LB liquid medium and culture it with shaking until the bacterial solution OD 600 The concentration of the culture medium is 0.8-1.2 to obtain seed solution, and the culture is expanded at an inoculation ratio of 1.8-2.2% to obtain bacterial solution. The bacterial solution is centrifuged, and the supernatant is discarded to obtain bacterial precipitation. The bacterial precipitation is resuspended and washed with sterile PBS to obtain bacterial suspension.

4. Use of the Paenibacillus dendriticus according to claim 1 and / or the bacterial suspension according to claim 2 in degrading lutein.

5. Use of the Paenibacillus dendriticus according to claim 1 and / or the bacterial suspension according to claim 2 in tobacco flavoring.

6. Use of the Paenibacillus dendriticus according to claim 1 and / or the bacterial suspension according to claim 2 in tobacco leaf fermentation, characterized in that: The bacterial suspension of Paenibacillus dendriticus is sprayed on the surface of tobacco shreds for fermentation.

7. Use of the Paenibacillus dendriticus according to claim 1 and / or the bacterial suspension according to claim 2 in improving the quality of tobacco leaves.

8. The use according to claim 6, characterized in that: The fermentation conditions are as follows: fermentation for 45-60 hours at a temperature of 32-40° C. and a humidity of 45-65%.

9. The use according to claim 6, characterized in that: The volume mass ratio of the bacterial suspension to the tobacco shreds is 1:45-55.

10. The use according to claim 6, characterized in that: The concentration of the bacterial suspension was 5×10 7 ~5×10 10 CFU / mL.

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