Bacillus vallismortis S7 and application of bacillus vallismortis S7 in improvement of tobacco leaf quality

Abstract

The invention discloses bacillus vallismortis S7 and application thereof to improvement of tobacco leaf quality, and belongs to the technical field of bioengineering. The strain is preserved in the China General Microbiological Culture Collection Center on September 6, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31894. The strain can produce cellulase at high yield, can inhibit tobacco mould, has the capability of reducing nicotine, and can degrade starch, protein, pectin, cellulose and other macromolecular compounds in tobacco. After the strain or the prepared microbial agent is applied to tobacco leaves, the quality of the tobacco leaves can be improved, and the use value of the tobacco leaves is improved.

Description

Technical Field

[0001] The invention relates to a Death Valley Bacillus S7 and application thereof in improving tobacco leaf quality, belonging to the technical field of bioengineering. Background Art

[0002] In the production of cigarette industry, tobacco leaves from different regions have their own unique characteristics. Cigarette factories purchase tobacco leaves from farmers in various regions according to demand. However, on the one hand, the quality of the purchased tobacco leaves varies, and on the other hand, some tobacco leaves may be abandoned due to long inventory years, which affects the availability of some tobacco leaves in cigarette formulas. In order to deal with this type of tobacco leaves, tobacco companies often need to spend a lot of money. The storage of tobacco leaves in warehouses takes up manpower and material resources, which seriously affects the benefits of enterprises.

[0003] At present, this type of tobacco with little aroma, poor aroma quality, strong impurities and strong irritation is collectively referred to as unsuitable or difficult-to-use tobacco. The production of this type of tobacco is mainly caused by the structural contradiction between tobacco agricultural planting and cigarette industrial production. Industrial enterprises currently have a large backlog of this type of tobacco. In addition, tobacco leaves are easily infected by mold during storage, which not only causes the tobacco leaves to deteriorate and reduce their quality, but may also produce mycotoxins that are harmful to the human body. Traditional prevention and control methods, such as the use of chemical fungicides, can inhibit the growth of mold, but may leave residual harmful substances and affect the safety of tobacco. Therefore, how to effectively use this type of unsuitable or difficult-to-use tobacco leaves and solve the storage problem of tobacco leaves has become a major problem that the cigarette industry needs to solve urgently.

[0004] Macromolecular compounds such as protein, starch, pectin, cellulose and lignin have an important impact on the quality of tobacco leaves by affecting the sensory quality, combustibility and health safety of tobacco leaves. Through the growth and metabolism of related microorganisms, the content of macromolecular compounds such as protein, pectin, cellulose and lignin in tobacco leaves can be effectively reduced, and the production of harmful substances can be reduced. It can also increase the content of small molecular substances in tobacco leaves, increase the content of aroma components, improve the sensory quality of tobacco leaves, and promote the aging process of tobacco leaves, thereby having a positive impact on the intrinsic quality of tobacco leaves. On the other hand, nicotine is an important component in tobacco, but its excessive content will increase the irritation of smoke and affect the taste of cigarettes. At the same time, from the perspective of safety, high nicotine content is also an unfavorable factor. With the increasing awareness of consumers about their own health and environmental protection, reducing the nicotine content in tobacco has become an important direction for the development of the tobacco industry.

[0005] In order to increase the proportion of unsuitable or difficult-to-use tobacco leaves in cigarettes, it is necessary to improve the smoking quality of these tobacco leaves. Therefore, using microbial fermentation to ferment tobacco leaves can effectively improve the smoking quality of tobacco leaves, which is of great value and significance for solving the problems of unsuitable or difficult-to-use tobacco leaves, improving the intrinsic quality of tobacco leaves, and storing tobacco leaves. Summary of the invention

[0006] The purpose of the present invention is to provide a Death Valley Bacillus and an application thereof. The Death Valley Bacillus can inhibit mold in leaves, reduce nicotine content, degrade the content of macromolecular compounds such as protein, cellulose, lignin, pectin, etc. in tobacco leaves, and improve the sensory quality of tobacco leaves. When the strain is used to ferment tobacco leaves, the fermentation time is short, the efficiency of tobacco production can be effectively improved, the production cost can be reduced, and the quality of tobacco can be improved.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The first object of the present invention is to provide a strain of Bacillus vallismortis S7, which has been deposited in the China General Microbiological Culture Collection Center on September 6, 2024, with a deposit number of CGMCC No.31894.

[0009] In one embodiment of the present invention, the Death Valley Bacillus comes from C2L-AB tobacco leaves from Longnan, Gansu Province in 2022, is identified as Death Valley Bacillus (Bacillus vallismortis), and is named Death Valley Bacillus S7.

[0010] In one embodiment of the present invention, Bacillus death valley S7 has the following biological characteristics:

[0011] (1) Bacterial characteristics: Gram-positive bacteria, with a strain size of 0.2-0.5 μm × 1.2-3.5 μm, thin rod-shaped, and without flagella;

[0012] (2) Colony characteristics: The colonies are large, with rough surfaces with protrusions, flat, and irregular edges;

[0013] (3) Growth characteristics: The growth temperature range of the Death Valley Bacillus S7 strain is 28-37°C, it can tolerate 0-12.5% ​​NaCl, and the growth pH range is 5.0-9.0;

[0014] (4) It is a facultative aerobe and grows well under both aerobic and anaerobic conditions;

[0015] (5) It can produce high amounts of cellulase and has the ability to reduce protein, starch, pectin, lignin, and cellulose in tobacco leaves;

[0016] (6) Can inhibit tobacco leaf mold;

[0017] (7) It has the ability to reduce nicotine, with a degradation rate of 69.5% for 1g / L nicotine concentration;

[0018] (8) Improve the smoking quality of tobacco leaves.

[0019] In one embodiment of the present invention, the nucleotide sequence of 16S rDNA of Bacillus dysvalleyi S7 is shown as SEQ ID NO.3.

[0020] The second object of the present invention is to provide a microbial agent containing the Death Valley Bacillus CGMCC No.31894 of the present invention.

[0021] In one embodiment of the present invention, the microbial agent uses Death Valley Bacillus CGMCC No.31894 as the main microorganism.

[0022] In one embodiment of the present invention, the number of cells of Bacillus death valley CGMCC No.31894 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 prepared by preparing the seed solution of Death Valley Bacillus CGMCC No.31894 and then expanding the culture.

[0024] In one embodiment of the present invention, the microbial agent contains living cells of the CGMCC No. 31894 strain of the present invention, dry cells of the CGMCC No. 31894 strain of the present invention obtained by freeze-drying, immobilized cells of the CGMCC No. 31894 strain of the present invention, a liquid agent of the CGMCC No. 31894 strain of the present invention, a solid agent of the CGMCC No. 31894 strain of the present invention, or the CGMCC No. 31894 strain of the present invention in any other form.

[0025] In one embodiment of the present invention, the microbial agent is prepared by the following steps:

[0026] (1) inoculating Death Valley Bacillus CGMCC No.31894 into LB liquid culture medium to obtain seed liquid after culturing;

[0027] (2) inoculating the seed liquid into LB liquid culture medium to obtain a fermentation liquid;

[0028] (3) The fermentation liquid is centrifuged, the supernatant is discarded, and then water is added to suspend the liquid, and the number of viable bacteria is adjusted to obtain a microbial agent containing Death Valley Bacillus CGMCC No. 31894.

[0029] In one embodiment of the present invention, the culture conditions in step (1) are 25-35° C., 100-250 r / min; and the culture time is 10-16 h.

[0030] In one embodiment of the present invention, the culture conditions in step (2) are 25-35° C. and 100-250 r / min; and the OD600 of the fermentation liquid is 0.8-3.

[0031] In one embodiment of the present invention, the centrifugation condition in step (3) is 3-7°C, 5000-7000 rpm, and 20-30 min.

[0032] The third object of the present invention is to provide a method for inhibiting tobacco mold, reducing nicotine, reducing the content of macromolecular compounds in tobacco or improving the quality of tobacco, wherein the method utilizes death valley bacillus CGMCC No.31894 or a microbial inoculant containing CGMCC No.31894 to ferment and improve the quality of tobacco.

[0033] In one embodiment of the present invention, the method comprises the following steps:

[0034] (1) spraying a suspension of Bacillus death valley CGMCC No.31894 onto the surface of tobacco leaves and then fermenting;

[0035] (2) Drying the fermented tobacco leaves to obtain fermented tobacco leaves.

[0036] In one embodiment of the present invention, in step (1), the bacterial suspension is (1.2-1.8)×10 8 CFU / g sprayed onto the tobacco leaf surface;

[0037] Preferably, the bacterial suspension is prepared at 1.2×10 8 CFU / g sprayed onto the surface of tobacco leaves.

[0038] In one embodiment of the present invention, the fermentation temperature in step (1) is 25-35° C., the humidity is 65%-75%, and the fermentation time is 16-50 h;

[0039] Preferably, the fermentation time is 24 hours.

[0040] 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%;

[0041] Preferably, the drying temperature is 50° C. and the water content is 1.2%.

[0042] In one embodiment of the present invention, the tobacco leaf macromolecular compound is protein, starch, pectin, lignin or cellulose.

[0043] The fourth object of the present invention is to provide the use of the death valley bacillus CGMCC No. 31894 or a microbial agent containing the CGMCC No. 31894 in the preparation of tobacco products.

[0044] In one embodiment of the present invention, the tobacco product is tobacco flavoring, tobacco extract, flue-cured tobacco leaves or cigar tobacco leaves.

[0045] Biomaterial Deposit

[0046] Bacillus vallismortis S7, taxonomic name: Bacillus vallismortis, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 6, 2024, with the deposit number CGMCC No.31894, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0047] Beneficial effects:

[0048] (1) The Death Valley Bacillus S7 screened and preserved by the present invention can produce high-yield cellulase and has the ability to degrade macromolecular substances such as protein, starch, pectin, lignin, cellulose, etc. in tobacco leaves. Specifically, the protein degradation rate is 22.94%, the starch degradation rate is 29.09%, and the cellulose degradation rate can reach 46.14%;

[0049] (2) The Death Valley Bacillus S7 provided by the present invention can inhibit tobacco leaf mold, and the inhibition rate can reach 56.25%;

[0050] (3) The Death Valley Bacillus S7 provided by the present invention has the ability to reduce nicotine, with a degradation rate of 69.5% for 1 g / L nicotine concentration, and can grow at a nicotine concentration of up to 5 g / L, with a degradation rate of 20.28% for 5 g / L nicotine concentration;

[0051] (4) The aroma quality, aroma volume, permeability and salivation sensation of tobacco leaves fermented by the strain of the present invention are improved, and the foreign smell and irritation are reduced;

[0052] (5) When the Death Valley Bacillus S7 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.

[0053] In summary, the application of this strain to tobacco leaves can improve their quality and enhance the use value of tobacco leaves. So far, no precedents have been found for the function and application of this microorganism in this regard. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] 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.

[0055] Figure 1 This is a plate map of Bacillus Death Valley;

[0056] Figure 2 This is an oil-immersion image of Bacillus of Death Valley;

[0057] Figure 3 This is the growth curve of Death Valley Bacillus. DETAILED DESCRIPTION

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 1. The culture medium involved in the following embodiments is as follows:

[0063] LB liquid medium: tryptone 10g / L, yeast powder 10g / L, sodium chloride 5g / L, sterilized at 121℃ for 20min

[0064] LB solid culture 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.

[0065] 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.

[0066] 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).

[0067] 2. The detection methods involved in the following embodiments are as follows:

[0068] The protein detection method was the BCA kit method (Suzhou Gres Biotechnology Co., Ltd., G0418W);

[0069] The starch detection method was the enzymatic kit method (Suzhou Grace Biotechnology Co., Ltd., G0551W);

[0070] The pectin detection method was the carbazole colorimetric kit method (Suzhou Gres Biotechnology Co., Ltd., G0717W);

[0071] The lignin detection method was the acetylation kit method (Suzhou Gres Biotechnology Co., Ltd., G0708W);

[0072] The cellulose detection method was anthrone kit method (Suzhou Gres Biotechnology Co., Ltd., G0715W).

[0073] 3. The sources of materials involved in the following embodiments are as follows:

[0074] C2L-AB tobacco leaves were sourced from Gansu Tobacco Industry Co., Ltd.

[0075] Example 1: Screening and identification of Death Valley Bacillus

[0076] The dominant strains were selected from the 2022 C2L-AB tobacco leaves in Longnan, Gansu. The specific operations are as follows:

[0077] 1. Isolation and screening of Death Valley Bacillus:

[0078] 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.

[0079] 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.

[0080] 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 8 different colonies were numbered, named S1 to S8, and a single colony was picked and inoculated into LB liquid culture medium for expansion and preservation.

[0081] 2. Molecular biological identification of Death Valley Bacillus:

[0082] 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 S7 strain was determined to be Bacillus vallismortis, and the strain was stored in a refrigerator at -80℃ for later use.

[0083] SEQ ID NO.3:

[0084] TACGGCTTACCCTTGTACGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAG

[0085] The Death Valley Bacillus S7 was deposited in the China General Microbiological Culture Collection Center on September 6, 2024, with the deposit number CGMCC No.31894.

[0086] 3. Morphological characteristics and growth characteristics:

[0087] The isolated Death Valley Bacillus S7 strain was streaked on LB solid medium at 30°C and its 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 0.2-0.5 μm × 1.2-3.5 μm, a thin rod shape, and no flagella.

[0088] The death valley Bacillus S7 slant-preserved strain was inoculated into LB liquid medium and cultured in a shaker at 30°C and 200 rpm for 12 h as seed liquid. The seed liquid was inoculated into LB 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.

[0089] The growth temperature range of Death Valley Bacillus S7 strain is 28-37°C, it can tolerate 0-12.5% ​​NaCl, and the growth pH range is 5.0-9.0; it is a facultative aerobe and grows well under aerobic and anaerobic conditions.

[0090] Example 2: Enzyme production capacity of Death Valley Bacillus S7

[0091] The death valley bacillus S7 was inoculated into LB liquid medium at an inoculation 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 production of amylase, protease, pectinase, and cellulase was determined by the following methods, respectively. The specific methods are as follows:

[0092] 1. Determination of amylase content: Use the method in GB / T 24401-2009.

[0093] 2. The protease content was determined using the method in GB / T 33410-2016.

[0094] 3. The pectinase content was determined using the method in QB / T 4482-2013.

[0095] 4. Determination of cellulase content: The method in GB 7300.403-2022 was used for determination.

[0096] The strains S1-S8 isolated in Example 1 were cultured in shake flasks in the same manner as above, and the yields of amylase, protease, pectinase and cellulase in the supernatant of the fermentation broth were determined. The results are shown in Table 1.

[0097] Table 1 Detection results of amylase, protease, pectinase and cellulase in the supernatant of the fermentation broth of the strain shake flask

[0098]

[0099] According to the results in the above table, Bacillus S1, Death Valley Bacillus S7 and Bacillus subtilis S8 have strong amylase, protease, pectinase and cellulase activities, among which the cellulase activity of Death Valley Bacillus S7 is more prominent.

[0100] Example 3: Ability of strains to inhibit tobacco mold

[0101] The tobacco molds involved in this embodiment are as follows:

[0102] The relatively high content of isolated C2L-AB tobacco leaves produced in Longnan, Gansu in 2022 was found to be Rhizopus arrhizus after molecular identification; the relatively high content of isolated C3F-AB tobacco leaves produced in Xuanwei, Yunnan in 2022 was found to be Aspergillus niger after molecular identification; the relatively high content of isolated C3F-AB tobacco leaves produced in Baoji, Shaanxi in 2022 was found to be Aspergillus oryzae after molecular identification; the above three fungi are all preserved in the laboratory of Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.

[0103] Bacillus S1, Bacillus death valley S7 and Bacillus subtilis S8 were inoculated on PDA solid culture medium, respectively. Three 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, Rhizopus arrhizus, Aspergillus niger and Aspergillus oryzae in tobacco leaves were inoculated at the center of the plate culture medium, respectively. The plate inoculated with only 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.

[0104] Calculation formula: Inhibition rate (%) = (control colony radius - treatment colony radius) / control colony radius * 100%

[0105] The results are shown in Table 2.

[0106] Table 2 Results of the inhibition rate of the strains on mold

[0107]

[0108] According to the results in the table, strains S1, S7, and S8 all have a certain inhibitory effect on the molds isolated from tobacco leaves, and strain S7 has a very significant inhibitory effect on the three molds, with inhibition rates of 56.25%, 65.71%, and 71.40% on Rhizopus arrhizus, Aspergillus niger, and Aspergillus oryzae, respectively, indicating that Death Valley Bacillus S7 is beneficial for application in the tobacco fermentation process.

[0109] Example 4: Ability of strains to reduce nicotine

[0110] Bacillus S1, Bacillus death valley S7 and Bacillus subtilis S8 glycerol tube preserved strains were inoculated with 1% inoculum into nicotine liquid medium shake flasks 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 at different nicotine concentrations was determined at 20 hours of culture, and 1% volume of sterile water was inoculated at different concentrations as a control group.

[0111] The detection method of nicotine degradation rate is as follows:

[0112] 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:

[0113] Degradation rate % = (control group OD 259 - Experimental group OD 259 ) / control group OD 259 ×100%

[0114] The results are shown in Table 3.

[0115] Table 3 Degradation rate results of strains at different nicotine concentrations

[0116]

[0117] According to the results in the above table, the degradation rate of nicotine at a concentration of 1 g / L of Death Valley Bacillus S7 was 69.5% after being cultured in nicotine liquid culture medium for 20 hours. The degradation rate of nicotine by this strain was significantly better than that of Bacillus S1 and Bacillus subtilis S8.

[0118] Example 5: Degradation of macromolecular compounds in redried tobacco leaves by strains

[0119] The shake flask fermentation broth was obtained by culturing according to the method described in Example 2. The fermentation broth was centrifuged at 7000 rpm for 25 min at 4°C to collect the fermented bacterial sludge, add sterile saline to fully resuspend the bacterial sludge, and dilute with sterile saline to make the biomass in the bacterial suspension 6.0×10 8CFU / 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% humidity for 24 hours. At the same time, spray 1 mL of sterile saline as a control group.

[0120] Weigh an appropriate amount of the tobacco leaves fermented for 24 hours and crush them into tobacco powder using a multifunctional crusher. Pass the powder through a 60-mesh sieve. Dry the sieved tobacco powder in an oven at 60°C for 3 hours, and then test the protein, starch, pectin, lignin and cellulose contents.

[0121] The results of the detection of the content of macromolecular compounds in fermented tobacco leaves are shown in Table 4.

[0122] Table 4 Degradation results of strains on macromolecular compounds in tobacco leaves

[0123]

[0124] According to the results in the table, the contents of five macromolecular compounds, protein, starch, pectin, lignin, and cellulose, were all reduced in tobacco leaves fermented with Death Valley Bacillus S7, among which the reduction in the contents of protein, starch, and cellulose was particularly significant. Even though the protease, amylase, pectin, and cellulase production capabilities of Bacillus S1 and Bacillus subtilis S8 strains in Example 2 were similar to those of Death Valley Bacillus S7, Death Valley Bacillus S7 was significantly better than other screened strains in the application of degrading tobacco leaf macromolecular compounds, which may be related to the strain's strong ability to metabolize and tolerate nicotine.

[0125] The use of the Death Valley Bacillus S7 to ferment tobacco shreds can effectively promote the degradation of macromolecular substances in tobacco to form various small molecular substances that are beneficial to improving tobacco quality, thereby improving the quality of tobacco leaves.

[0126] Example 6: Sensory evaluation of tobacco leaves fermented with Death Valley Bacillus S7

[0127] The smoking quality of tobacco leaves fermented with the Death Valley Bacillus of the present invention is evaluated by the following specific method:

[0128] The strain 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, resuspended in sterile saline, and diluted with sterile saline to make the biomass in the bacterial suspension 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 8CFU / g, placed in a constant temperature and humidity chamber at 30°C and 70% humidity for 24h (experimental group 1) or 48h (experimental group 2). At the same time, sprayed with 1mL of sterile saline as a control group.

[0129] 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.

[0130] The results of the suction test are shown in Table 5.

[0131] Table 5 Smoking evaluation of tobacco leaves in the experimental group and the control group

[0132]

[0133] The results showed that after the redried tobacco leaves were fermented with Bacillus vallismortis for 24 hours and 48 hours, the sensory evaluation results showed that the aroma quality and aroma quantity of the tobacco leaves were significantly improved, and the impurities and irritation were significantly reduced; in addition, the permeability and concentration of the fermented tobacco leaves were also slightly improved, and the satisfaction and salivation during smoking were also significantly increased. There was no significant difference in the results after 24 hours and 48 hours of fermentation, so the ideal effect can be achieved in just 24 hours.

[0134] 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 vallismortis, characterized in that: It was deposited in the China General Microbiological Culture Collection Center on September 6, 2024, with the deposit number CGMCC No.31894.

2. A microbial agent, characterized in that: Contains the Death Valley Bacillus CGMCC No.31894 of the present invention.

3. The microbial agent according to claim 2, characterized in that: The number of cells of Bacillus death valley CGMCC No. 31894 in the bacterial agent is not less than 3.0×10 8 CFU / mL.

4. The microbial agent according to claim 2, characterized in that: The microbial agent contains living cells of the CGMCC No. 31894 strain of the present invention, dry cells of the CGMCC No. 31894 strain of the present invention obtained by freeze drying, immobilized cells of the CGMCC No. 31894 strain of the present invention, a liquid agent of the CGMCC No. 31894 strain of the present invention, a solid agent of the CGMCC No. 31894 strain of the present invention, or the CGMCC No. 31894 strain of the present invention in any other form.

5. A method for inhibiting tobacco leaf mold, reducing nicotine, reducing the content of macromolecular compounds in tobacco leaves, or improving the quality of tobacco leaves, characterized in that: The method comprises fermenting tobacco leaves using the death valley bacillus CGMCC No. 31894 or a microbial agent containing the CGMCC No. 31894 as claimed in claim 1.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) Spraying CGMCC No.31894 bacterial suspension onto the surface of tobacco leaves and then fermenting; (2) Drying the fermented tobacco leaves to obtain fermented tobacco leaves.

7. The method according to claim 6, characterized in that The bacterial suspension in step (1) is prepared according to (1.2-1.8)×10 8 CFU / g tobacco leaf is sprayed onto the surface of the tobacco leaf; and the fermentation time is 16 to 50 hours.

8. The method according to claim 6, characterized in that The drying temperature in step (2) is 45-55° C.; the moisture content of the fermented tobacco leaves is 1.0%-1.2%.

9. The method according to claim 5, characterized in that The tobacco leaf macromolecular compound is protein, starch, pectin, lignin or cellulose.

10. Use of the death valley bacillus CGMCC No. 31894 of claim 1 or a microbial agent containing CGMCC No. 31894 of claim 1 in the preparation of tobacco products.

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