Bacillus siamensis G12, microbial inoculum, preparation method and application of bacillus siamensis G12 in improvement of tobacco leaf quality

Abstract

The invention discloses bacillus siamensis G12, a microbial inoculum, a preparation method and application of the bacillus siamensis G12 in improvement of tobacco leaf quality, and belongs to the technical field of bioengineering. The bacillus siamensis provided by the invention can inhibit mould in tobacco leaves, can degrade nicotine, and can well survive and metabolize in a tobacco leaf environment; a very good degradation effect is achieved on macromolecular compounds including protein, pectin, cellulose, lignin and starch which influence the quality, combustibility, health and safety of the tobacco leaves; the tobacco leaf smoking quality can be effectively improved, tobacco leaf fermentation is accelerated, the tobacco leaf fermentation time is saved, and the usability of tobacco leaves, especially low-quality tobacco leaves, is greatly improved.

Description

Technical Field

[0001] The invention relates to a siam bacillus G12, a bacterial agent, a preparation method, and application of the bacterial agent in improving tobacco leaf quality, and belongs to the technical field of bioengineering. Background Art

[0002] In the field of cigarette industrial production, tobacco leaves from various regions are purchased on demand by cigarette factories for their unique regional characteristics. However, due to the uneven quality of tobacco leaves when they are purchased, some tobacco leaves are often difficult to be included in cigarette formulas. Tobacco companies need to bear high costs to deal with these unsuitable or difficult-to-use tobacco leaves, and these tobacco leaves may face the risk of being abandoned due to long-term inventory, or consume a lot of manpower and material resources due to hoarding in warehouses, which seriously restricts the economic benefits of the company. At present, industrial enterprises have a large backlog of such tobacco leaves in their inventory. How to efficiently use these difficult-to-use tobacco leaves has become an urgent issue that the cigarette industry needs to solve.

[0003] In addition, tobacco leaves are susceptible to mold during storage, which can lead to quality degradation, poor taste, and even the production of harmful toxins, which seriously threatens the health of consumers. Traditional prevention and control methods, such as the use of chemical fungicides, can effectively control mold, but may cause environmental pollution and safety hazards.

[0004] Nicotine is the main alkaloid in tobacco, and its content directly affects the taste and irritation of tobacco. High nicotine content may not only cause health problems, but also limit the scope of application of tobacco products. Tobacco leaves contain a large amount of macromolecular compounds such as protein, starch, pectin, cellulose and lignin. The presence of these substances affects the combustion performance and sensory quality of tobacco. Through the degradation of related microorganisms, these macromolecular substances can be converted into small molecular compounds, increasing the content of aroma components, thereby improving the quality of tobacco leaves, reducing the production of harmful substances, and improving the hygroscopicity, combustibility and taste of tobacco, while facilitating subsequent processing.

[0005] Microbial fermentation is used to reduce the irritation of tobacco leaves and increase their softness and aroma complexity. This process usually takes a long time. The fermentation method in the prior art of microbial fermentation of tobacco leaves usually takes at least 48 hours or even longer, which not only increases production costs, but also limits the rapid improvement of production efficiency and capacity.

[0006] In summary, although some studies have tried to introduce microbial fermentation technology to improve the tobacco fermentation process, most methods still face problems such as unstable fermentation effect, long fermentation time, and low production efficiency. Therefore, screening and cultivating a new type of microbial strain to inhibit mold on the surface of tobacco leaves, reduce nicotine content, efficiently catalyze the transformation of key chemical components inside tobacco leaves, improve the aroma and taste of tobacco leaves, and significantly shorten the fermentation time can not only significantly improve the efficiency of tobacco production, reduce production costs, and improve tobacco quality, but also bring a more environmentally friendly and efficient production model to the tobacco industry. Summary of the invention

[0007] The object of the present invention is to provide a strain of Bacillus siamensis, a bacterial agent thereof, and an application thereof in improving the quality of tobacco leaves, wherein the Bacillus siamensis can inhibit tobacco leaf mold, reduce nicotine content, reduce the content of macromolecular compounds such as protein, cellulose, lignin, and pectin in tobacco leaves, and significantly improve the sensory quality of tobacco leaves; when fermenting tobacco leaves, the fermentation time can be greatly shortened, and the efficiency of tobacco production can be significantly improved, and the production cost can be reduced. The Bacillus siamensis provided by the present invention can effectively utilize tobacco leaves that are not suitable or difficult to use, and solve the quality and storage problems of tobacco leaves.

[0008] The technical solution adopted by the present invention is:

[0009] The first object of the present invention is to provide a strain of Bacillus siamensis, which has been deposited in the China General Microbiological Culture Collection Center on September 6, 2024, with a deposit number of CGMCC No.31893.

[0010] In one embodiment of the present invention, the Siamese Bacillus is isolated from C3F-AB tobacco leaves in Xuanwei, Yunnan in 2022 and is named Siamese Bacillus G12.

[0011] In one embodiment of the present invention, the nucleotide sequence of 16S rDNA of Bacillus siamensis provided by the present invention has the sequence shown in SEQ ID NO.3.

[0012] In one embodiment of the present invention, the siam bacillus CGMCC No.31893 provided by the present invention has the following biological characteristics:

[0013] (1) Gram-positive bacteria, with a strain size of 0.5-0.8 μm × 1.2-1.8 μm, rod-shaped, without flagella, with large colonies on the plate, a rough surface with protrusions, flat, and irregular edges;

[0014] (2) Growth temperature range is 28-37°C, can tolerate 0-14.5% NaCl, and growth pH range is 4.5-9.5;

[0015] (3) It is a facultative aerobe and grows well under both aerobic and anaerobic conditions;

[0016] (4) It can inhibit mold on the surface of tobacco leaves, with an inhibition rate of up to 80%;

[0017] (5) It has the ability to reduce nicotine, with a degradation rate of up to 98.6% for 1g / L nicotine concentration;

[0018] (6) It can reduce the content of protein, starch, pectin, lignin and cellulose in tobacco leaves;

[0019] (7) Improve the smoking quality of tobacco leaves.

[0020] The second object of the present invention is to provide a microbial agent containing the siam bacillus CGMCC No.31893 of the present invention.

[0021] In one embodiment of the present invention, the microbial agent uses Bacillus siamese CGMCC No.31893 as the main microorganism.

[0022] In one embodiment of the present invention, the number of cells of Bacillus siamensis CGMCC No.31893 in the bacterial agent is not less than 3.0×10 8 CFU / mL.

[0023] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Bacillus siamensis CGMCC No.31893 and then expanding the culture.

[0024] In one embodiment of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0025] In one embodiment of the present invention, the microbial agent contains living cells of the CGMCC No. 31893 strain of the present invention, dry cells of the CGMCC No. 31893 strain of the present invention obtained by freeze-drying, immobilized cells of the CGMCC No. 31893 strain of the present invention, or the CGMCC No. 31893 strain of the present invention in any other form.

[0026] The third object of the present invention is to provide a method for inhibiting tobacco leaf mold, reducing nicotine, reducing macromolecular compounds in tobacco leaves or improving tobacco leaf quality, wherein the method utilizes siam bacillus CGMCC No.31893 or a microbial inoculant containing CGMCC No.31893 to ferment tobacco leaves to improve tobacco leaf quality.

[0027] In one embodiment of the present invention, the method comprises the following steps:

[0028] (1) spraying a suspension of Bacillus siamese CGMCC No.31893 onto the surface of tobacco leaves and then fermenting;

[0029] (2) Drying the fermented tobacco leaves to obtain fermented tobacco leaves.

[0030] In one embodiment of the present invention, the bacterial suspension in step (1) is (1.0-1.8)×10 8 CFU / g sprayed onto the tobacco leaf surface;

[0031] Preferably, the bacterial suspension is prepared at 1.2×10 8 CFU / g sprayed onto the surface of tobacco leaves.

[0032] In one embodiment of the present invention, the fermentation temperature is 30-37°C, the humidity is 60%-80%, and the fermentation time is 4-48h;

[0033] Preferably, the fermentation time is 24 hours.

[0034] In one embodiment of the present invention, the drying temperature in step (2) is 45-55° C.; the moisture content of the fermented tobacco leaves is 1.0%-1.2%.

[0035] In one embodiment of the present invention, the macromolecular compound in the tobacco leaf is protein, starch, pectin, lignin or cellulose.

[0036] The fourth object of the present invention is to provide the use of the siam bacillus CGMCC No. 31893 or a microbial agent containing the CGMCC No. 31893 in the preparation of tobacco products.

[0037] In one embodiment of the present invention, the tobacco product is tobacco flavoring, tobacco extract, flue-cured tobacco leaves or cigar tobacco leaves.

[0038] The fifth object of the present invention is to provide a fermentation medium for the siam Bacillus CGMCC No.31893.

[0039] In one embodiment of the present invention, the fermentation medium formula is: 5g / L-15g / L carbon source, 10g / L-20g / L complex nitrogen source, 2.5g / L-7.5g / L (NH4)2SO4, 3g / L-4g / L KH2PO4, 5g / L-6g / L K2HPO4; the fermentation medium solvent is water.

[0040] In one embodiment of the present invention, the fermentation medium formula is: 15 g / L carbon source, 10 g / L complex nitrogen source, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 5.5 g / L KH2PO4; the fermentation medium solvent is water.

[0041] In one embodiment of the present invention, the carbon source is one or more of glucose, maltodextrin, arabinose and sucrose.

[0042] Preferably, the carbon source is maltodextrin.

[0043] In one embodiment of the present invention, the composite nitrogen source is a combination of two or more of yeast powder, tryptone, soy peptone, yeast peptone, and acid-hydrolyzed casein.

[0044] Preferably, the composite nitrogen source consists of yeast powder and soy peptone in a weight ratio of 1:1.

[0045] The sixth object of the present invention is to provide a drying method for the Siamese Bacillus CGMCC No.31893, the specific implementation steps comprising: adding water to the Siamese Bacillus bacterial sludge, resuspending and centrifuging to obtain washed bacterial sludge; resuspending the bacterial sludge again and adding a freeze-drying protective agent, freeze-drying, and obtaining freeze-dried bacterial powder.

[0046] In one embodiment of the present invention, the lyoprotectant is one or more of sucrose, glucose, trehalose, lactose, and galactose;

[0047] Preferably, the lyoprotectant is sucrose.

[0048] In one embodiment of the present invention, the mass ratio of the bacterial mud to the freeze-drying protective agent is 10:(1-4);

[0049] Preferably, the mass ratio is 10:1.

[0050] Biomaterial Deposit

[0051] Bacillus siamensis G12, taxonomic name: Bacillus siamensis, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 6, 2024, with the deposit number CGMCC No.31893, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0052] Beneficial effects:

[0053] The present invention provides a siam bacillus, which can inhibit mold in tobacco leaves, degrade nicotine, and survive and metabolize well in a tobacco leaf environment; it has a very good degradation effect on macromolecular compounds that affect the quality, combustibility and health safety of tobacco leaves that are difficult to use or unsuitable, and can effectively improve the smoking quality of tobacco leaves, accelerate tobacco leaf fermentation, save tobacco leaf fermentation time, and greatly improve the usability of tobacco leaves, especially low-quality tobacco leaves. Specifically:

[0054] (1) The Siamese Bacillus G12 screened, obtained and preserved by the present invention has the ability to degrade macromolecular substances such as protein, starch, pectin, lignin, cellulose, etc. in tobacco leaves, with a degradation rate of up to 32.81% for protein, 28.41% for pectin, and 25.48% for lignin;

[0055] (2) The Siamese Bacillus G12 provided by the present invention can inhibit mold on the surface of tobacco leaves, and the inhibition rate can reach 80%;

[0056] (3) The Siamese Bacillus G12 provided by the present invention has the ability to reduce nicotine, with a degradation rate of 98.6% for 1 g / L nicotine concentration, and can grow at a nicotine concentration of up to 5 g / L, with a degradation rate of 66.8% for 2.5 g / L nicotine concentration;

[0057] (4) The aroma, permeability and salivation of tobacco leaves fermented by the strain of the present invention are improved, the foreign smell and irritation are reduced, and the quality of tobacco leaves is significantly improved;

[0058] (5) When the siam bacillus G12 provided by the present invention is used for tobacco leaf fermentation, the fermentation time is greatly shortened, and the ideal smoking standard can be achieved in only 24 hours, thereby improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0060] Figure 1 is a plate map of Bacillus siamensis G12;

[0061] Figure 2 This is an oil microscope image of Bacillus siamensis G12;

[0062] Figure 3 This is the growth curve of Bacillus siamese G12. DETAILED DESCRIPTION

[0063] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0064] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0065] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] In addition to the tobacco leaf types used in the following embodiments, the tobacco leaves in the present application may also be selected from various other known or new varieties of tobacco leaves. For other tobacco leaf varieties, those skilled in the art can use conventional technical means in the art to adjust the fermentation process.

[0067] 1. The culture medium involved in the following embodiments is as follows:

[0068] LB liquid medium: tryptone 10g / L, yeast powder 10g / L, sodium chloride 5g / L, sterilized at 121℃ for 20min;

[0069] LB solid medium: 10 g / L tryptone, 10 g / L yeast powder, 5 g / L sodium chloride, 15 g / L agar, sterilize at 121°C for 20 min, and pour into the plate after cooling;

[0070] PDA solid culture medium: purchased from Haibo Biotechnology, weigh 46 g of the finished culture medium, dissolve it in 1000 mL of purified water, divide it into portions, sterilize it under high pressure at 121°C for 15 min, and pour it into a plate after cooling.

[0071] Nicotine liquid culture medium: first prepare trace element solution (0.4g MnSO4·7H2O, 0.2g FeSO4·7H2O, 0.2g CaCl2·2H2O), and then make up to 100mL with 0.1mol / L HCl; then prepare nicotine culture medium: 13.3g K2HPO4, 4.0g KH2PO4, 1.0g yeast powder, 10mL trace element solution, add ddH2O to make up to 1000mL, pH is 7.0, sterilize at 121℃ for 20min, and add a certain amount of nicotine (filtered with 0.22μm membrane).

[0072] 2. The detection methods involved in the following embodiments are as follows:

[0073] The protein detection method was the BCA kit method (Suzhou Gres Biotechnology Co., Ltd., G0418W);

[0074] The starch detection method was the enzymatic kit method (Suzhou Grace Biotechnology Co., Ltd., G0551W);

[0075] The pectin detection method was the carbazole colorimetric kit method (Suzhou Gres Biotechnology Co., Ltd., G0717W);

[0076] The lignin detection method was the acetylation kit method (Suzhou Gres Biotechnology Co., Ltd., G0708W);

[0077] The cellulose detection method was anthrone kit method (Suzhou Gres Biotechnology Co., Ltd., G0715W).

[0078] 3. The sources of materials involved in the following embodiments are as follows:

[0079] The 2022 C3F-AB tobacco leaves from Xuanwei, Yunnan come from Gansu Tobacco Industry Co., Ltd.

[0080] Example 1: Screening and identification of Siamese Bacillus

[0081] The dominant strains were selected from the 2022 C3F-AB tobacco leaves of Xuanwei, Yunnan. The specific operations are as follows:

[0082] 1.1 Isolation of Bacillus siamensis

[0083] In the clean bench, weigh 9 g of C2L-AB tobacco leaf sample stored at 4°C (cut into pieces with sterile scissors) and 1 g of sterile glass beads, place them in a sterile Erlenmeyer flask containing 200 mL of sterile saline, shake at 200 r / min and 20°C for 60 min, filter through 2 layers of gauze to remove impurities in the tobacco leaves, and retain the filtrate sample rich in microorganisms on the surface of the tobacco leaves.

[0084] The collected filtrate sample was transferred into a sterile 50 mL centrifuge tube, centrifuged at 12000 rpm and 4°C for 15 min, the supernatant was removed, the precipitate was retained, and finally the precipitate was resuspended with 5 mL of sterile saline to obtain the enrichment solution.

[0085] 100 μL of enrichment solution was evenly spread on the surface of LB solid culture medium, and then cultured at 25°C for 24 hours. The colony morphology was recorded and different colonies were numbered from G1 to G30, and single colonies were picked and inoculated into LB liquid culture medium for expansion and preservation.

[0086] 1.2 Molecular biological identification of Bacillus siamensis

[0087] The selected strains were inoculated into LB liquid medium, cultured overnight at 37°C and 200 rpm, centrifuged at 12000 r / min for 5 min, and the supernatant was discarded to obtain the bacterial cells; the bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit ( The genomic DNA of the bacterium was extracted using a Bacteria Genomic DNA Kit (Beijing Qingke Biotechnology Co., Ltd.), and PCR amplification was performed using universal primers for 16S rDNA (primer F (SEQ ID NO.1): AAGGAGGTGATCCAGCCGCA; primer R (SEQ ID NO.2): AGAGTTTGATCCTGGCTCAG). The PCR reaction system was: 19 μL double distilled water, 10× PCR buffer (MgSO4 / ... 2+ 15mM) 2.5μL, forward and reverse primers 1μL each, dNTP 1μL, Taq enzyme 0.5μL; PCR reaction program: 94℃3min; 30 cycles of 94℃30s, 55℃30s, 72℃2min; finally 72℃10min; the amplified product was analyzed by 0.8% agarose gel electrophoresis, and the successfully amplified DNA fragment was detected, and sequenced by Beijing Qingke Biotechnology Co., Ltd. to obtain the 16S rDNA sequence of the bacteria as shown in SEQ ID No.3; the 16S rDNA sequence was compared and analyzed in the NCBI database by Blast, and the G12 strain was identified as Bacillus siamensis, and stored in a refrigerator at -80℃ for later use.

[0088] The colony morphologies of G11, G12 and G17 obtained in step 1.1 are relatively similar and they are identified as different species of Bacillus.

[0089] SEQ ID NO.3:

[0090] ATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGA

[0091] GTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATA

[0092] CCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTA

[0093] CAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGAT

[0094] GCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTC

[0095] CTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG

[0096] CCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTG

[0097] CCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGT

[0098] GCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAG

[0099] GGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGT

[0100] CATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGG

[0101] TGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAA

[0102] CTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCA

[0103] CGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAA

[0104] CGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGA

[0105] CGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT

[0106] TACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGA

[0107] GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG

[0108] CAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACT

[0109] GCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACC

[0110] TGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAG

[0111] CCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGC

[0112] TGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTAC

[0113] ACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTAT

[0114] GGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT

[0115] The Siamese Bacillus G12 strain was deposited in the China General Microbiological Culture Collection Center on September 6, 2024, with the deposit number CGMCC No.31893.

[0116] 1.3 Morphological characteristics and growth characteristics

[0117] The isolated Siamese Bacillus G12 strain was streaked on LB solid plate medium and cultured at 30°C. The colony morphology was as follows Figure 1 As shown in the figure, it is a round single colony, milky white, with a slightly raised center. The smear of the bacteria was gram-stained, and the bacterial morphology was as follows Figure 2 As shown, the bacteria are Gram-positive bacteria with a strain size of 0.5-0.8μm×1.2-1.8μm, thin rod-shaped, and without flagella.

[0118] The slant-preserved strain of Bacillus siameensis G12 was inoculated into LB liquid medium and cultured in a shaker at 30°C and 200 rpm for 12 h as seed solution. The seed solution was inoculated into LB liquid medium at 10% inoculation volume and cultured at 30°C and 200 rpm. The OD was detected at different culture times. 600 The growth curve was obtained. Figure 3 shown.

[0119] The growth temperature range of Siamese Bacillus G12 strain is 28-37°C, it can tolerate 0-14.5% NaCl, and the growth pH range is 4.5-9.5; it is a facultative aerobic bacterium and grows well under aerobic and anaerobic conditions.

[0120] Example 2: Enzyme production capacity of Bacillus siamese G12

[0121] Siamese Bacillus G12 was inoculated into LB liquid medium at an inoculum rate of 1%, and cultured in a shaker at 30°C and 200 rpm for 36 hours to obtain a fermentation liquid; the fermentation liquid was centrifuged at 7000 rpm for 25 minutes at 4°C, and the supernatant of the fermentation liquid was collected. The yields of amylase, protease, pectinase, and cellulase were determined by the following methods, respectively. The specific methods are as follows:

[0122] 1. Determination of amylase content: Use the method in GB / T 24401-2009.

[0123] 2. The protease content was determined using the method in GB / T 33410-2016.

[0124] 3. The pectinase content was determined using the method in QB / T 4482-2013.

[0125] 4. Determination of cellulase content: The method in GB 7300.403-2022 was used for determination.

[0126] The strains isolated in Example 1, Bacillus G11 and Bacillus Velez G17, were cultured in shake flasks according to the same method as above, and the production of amylase, protease, pectinase and cellulase in the supernatant of the fermentation broth was determined. The results are shown in Table 1.

[0127] Table 1 Detection results of amylase, protease, pectinase and cellulase in the supernatant of the fermentation broth of the strain shake flask

[0128] Screening strain number Amylase activity (U / ml) Protease activity (U / ml) Pectinase activity (U / ml) Cellulase activity (U / ml) G12 0.4936 1.62 0.907 0.0375 G11 0.1765 1.24 0.715 0.0293 G17 0.5278 1.53 1.214 0.0316

[0129] According to the results in the above table, the activities of amylase and pectinase contained in the shake flask fermentation broth of Bacillus Velez G17 are the highest, and the activities of protease and cellulase contained in the shake flask fermentation broth of Bacillus siameensis G12 are the highest.

[0130] Example 3: Ability of Bacillus siamese G12 to inhibit mold on tobacco leaf surface

[0131] The tobacco leaf molds involved in this embodiment are as follows:

[0132] The relatively high content of the isolated strain from the C2L-AB tobacco leaves produced in Longnan, Gansu in 2022 was identified as Rhizopus arrhizus by molecular identification; the relatively high content of the isolated strain from the C3F-AB tobacco leaves produced in Xuanwei, Yunnan in 2022 was identified as Aspergillus niger by molecular identification; the relatively high content of the isolated strain from the C3F-AB tobacco leaves produced in Baoji, Shaanxi in 2022 was identified as Aspergillus oryzae by molecular identification; the three strains were preserved in the laboratory of Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.

[0133] The siam Bacillus G12, Bacillus G11 and Bacillus Velezii G17 isolated in Example 1 were inoculated on PDA solid culture medium respectively, and 3 strains were inoculated in an equilateral triangle near the edge of the culture dish and cultured at 25°C for 24 hours. After the colony band grew out, the mold in the tobacco leaves was inoculated at the center point of the plate culture medium, and the plate inoculated with only the mold was used as the control. When the control colony filled the culture dish, the mold colony radius and the control colony radius were measured, and the inhibition rate of the strain on the mold was calculated according to the radius.

[0134] Calculation formula: Inhibition rate (%) = (control colony radius - treatment colony radius) / control colony radius * 100%

[0135] The results are shown in Table 2.

[0136] Table 2 Results of the inhibition rate of the strains on mold

[0137]

[0138] According to the results in the table, the three strains of Bacillus G11, G12 and G17 all have certain inhibitory effects on different molds isolated from the surface of tobacco leaves, but among them, G12 has a very significant inhibitory effect on the three molds, with inhibition rates of 80%, 85.71% and 65.71% on Rhizopus arrhizus, Aspergillus niger and Aspergillus oryzae, respectively. It can be better applied to the tobacco fermentation process to inhibit the mildew of tobacco leaves.

[0139] Example 4: Ability of Bacillus siamese G12 to reduce nicotine

[0140] The glycerol tube-preserved strain of Bacillus siamese G12 was inoculated into LB liquid medium at a rate of 0.1%, and cultured at 30°C with shaking until the OD 600 When the inoculum reached 0.6, the inoculum was inoculated with 1% of the inoculum into nicotine liquid medium with nicotine content of 0.25, 0.5, 1, 2, 2.5, and 5 g / L, respectively, and the culture conditions were 30°C, pH 7.0, and 200 r / min. The nicotine degradation rate under different nicotine concentrations was determined after culturing for 20 hours, and 1% of the volume of sterile water was inoculated as the control group under different concentrations.

[0141] Bacillus G11 and Bacillus Velez G17 were cultured according to the above method.

[0142] The fermentation broth of the control group and the experimental group after culturing for 20 hours was centrifuged at 12000 rpm for 5 minutes, and the supernatant was measured with an ultraviolet spectrophotometer at 259 nm to measure the OD value. The degradation rate calculation formula under different nicotine concentrations is as follows:

[0143] Degradation rate % = (control group OD 259 - Experimental group OD 259 ) / control group OD 259 ×100%

[0144] The results are shown in Table 3.

[0145] Table 3 Degradation rate of strains at different nicotine concentrations (%)

[0146]

[0147] According to the results in the above table, after being cultured in nicotine liquid culture medium for 20 hours, the degradation rate of nicotine at a concentration of 1g / L of Bacillus siamese G12 was as high as 98.6%. The degradation rate of nicotine by this strain was significantly better than that of Bacillus G11 and Bacillus Velezii G17, which fully demonstrated that this strain can survive in a nicotine environment and can also have a significant degradation effect on nicotine in tobacco.

[0148] Example 5: Degradation of macromolecular compounds in redried tobacco leaves by strains

[0149] The fermentation liquid was obtained by culturing according to the method described in Example 2, and the fermentation liquid was centrifuged at 7000 rpm for 25 min at 4°C to collect the fermentation mud, and sterile saline was added to fully resuspend the mud. The bacterial concentration in the resuspended bacterial liquid was adjusted to 9.0×10 8 CFU / mL. The bacterial suspension was evenly sprayed on low-grade tobacco leaves. 1 mL of bacterial suspension was sprayed on every 5 g of tobacco shreds, which means that the biomass acting on the tobacco leaves was 1.8×10 8 CFU / g, and fermented in a constant temperature and humidity chamber at 30°C and 70% humidity for 24 hours. At the same time, spray 1 mL of sterile saline as a control group.

[0150] Weigh an appropriate amount of the fermented tobacco leaves mentioned above and crush them into tobacco powder using a multifunctional pulverizer, and pass them through a 60-mesh sieve. The protein, starch, pectin, lignin and cellulose contents of the sieved tobacco leaf powder are tested.

[0151] The results of the detection of the content of macromolecular compounds in fermented tobacco leaves are shown in Table 4.

[0152] Table 4 Degradation results of strains on macromolecular compounds in tobacco leaves

[0153]

[0154] According to the results in the table, even though the activities of protease, amylase and pectinase of the G17 strain in Example 2 were similar to those of the Siamese Bacillus G12, the effect of Siamese Bacillus G12 in degrading macromolecular compounds in tobacco leaves was significantly better than that of the G17 and G11 strains.

[0155] Therefore, the application of Siamese Bacillus G12 in tobacco fermentation can effectively reduce the content of macromolecular compounds such as protein, pectin, cellulose, lignin and starch that affect the sensory quality, combustibility and health safety of tobacco through growth and metabolism, thereby improving the quality of tobacco.

[0156] Example 6: Application of Bacillus siamensis G12 in improving tobacco leaf quality

[0157] The method of fermenting tobacco leaves with Bacillus siamense G12 to improve the quality of tobacco leaves is as follows:

[0158] Siamese Bacillus G12 was inoculated into LB liquid medium and cultured according to the method of Example 2 to obtain fermentation broth. The bacterial sludge was collected by centrifugation and resuspended in sterile saline. The bacterial cell concentration in the resuspended bacterial broth was adjusted to 6.0×10 8 CFU / mL. The bacterial suspension was evenly sprayed on low-grade tobacco leaves. 1 mL of bacterial suspension was sprayed on every 5 g of tobacco shreds, which means that the biomass acting on the tobacco leaves was 1.2×10 8 CFU / g, and fermented in a constant temperature and humidity chamber at 30°C and 70% for 24h or 48h. At the same time, spray 1mL of sterile saline as a control group.

[0159] The fermented tobacco leaves were cut into shreds according to industry standards and rolled into cigarettes. The cigarettes were placed in a constant humidity chamber at a temperature of 22°C and a relative humidity of 60% for 48 hours for balancing in a smoking evaluation experiment.

[0160] The results of the suction test are shown in Table 5:

[0161] Table 5 Smoking results of tobacco leaves in the experimental group and the control group

[0162]

[0163] The results in Table 5 show that after fermenting the redried tobacco leaves with Siamese Bacillus G12 for 24 hours and 48 hours, the aroma of the tobacco leaves was significantly increased, the foreign smell and irritation were reduced, the permeability and concentration were increased, and the quality of the tobacco leaves was significantly improved; and the ideal effect of improving the evaluation quality of tobacco leaves can be achieved after fermenting for 24 hours.

[0164] Example 7: Optimization of fermentation medium composition

[0165] Scheme 1: A fermentation culture method for Siamese Bacillus G12 is provided, comprising the following steps:

[0166] (1) The preserved Siamese Bacillus glycerol strain was inoculated into a seed culture medium at an inoculum rate of 0.1%, and cultured at 30°C and 180 r / min for 16 h to obtain a seed solution. The composition of the seed culture medium is: peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 5.0 g / L;

[0167] (2) Prepare 1L fermentation medium in a 2.5L fermenter. Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2%. Fermentation was started at 30°C, a speed of 200rpm, and a ventilation ratio of 1.0vvm. The pH of the fermentation process was adjusted to 7.0 by ammonia water. The dissolved oxygen (DO) was maintained at 30%±10% by adjusting the speed and ventilation. The composition of the fermentation medium was: carbon source (maltodextrin) 15g / L, composite nitrogen source (composed of yeast powder and soy peptone in a mass ratio of 1:1) 10g / L, (NH4)2SO4 5g / L, 3g / LKH2PO4, 5.5g / L K2HPO4. When the DO value of the medium began to rise, 50% glucose solution was added at a rate of 5-10g / L / h. The DO value was controlled at 30%±10% during the feeding period. The fermentation was stopped after 36h of fermentation.

[0168] Scheme 2, specific implementation method is the same as scheme 1, except that the composition of the complex nitrogen source in the fermentation medium is adjusted:

[0169] The composite nitrogen source consists of yeast powder and soy peptone in a mass ratio of 3:1.

[0170] Scheme 3, specific implementation method is the same as scheme 1, except that the composition of the complex nitrogen source in the fermentation medium is adjusted:

[0171] The composite nitrogen source is composed of yeast powder and soy peptone in a mass ratio of 1:3.

[0172] Scheme 4: The specific implementation method is the same as that of Scheme 1, except that the composition of the complex nitrogen source in the fermentation medium is adjusted:

[0173] The composite nitrogen source consists of yeast powder and trypsin in a mass ratio of 1:1.

[0174] Scheme 5: The specific implementation method is the same as that of Scheme 1, except that the composition of the complex nitrogen source in the fermentation medium is adjusted:

[0175] The composite nitrogen source consists of yeast powder and yeast peptone in a mass ratio of 1:1.

[0176] Scheme 6, the specific implementation method is the same as scheme 1, except that the composition of the complex nitrogen source in the fermentation medium is adjusted:

[0177] The composite nitrogen source is composed of yeast powder and acid hydrolyzed casein peptone in a mass ratio of 1:1.

[0178] Scheme 7, the specific implementation method is the same as scheme 1, except that the carbon source in the fermentation medium is adjusted:

[0179] The carbon source is 10 g / L maltodextrin.

[0180] Scheme 8: The specific implementation method is the same as that of Scheme 1, except that the carbon source in the fermentation medium is adjusted:

[0181] The carbon source is 15 g / L glucose.

[0182] Scheme 9: The specific implementation method is the same as scheme 1, except that the carbon source in the fermentation medium is adjusted:

[0183] The carbon source is 15 g / L arabinose.

[0184] Scheme 10, the specific implementation method is the same as scheme 1, except that the carbon source in the fermentation medium is adjusted:

[0185] The carbon source is 15 g / L sucrose.

[0186] After the fermentation, the fermentation liquid was diluted 200 times, and the OD value of the diluted fermentation liquid was measured using a spectrophotometer at a wavelength of 600 nm; at the same time, the fermentation liquid was diluted to 10 -10 , select 10 -8 , 10 -9 , 10 -10 The LB plates were coated with three gradients, and after coating, the plates were placed in a 30°C incubator for 24 h, and then counted to calculate the number of live bacteria.

[0187] The test results of schemes 1 to 10 are shown in Table 6:

[0188] Table 6 The number of viable bacteria of Bacillus siamese G12 fermented in different fermentation media

[0189]

[0190]

[0191] The results in Table 6 show that a large number of microorganisms can be obtained in a short time by fermenting Bacillus subtilis G12 using the fermentation medium described in Scheme 1 (15 g / L carbon source (maltodextrin), 10 g / L composite nitrogen source (composed of yeast powder and soy peptone in a mass ratio of 1:1), 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 5.5 g / LK2HPO4).

[0192] Example 8: Screening of freeze-drying protective agent formulation

[0193] The fermentation medium described in Scheme 1 of Example 2 was used to ferment Bacillus siamese G12 for 24 h. The fermentation broth was centrifuged at 7000 rpm for 25 min at 4 °C. The resulting fermentation sludge (viable count of 3.20 × 10 10A freeze-drying method for the fermentation broth of Bacillus siamese G12 is provided, comprising the following steps:

[0194] Solution 1:

[0195] Weigh 3 g of sucrose, a freeze-drying protective agent component, and add sterile water to dissolve and make up to 50 mL; then add the above 30 g of fermented bacterial sludge, resuspend and then freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 10:1.

[0196] Solution 2

[0197] Weigh 6 g of sucrose, a freeze-drying protective agent component, and add sterile water to dissolve and make up to 100 mL; then add the above-mentioned 30 g fermented bacterial sludge, resuspend and then freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 5:1.

[0198] Solution 3

[0199] Weigh 9 g of sucrose, a freeze-drying protective agent component, and add sterile water to dissolve and adjust the volume to 150 mL; then add the above 30 g of fermented bacterial sludge, resuspend and then freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 10:3.

[0200] Solution 4:

[0201] Weigh 3 g of trehalose, a freeze-drying protective agent, and add sterile water to dissolve and adjust the volume to 50 mL; then add the above 30 g of fermented bacterial sludge, resuspend and then freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 10:1.

[0202] Solution 5:

[0203] Weigh 6 g of trehalose, a freeze-drying protective agent, and add sterile water to dissolve and make up to 100 mL; then add the above 30 g of fermented bacterial sludge, resuspend and then freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 5:1.

[0204] Solution 6:

[0205] Weigh 9 g of trehalose, a freeze-drying protective agent, and add sterile water to dissolve and adjust the volume to 150 mL; then add the above 30 g of fermented bacterial mud, resuspend and freeze-dry, that is, the mass ratio of bacterial mud to protective agent is 10:3.

[0206] Solution 7:

[0207] Weigh 3 g of trehalose and 3 g of sucrose as freeze-drying protective agents, add sterile water to dissolve and make up to 100 mL; then add the above 30 g of fermented bacterial mud, resuspend and then freeze-dry, that is, the mass ratio of bacterial mud: trehalose: sucrose is 10:1:1.

[0208] Solution 8:

[0209] Weigh 3 g of trehalose and 3 g of glucose as freeze-drying protective agents, add sterile water to dissolve and make up to 100 mL; then add the above 30 g of fermented bacterial mud, resuspend and freeze-dry, that is, the mass ratio of bacterial mud: trehalose: glucose is 10:1:1.

[0210] Solution 9:

[0211] Weigh 3 g of trehalose and 3 g of lactose as freeze-drying protective agents, add sterile water to dissolve and make up to 100 mL; then add the above 30 g of fermented bacterial mud, resuspend and freeze-dry, that is, the mass ratio of bacterial mud: trehalose: lactose is 10:1:1.

[0212] Solution 10:

[0213] Weigh 9 g of glucose as a freeze-drying protective agent, add sterile water to dissolve and make up to 150 mL; then add the above 30 g of fermented bacterial sludge, resuspend and freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 10:3.

[0214] Solution 11:

[0215] Weigh 9 g of lactose, a freeze-drying protective agent, and add sterile water to dissolve and adjust the volume to 150 mL; then add the above 30 g of fermented bacterial sludge, resuspend and then freeze-dry, that is, the mass ratio of bacterial sludge to protective agent is 10:3.

[0216] Solution 12:

[0217] Weigh 9 g of galactose, a freeze-drying protective agent, and add sterile water to dissolve and adjust the volume to 150 mL; then add the above-mentioned 30 g of fermented bacterial mud, resuspend and then freeze-dry, that is, the mass ratio of bacterial mud to protective agent is 10:3.

[0218] After freeze-drying, the freeze-dried powder was collected and weighed. The bacterial solution before freeze-drying, the resuspended bacterial solution and the freeze-dried bacterial agent were taken for viable bacterial count by dilution plate method, and the freeze-drying survival rate was calculated.

[0219] Freeze-drying survival rate (%) = (number of live bacteria per unit freeze-dried powder after freeze-drying * mass of freeze-dried powder) / (number of live bacteria per unit of bacterial mud before freeze-drying * mass of bacterial mud) * 100%

[0220] The freeze-dried protective agent formula and the survival rate of the microorganisms after freeze-drying are shown in Table 7 below:

[0221] Table 7 Survival rate of lyophilized siam Bacillus G12 fermentation broth

[0222]

[0223]

[0224] According to the results shown in Table 7, the optimal freeze-drying protective agent component for producing Siamese Bacillus G12 inoculum in this embodiment is sucrose, and the optimal mass ratio of bacterial mud to protective agent is 10:1. When this formula is used as a freeze-drying protective agent, the number of viable bacteria in the freeze-dried Siamese Bacillus G12 inoculum can reach 1.00×10 11 CFU / g, and the freeze-dried survival rate was 96.00%.

[0225] Finally, although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacillus siamensis, characterized in that: The strain was deposited in the China General Microbiological Culture Collection Center on September 6, 2024, with the deposit number CGMCC No.31893.

2. A microbial agent, characterized in that: Containing the Siamese Bacillus CGMCC No.31893 of the present invention; Preferably, the microbial agent uses Bacillus siamese CGMCC No.31893 as the main microorganism.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

4. The microbial agent according to claim 2, characterized in that: The microbial agent contains living cells of the CGMCC No. 31893 strain of the present invention, dry cells of the CGMCC No. 31893 strain of the present invention obtained by freeze drying, immobilized cells of the CGMCC No. 31893 strain of the present invention, or the CGMCC No. 31893 strain of the present invention in any other form.

5. A method for inhibiting tobacco mold, reducing nicotine, reducing macromolecular compounds in tobacco or improving tobacco quality, characterized in that: The method uses the Siamese Bacillus CGMCC No. 31893 of claim 1 or a microbial agent containing CGMCC No. 31893 of claim 1 to ferment tobacco leaves to improve the quality of tobacco leaves; Optionally, the macromolecular compound in the tobacco leaf is protein, starch, pectin, lignin or cellulose.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) spraying a suspension of Bacillus siamese CGMCC No.31893 onto the surface of tobacco leaves and then fermenting; (2) drying the fermented tobacco leaves to obtain fermented tobacco leaves; Preferably, the bacterial suspension in step (1) is (1.0-1.8)×10 8 CFU / g sprayed onto the tobacco leaf surface; Preferably, the fermentation temperature is 30-37°C, the humidity is 60%-80%, and the fermentation time is 24-48h; preferably, the fermentation time is 24h; Preferably, the drying temperature in step (2) is 45-55° C.; and the moisture content of the fermented tobacco leaves is 1.0%-1.2%.

7. Use of the siam bacillus CGMCC No. 31893 of claim 1 or a microbial agent containing the CGMCC No. 31893 of claim 1 in the preparation of tobacco products.

8. The use according to claim 7, characterized in that: The tobacco products are tobacco spices, tobacco extracts, flue-cured tobacco leaves or cigar tobacco leaves.

9. A fermentation medium for the siam Bacillus CGMCC No.31893 of claim 1, characterized in that: The fermentation medium formula is: 5g / L-15g / L carbon source, 10g / L-20g / L composite nitrogen source, 2.5g / L-7.5g / L (NH4)2SO4, 3g / L-4g / L KH2PO4, 5g / L-6g / L K2HPO4; the fermentation medium solvent is water; Preferably, the fermentation medium formula is: carbon source 15g / L, complex nitrogen source 10g / L, (NH4)2SO4 5g / L, 3g / LKH2PO4, 5.5g / L KH2PO4; the fermentation medium solvent is water; Preferably, the carbon source is one or more of glucose, maltodextrin, arabinose, and sucrose; Preferably, the carbon source is maltodextrin; Preferably, the composite nitrogen source is a combination of two or more of yeast powder, tryptone, soy peptone, yeast peptone, and acid-hydrolyzed casein; Preferably, the composite nitrogen source consists of yeast powder and soy peptone in a weight ratio of 1:

1.

10. A method for drying the siam bacillus CGMCC No.31893 according to claim 1, characterized in that: The specific implementation steps include: adding water to the CGMCC No.31893 bacterial sludge, resuspending and centrifuging to obtain cleaned bacterial sludge; resuspending the bacterial sludge again and adding a lyophilization protective agent, freeze-drying, and obtaining lyophilized bacterial powder; Preferably, the lyoprotectant is one or more of sucrose, glucose, trehalose, lactose, and galactose; Preferably, the lyoprotectant is sucrose; Preferably, the mass ratio of the bacterial mud to the freeze-drying protective agent is 10:(1-4); Preferably, the mass ratio is 10:1.

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