Bacillus velezensis and application thereof

Abstract

The invention relates to bacillus velezensis and application thereof, and belongs to the technical field of microorganisms. According to the invention, a strain TB24016 capable of efficiently antagonizing various moulds is separated from cigar tobacco leaves, and through morphological and molecular biological identification, the strain is bacillus velezensis, has an antagonistic effect on aspergillus mongolicus, aspergillus cristatus, conehead mononuclear, gliocladium brachypodium and aspergillus shermangii which are relatively strong in mildewing ability and relatively heavy in harmfulness, and can be used for efficiently antagonizing various moulds, so that the strain TB24016 can be used for efficiently antagonizing various moulds. The strain is preserved in the China Center for Type Culture Collection on October 14, 2024, and the preservation number is CCTCC NO: M 20242186. The invention provides a strain capable of efficiently antagonizing various moulds with strong mildew-causing ability, and a new microbial resource is provided for preventing and treating the moulds of tobacco products.

Description

Technical Field

[0001] The invention relates to Bacillus Velez and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Tobacco belongs to the genus Nicotiana of the Solanaceae family. It is an annual or limited perennial herb, with glandular hairs all over, strong roots, stems 0.7-2 meters high, slightly lignified at the base, and oblong-lanceolate, lanceolate, oblong or ovate leaves, gradually pointed at the top, gradually narrowed at the base to the stem into an ear-shaped and semi-amplexicaulous stem, 10-30 (-70) cm long, 8-15 (-30) cm wide, with an inconspicuous or wing-shaped petiole. After the tobacco leaves mature, they are picked and fermented. Through the action of catalytic factors, the basic substances in the original tobacco leaves undergo a series of biochemical changes, making the chemical composition in the tobacco leaves more coordinated. However, due to the long fermentation and aging cycle of tobacco leaves, they are easily disturbed by the external environment (temperature and humidity), and tobacco leaves are rich in nutrients such as organic acids and sugars. Once stored in suitable environmental conditions (temperature and humidity, etc.), tobacco leaves are more susceptible to microbial (mainly mold) infection and cause economic losses. Fungal infection not only affects the appearance and sensory quality of tobacco, causing serious economic losses, but may also produce mycotoxins, which directly affect the quality and safety of tobacco products. Therefore, the control of tobacco leaf mildew is an important research direction during the storage period of tobacco leaves. Solving the problem of tobacco leaf mildew and preventing and controlling mildew are of great importance to the tobacco industry.

[0003] Cigar mildew is mainly caused by mold, but the types of mold in different tobacco producing areas show obvious differences due to the influence of geographical environment and climatic conditions. Zhang Daiyuan et al. found that the main mold-causing fungi in Hubei production area are Penicillium chrysogenum, Aspergillus polydorum, Trichoderma yunnanensis and Talaromyces fusca; Bai Xinyi et al. found that the main mold-causing fungi in Hunan production area are Penicillium citrinum and Penicillium isletum; Song Jiabao et al. found that the main mold-causing fungi in Hainan production area are Talaromyces fusca, Scopulariopsis short-handled and Aspergillus.

[0004] At present, the factory's anti-mildew measures include adjusting the environmental humidity, vacuum rehumidification, hot air moisturizing leaves, alcohol wiping, and drying sheets to treat moldy tobacco leaves, but all of these may affect the quality or processing performance of tobacco leaves, and may also kill the beneficial microorganisms in the tobacco leaves. A wide range of mold prevention and control methods include physical, chemical, and biological methods. Among them, physical methods mainly include low temperature, heating, ventilation, drying, vacuum, irradiation, and adsorption. Radiation control of mold and toxins has been proven to be effective (both x-rays and gamma rays can be used), and ionizing radiation can also play a certain inhibitory role; however, this method may affect the quality of tobacco leaves, and there are problems such as high energy consumption and high cost, so it has not been promoted. The chemical method uses different chemical synthetic preservatives, including acids, alkalis, oxidants, chlorinating agents and other inhibitors. Usually, zinc salicylate and zinc benzoate, sodium silicate, sodium sulfate, sodium carbonate, sodium bicarbonate, iron chelate, iron sulfate, ammonium bicarbonate, methyl paraben, ethyl paraben, barium metaborate, zinc oxide, etc. are used to inhibit the growth of fungi; chemical fungicides have good effects, but may cause a significant reduction in tobacco aroma quality or produce toxic side effects. Considering the safety issues of physical and chemical methods, it is necessary to find and develop safer biological agents to control mold contamination. The biological method mainly uses microorganisms or microbial metabolites to inhibit or kill mold, thereby preventing the occurrence of tobacco leaf mildew. Due to its safety, environmental protection, high efficiency and pollution-free advantages, it has broad application prospects.

[0005] However, there are few reports on biocontrol bacteria for effective prevention and control of tobacco mildew, and the control effect is poor. Therefore, it is urgent to find or develop efficient and safe biological agents to provide important reference for ensuring the quality and safety of tobacco leaves and the sustainable development of tobacco in my country. Summary of the invention

[0006] The first object of the present invention is to provide a Bacillus Velezii that can effectively antagonize multiple fungi.

[0007] The second object of the present invention is to provide an application of Bacillus Velezii in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products, and to provide a method for preventing mildew of tobacco products with good control effect and high biological safety.

[0008] In order to achieve the above object, a technical solution of Bacillus Velezii in the present invention is:

[0009] A Bacillus Velez subtilis, wherein the deposit number of the Bacillus Velez subtilis is CCTCC NO: M 20242186.

[0010] The beneficial effect of the above technical scheme is that the Bacillus velezensis of the present invention is a pioneering invention. The present invention isolates a strain that can effectively antagonize molds with strong mildew-causing ability from cigar tobacco leaves, named TB24016. After morphological and molecular biological identification, the bacterium is Bacillus velezensis, which has antagonistic effects on Aspergillus montenegro, Aspergillus cristatus, Aspergillus unicellularis, Scopulariopsis short-handled, and Aspergillus shevarium with strong mildew-causing ability.

[0011] In order to achieve the above-mentioned object, the technical scheme of using Bacillus Velezii in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products in the present invention is:

[0012] The invention discloses an application of Bacillus Velezii in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products.

[0013] The beneficial effect of the above technical scheme is that: the present invention isolates a strain of Bacillus Velezii TB24016 from cigar tobacco leaves, and uses the Oxford cup method to analyze the antibacterial ability of Bacillus Velezii TB24016, and finds that the fungus can inhibit the growth of Aspergillus montenegro, Aspergillus crown, single cell co-head mold, short handle Scopulariopsis, and Aspergillus shevatus with strong mildew ability. Further, by inoculating it on cigar tobacco leaves for fermentation, it is found that it can inhibit the mildew effect of the above fungi on cigar tobacco leaves. The present invention provides a Bacillus Velezii that can effectively antagonize a variety of molds with strong mildew-causing ability, provides a new microbial resource for the mildew control of tobacco products, and provides relatively specific microbial resources for mildew of tobacco products in different production areas, which not only enriches the microbial resource library for preventing and controlling tobacco mildew, but also lays a foundation for improving the specificity of preventing and controlling tobacco mildew in different production areas.

[0014] As a further improvement, the fungus is Aspergillus monteridium, Aspergillus cristatus, Aspergillus unicellularis, Scopulariopsis brevicaulis and / or Aspergillus shevarium.

[0015] As a further improvement, the bacterial liquid of Bacillus Velez is sprayed into tobacco products.

[0016] As a further improvement, when the fungus is Aspergillus monteri, Aspergillus cristatus, Scopulariopsis brevis and / or Aspergillus shevarium, the concentration of the bacterial solution is not less than 10 8 CFU / mL.

[0017] As a further improvement, when the fungus is a single cell symphysema, the concentration of the bacterial solution is not less than 10 7 CFU / mL.

[0018] As a further improvement, the tobacco products include cigar tobacco leaves, cigarettes and tobacco sheets. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology diagram of strain TB24016 in Example 2 of the present invention on solid LB medium;

[0020] Figure 2 This is a microscopic morphology of strain TB24016 in Example 2 of the present invention;

[0021] Figure 3 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence of strain TB24016 in Example 2 of the present invention;

[0022] Figure 4 This is a diagram showing the anti-mildew effect of Bacillus Velezii TB24016 in cigar tobacco leaves in Example 4 of the present invention. DETAILED DESCRIPTION

[0023] Existing factory mildew prevention technology may have an impact on tobacco leaf quality or processing performance, and may kill the beneficial microorganisms in tobacco leaves at the same time, which will affect the quality of tobacco leaves, and there are problems such as large energy consumption, high cost, significantly reduced nutritional value, and toxic and side effects, so it has not been promoted. And biological control methods have the advantages of full environmental protection, high efficiency and pollution-free, and are increasingly valued. However, there are fewer strains of mildew-proof tobacco products that have been disclosed, and the mildew-proof ability of the mildew-proof strains that have been reported still needs to be strengthened. The present invention has found through a large number of previous studies that the main molds that cause cigar tobacco leaves in Sichuan production areas to mildew are Aspergillus monteri, Aspergillus crown, single cell co-head mold, short-handled Scopulariopsis, and Aspergillus sheva, and some of them are not common in other production areas, and the mildew-causing ability of these molds is strong. Based on this, the present invention provides a kind of Bacillus velez.

[0024] The present invention isolates a strain that can efficiently antagonize multiple fungi from cigar tobacco leaves. After physiological, biochemical and molecular biological identification, the bacterium is Bacillus velezensis, named TB24016, and the bacterium has antagonistic effects on Aspergillus montenegro, Aspergillus crown, single cell co-head mold, short handle Scopulariopsis, and Aspergillus shevatus with strong mildew ability. And the strain that can efficiently antagonize multiple fungi screened by the present invention is Bacillus velezensis, which is a gram-positive bacterium of the genus Bacillus Cohn of the family Bacillaceae, widely distributed in nature, and is derived from the biocontrol bacteria in tobacco field soil and tobacco leaves, and has the characteristics of being environmentally friendly and not easy to produce drug resistance, and plays an important role in protecting tobacco quality and ecological environment safety.

[0025] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0026] The culture medium formula and preparation process used in the embodiments of the present invention are as follows:

[0027] Potato glucose agar medium: prepare according to the ratio of 300g / L potato, 20g / L glucose, 15g / L agar, and 0.1g / L chloramphenicol, and finally place it in an autoclave for sterilization at 121℃ for 15min.

[0028] Sabouraud medium: Prepare according to the ratio of 10g / L tryptone, 20g / L agar, and 40g / L glucose, and finally place it in an autoclave for sterilization at 115℃ for 20min.

[0029] LB liquid culture medium: Prepare according to the ratio of 10 g / L tryptone, 5 g / L yeast powder, 5 g / L sodium chloride, and 1 g / L glucose, and finally place it in a high pressure sterilizer at 121°C for 15 min.

[0030] LB agar medium: prepare according to the proportion of 10 g / L tryptone, 5 g / L yeast powder, 5 g / L sodium chloride, 1 g / L glucose, and 15 g / L agar, and finally place it in a high pressure sterilizer at 121°C for 15 min.

[0031] Biomaterials used in the embodiments of the present invention:

[0032] Strains: Aspergillus monteri, Aspergillus cristatus, Aspergillus unicellularis, Aspergillus short-handled and Aspergillus shevarium were isolated from the surface of cigar tobacco leaves produced in Sichuan.

[0033] 1. A specific embodiment of a Bacillus Velezii of the present invention:

[0034] The Bacillus velezensis of the present invention is Bacillus velezensis TB24016, with a deposit number of CCTCC NO: M 20242186; the deposit date is October 14, 2024; the deposit unit is China Center for Type Culture Collection; the deposit address is China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The specific implementation operation is as follows:

[0035] Example 1 Isolation and screening of strain TB24016

[0036] In this example, a strain TB24016 capable of antagonizing multiple fungi was isolated from cigar tobacco leaves. The specific implementation operation is as follows:

[0037] Take 3g of cigar tobacco leaves from different production areas, add them into a sterilized test tube filled with 27mL of sterile water, seal it with a sterile sealing film, shake it for 30min, and culture it in a shaker at 180rpm and 37℃ for 2h. Then take 150μL of it into 5mL of LB liquid culture medium and culture it in a shaker at 180rpm and 37℃ for 12h. -2 ~10 -5 10 -2 , 10 -3 , 10 -4 and 10 -5 The gradient dilutions were spread on LB medium plates and cultured at 37°C for 12 hours. Single colonies on each plate were picked and purified to obtain further purified single colonies, and the obtained strain single colonies were numbered for further study. The Oxford cup method was used to explore the antagonistic ability of the numbered strains to different fungi (Aspergillus montenegro, Aspergillus crown, single cell co-head mold, short-handled Scopulariopsis, Aspergillus shevastatus), and the diameter of the inhibition zone appearing around the Oxford cup was measured, and strains with large diameters and broad-spectrum antifungal ability were selected. That is, the strain TB24016 that can effectively antagonize Aspergillus montenegro, Aspergillus crown, single cell co-head mold, short-handled Scopulariopsis, and Aspergillus shevastatus was obtained.

[0038] Example 2 Identification of strain TB24016

[0039] In this example, the strain TB24016 isolated and obtained in Example 1 was identified by morphology and molecular biology as Bacillus Velezii. The specific implementation operation is as follows:

[0040] 1. Morphological identification

[0041] The colony diameter of strain TB24016 cultured in LB medium is 1.57-6.3 mm, round, white, with concave lens protrusions, dry and wrinkled surface and complete edges, spreading; the bacteria are short rod-shaped, about 5.33-12.47 μm long, with spores, and Gram-positive bacteria. Figure 1 and Figure 2 The optimum growth temperature is 30-37℃ and the suitable pH is 6.8-7.2.

[0042] 2. Molecular Biology Identification

[0043] The strain TB24016 was classified and identified by 16S rDNA gene sequencing. The primers for PCR amplification of the 16S rDNA gene were 27F and 1492R, wherein the nucleotide sequence of 27F was: 5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO.1), and the nucleotide sequence of 1492R was: 5'-TACGGCTACCTTGTTACGACTT-3' (shown in SEQ ID NO.2).

[0044] Amplification program: pre-denaturation at 96°C for 5 min; denaturation at 96°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, for 35 cycles; extension at 72°C for 5 min.

[0045] The gene amplification product was purified and sequenced by BGI. The 16S rDNA sequence of the TB24016 strain was PCR amplified to obtain a 1500bp band. Its gene sequence was compared with the sequences of related strains in the NCBI database by BLAST, and a phylogenetic tree was constructed with the 16S rDNA sequences of related species. The 16S nucleotide sequence of the bacterial strain TB24016 measured by sequencing is shown in SEQ ID NO.3. The 16S rDNA gene sequence of the obtained strain TB24016 has 1414 bases. The results of homology analysis showed that the 16S rDNA gene sequence of the strain TB24016 is 96% similar to the 16S rDNA (ON054039.1) of Bacillus velezensis, and the phylogenetic tree was constructed by MEGA11.0 software. The phylogenetic tree is shown in Figure 3 As shown, it was shown that strain TB24016 belongs to Bacillus velez.

[0046] Combined with morphological characteristics and 16S rDNA gene sequence analysis, the strain was identified as Bacillus velezensis and named Bacillus velezensis TB24016. It was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit number CCTCC NO: M 20242186.

[0047] 2. Specific examples of the use of Bacillus Velezii of the present invention in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products:

[0048] Example 3 Application of Bacillus Velezii TB24016 in inhibiting fungal growth

[0049] In this example, the antibacterial activity of Bacillus velez TB24016 was analyzed by the Oxford cup method, and the specific implementation operation is as follows:

[0050] 1. Preparation of mold spore suspension

[0051] Inoculate 5 mold strains into potato dextrose agar (PDA) medium, place in a constant temperature and humidity incubator at 28°C for 5 days, wash the plate with 5mL PBS buffer to obtain a mixture of mycelium and spores, filter with 4 layers of sterile gauze to obtain a stock solution of bacterial suspension without mycelium. Cover the blood cell counting plate with a coverslip, and then use a sterile capillary dropper to drop a small drop of the shaken bacterial suspension from the edge of the coverslip, allowing the bacterial liquid to automatically enter the counting chamber along the gap by capillary penetration, and absorb the excess liquid with absorbent paper. Let it stand for 1 minute, count under an optical microscope, and dilute the spore suspension to 10 according to the counting results. 6 The concentration of CFU / mL. If the concentration is too high, dilute it with sterile water to the required concentration. If the concentration is too low, concentrate it by centrifugation.

[0052] 2. Oxford cup antibacterial experiment

[0053] The Velez Bacillus TB24016 was inoculated on LB solid medium plates and cultured at 37°C for 24-36 hours to activate it; the activated bacteria TB24016 was transferred to LB liquid medium and cultured at 37°C and 180 rpm for 14-16 hours until the middle growth period, and the optimal concentration was 10 8 CFU / mL. Pipette 200 μL of fungal spore suspension (10 6 CFU / mL) in a petri dish, then draw 30mL of sterilized Sabouraud medium and mix the suspension evenly, wait for the medium to solidify, place a corresponding number of Oxford cups to punch holes in the petri dish, and finally pour in 30mL of Sabouraud medium, wait for the medium to solidify, take out the Oxford cup, draw 200μL (Bacillus Velezii TB24016, concentration 10 8 CFU / mL) bacterial antagonist solution was added to the agar wells and placed at 28℃±1℃. After culturing for 5 days, the size of the inhibition zone was observed.

[0054] The antibacterial Bacillus lysininus preserved in the laboratory and the reported antibacterial Bacillus Velezii DY-7 were used as control bacteria, and the sizes of the inhibition zones are shown in Table 1. Compared with the control bacteria and DY-7, the inhibitory activity of Bacillus Velezii TB24016 against Aspergillus montegrillus, Aspergillus cristatus, Aspergillus unicellularis, Scopulariopsis brevis, and Aspergillus shevastatin was significantly improved.

[0055] Table 1 Diameters of inhibition zones of strain TB24016 against different molds (cm)

[0056]

[0057] Example 4 Application of Bacillus Velezii TB24016 in preventing and controlling mildew of tobacco products

[0058] In this embodiment, the fermentation liquid of Bacillus Velezii TB24016 is sprayed into tobacco products to effectively prevent and control fungal contamination. The specific implementation operation is as follows:

[0059] 1. Bacteria activation

[0060] The frozen and stored strain Bacillus velezensis TB24016 was inoculated into LB solid culture medium, and the bacterial cells were scraped and inoculated into LB liquid culture medium and cultured at 37°C, 180rpm for 16h; a certain volume of bacterial liquid was taken and stored in a glycerol storage tube, and the final glycerol volume in the tube was 20%; and it was frozen and stored at -80°C.

[0061] 2. Liquid culture

[0062] The frozen and stored strains were transferred to LB solid culture medium plates and cultured at 37°C for 24-36 hours to activate them; the activated seeds were transferred to LB liquid culture medium and cultured at 37°C, 180 rpm for 14-16 hours until the mid-logarithmic growth period to obtain the Bacillus Velezii TB24016 bacterial solution.

[0063] 3. Anti-mildew experiment in cigar tobacco leaves

[0064] The same part of the cigar tobacco leaf was cut into 13 cm × 13 cm size, sprayed with an appropriate amount of sterile water, and placed in a constant temperature and humidity incubator to balance the moisture for 48 h. After the moisture balance, the leaf was placed in an autoclave at 121 ° C for 30 min. The tobacco leaf was spread flat and placed in a 130 mm × 130 mm square culture dish. 50 μL of each spore suspension (10 6 CFU / mL, and the control strain was Bacillus Velezii DY-7 suspension (10 9 CFU / mL, 50 μL), and each group was sprayed with 50 μL of bacterial suspension of Bacillus Velezii TB24016 of different concentrations (10 6 , 10 7 , 10 8 , 10 9 CFU / mL), and then sealed and placed in a constant temperature incubator at 28℃ for 4 days to observe the anti-mildew effect.

[0065] The anti-mold effect of cigar tobacco leaves is as follows Figure 4 The results showed that the control strain DY-7 9At a higher bacterial suspension concentration of 10 CFU / mL, the mildew prevention effect on Aspergillus monteri, Scopulariopsis brevis, Aspergillus cristatus, Aspergillus unicellularis and Aspergillus chevalieri, which are the main causes of mildew in cigars produced in Sichuan, was not ideal. However, the mildew prevention effect of Bacillus Velezii TB24016 increased with the increase of bacterial suspension concentration. When the bacterial suspension concentration reached 10 8 CFU / mL, it showed good antifungal effect against Aspergillus monteri, Scopulariopsis brevis, Aspergillus cristatus, and Aspergillus shevastatin. 7 CFU / mL, it showed good anti-mildew effect against single-celled sympodial fungi.

[0066] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Bacillus Velez, characterized in that: The deposit number of the Bacillus Velez is CCTCC NO: M20242186.

2. Use of the Bacillus Velezii as claimed in claim 1 in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products.

3. The use of Bacillus Velezii according to claim 2 in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products, characterized in that: The fungus is Aspergillus monteri, Aspergillus cristatus, Aspergillus unicellularis, Scopulariopsis brevicaulis and / or Aspergillus shevarium.

4. The use of Bacillus Velezii according to claim 2 or 3 in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products, characterized in that: The bacterial liquid of Bacillus Velez is sprayed into tobacco products.

5. The use of Bacillus Velezii according to claim 4 in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products, characterized in that: When the fungus is Aspergillus monteri, Aspergillus cristatus, Scopulariopsis brevis and / or Aspergillus shevarium, the concentration of the bacterial solution is not less than 10 8 CFU / mL.

6. The use of Bacillus Velezii according to claim 4 in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products, characterized in that: When the fungus is a single cell symphysema, the concentration of the bacterial solution is not less than 10 7 CFU / mL.

7. The use of Bacillus Velezii according to claim 2 or 3 in inhibiting fungal growth and / or preventing and controlling mildew of tobacco products, characterized in that: The tobacco products include cigar tobacco leaves, cigarettes and strip tobacco.

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