Bacillus velezensis, application thereof and tobacco product mildew preventive

Abstract

The invention relates to bacillus velezensis and application thereof and a tobacco product mildew preventive, and belongs to the technical field of microorganisms. The bacillus velezensis disclosed by the invention is DY-4, the preservation number of the bacillus velezensis is CCTCC NO: M 20232640, and the bacillus velezensis is obtained by screening from cigar tobacco leaves. The strain can inhibit the growth of main mould in tobacco, and can inhibit the mould of tobacco. Experiments prove that the inhibition rates of the fermentation supernatant of the bacillus velezensis DY-4 on penicillium chrysogenum, aspergillus fumigatus and aspergillus niger are 28%, 23% and 18% respectively; the inhibition rates of the generated volatile substances on penicillium chrysogenum, aspergillus fumigatus and aspergillus niger are 51.47%, 51.52% and 12.27% respectively. The DY-4 is utilized to provide two methods for inhibiting the growth of the mould, and the application potential of the strain DY-4 in inhibiting the mould is expanded.

Description

Technical Field

[0001] The invention relates to Bacillus velez, application thereof and a tobacco product mildew preventive, belonging to the technical field of microorganisms. Background Art

[0002] Tobacco mildew refers to the growth and reproduction of mold on tobacco leaves or cigarettes, which leads to the destruction of their tissue structure, darkening of color, disappearance of aroma, and deterioration of the taste. The growth of mold requires suitable temperature and humidity conditions. Usually, mold is easy to reproduce when the temperature ranges from 20℃ to 37℃ and the relative humidity is between 70% and 80%. Moldy tobacco will not only destroy the original aroma and taste, but also lead to deterioration of the taste, increased irritation, and may even cause health problems. Moldy tobacco will produce harmful substances when burning, posing a potential threat to the health of smokers.

[0003] Cigars are special tobacco products made from fermented and aged tobacco leaves. The production process is complex, with many processes and high humidity. Fermented tobacco leaves are rich in organic acids, sugars and other nutrients, which are very conducive to the growth and reproduction of mold, thus causing mildew. With the increasing scale of cigar production, the problem of mildew has gradually become prominent, and the prevention and control of mildew has become a common problem that needs to be solved urgently in the entire industry.

[0004] Cigar production is a process in which microorganisms are involved from beginning to end. The use of biological control and the strategy of "using bacteria to treat bacteria" have very broad application prospects in the prevention and control of cigar mildew. At present, the biological control of cigar mildew is at a very basic stage, and there are few effective biological control strains. Therefore, screening highly efficient, widely used and effective biological control bacteria plays a very important role in the research and application of the prevention and control of cigar mildew.

[0005] The Chinese invention patent application document with a publication date of February 9, 2021 and publication number CN112342169A discloses a strain of Bacillus altitudinis and its application in preventing and controlling cigar fermentation and mildew. It specifically discloses Bacillus altitudinis YC-9 with a preservation number of CGMCC No. 20884, which can inhibit the growth of fungi and reduce tobacco leaf mildew. Combining this strain with Bacillus velezensis ACCC02735 can improve the appearance quality of cigar fermentation.

[0006] The Chinese invention patent application document with a publication date of August 30, 2024 and a publication number of CN116121104A discloses a strain of Bacillus velezensis and its application, and a tobacco product mildew inhibitor. Specifically, Bacillus velezensis DY-7 with a preservation number of CCTCC NO: M2022906 is disclosed, which has a significant antagonistic effect on Aspergillus niger, Bacillaceae, Trichoderma yunnanensis, and Penicillium chrysogenum.

[0007] The Chinese invention patent application document with a publication date of August 30, 2024 and a publication number of CN118562655A discloses a Bacillus licheniformis preparation and its application, specifically disclosing a Bacillus licheniformis preparation with a deposit number of GDMCC NO: 64425, which has a significant antagonistic effect on Aspergillus fumigatus, Aspergillus paper peel, Penicillium olsenii, Halodactylum, Penicillium TFLel, and Torula mackenziei.

[0008] Although the several Bacillus disclosed in the above patent application documents can inhibit certain tobacco molds, they are all applied by inoculating Bacillus into cigar tobacco leaves for fermentation. However, for some cigars that cannot be inoculated and fermented with exogenous strains, this inoculation and fermentation method is obviously not applicable. Therefore, it is of great significance to develop a method that can be used for both fermentation and fumigation to prevent and control mold in cigar tobacco leaves, which is suitable for different cigar production processes. Summary of the invention

[0009] The first object of the present invention is to provide a Bacillus Velez, which can be used for inoculation and fermentation to prevent and control molds, and can also be used for fumigation treatment to prevent and control molds.

[0010] The second object of the present invention is to provide an application of Bacillus Velezii in inhibiting fungal growth and / or mildew of tobacco products, and to provide a method for preventing mildew of tobacco products with good control effect and high biological safety.

[0011] The third object of the present invention is to provide a tobacco product mildew inhibitor, and to provide a tobacco product mildew inhibitor with good prevention and control effect and high biological safety.

[0012] In order to achieve the above object, a technical solution of Bacillus Velezii in the present invention is:

[0013] A Bacillus Velez subtilis, wherein the deposit number of the Bacillus Velez subtilis is CCTCC NO: M 20232640.

[0014] 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 strain DY-4 from cigar tobacco leaves, and the bacteria are identified as Bacillus velezensis through physiological, biochemical and molecular biological identification. The antibacterial ability of Bacillus velezensis DY-4 was analyzed by the Oxford cup method, and it was found that the fermentation supernatant and the volatile substances produced by the bacteria can inhibit Penicillium chrysogenum, Aspergillus fumigatus and Aspergillus niger; and, the bacterial liquid of DY-4 was subsequently sprayed on the surface of cigar tobacco for fermentation and culture, and it was found that the bacterial suspension can inhibit the mildew of cigar tobacco leaves. This fully shows that the Bacillus velezensis DY-4 provided by the present invention can not only prevent and control mold by inoculation and fermentation, but also prevent and control mold by fumigation, providing a new and available microbial resource for the mildew prevention and control of tobacco products.

[0015] In order to achieve the above-mentioned object, the technical scheme of the application of Bacillus Velez subtilis in inhibiting fungal growth and / or mildew of tobacco products in the present invention is:

[0016] The invention discloses an application of Bacillus Velez subtilis in inhibiting fungal growth and / or mildew of tobacco products.

[0017] The beneficial effect of the above technical scheme is that: the present invention isolates a strain of Bacillus Velez DY-4 from cigar tobacco leaves, and uses the Oxford cup method to analyze the antibacterial ability of Bacillus Velez DY-4, and finds that the fermentation supernatant of the bacteria and the volatile substances produced can inhibit Penicillium chrysogenum, Aspergillus fumigatus and Aspergillus niger. Therefore, the Bacillus Velez DY-4 provided by the present invention can be used as a biological mildew preventer for preventing and treating mold contamination of tobacco products. The present invention provides a Bacillus Velez that can effectively antagonize a variety of mold-causing molds with strong mildew-causing ability, and provides a new microbial resource for the prevention and control of mold of tobacco products. In addition, two methods for inhibiting the growth of Penicillium chrysogenum, Aspergillus niger and Aspergillus fumigatus by Bacillus Velez DY-4 are provided, which expands the application potential of DY-4 in inhibiting mold, and has a large application prospect in the biological control of cigar mildew.

[0018] As a further improvement, the fungus is Penicillium chrysogenum, Aspergillus fumigatus and / or Aspergillus niger.

[0019] As a further improvement, the Bacillus Velezii is used to perform fumigation treatment on tobacco products.

[0020] As a further improvement, the bacterial liquid or fermentation supernatant of the Bacillus Velez is sprayed into the tobacco product.

[0021] As a further improvement, the concentration of Bacillus Velezii in the bacterial solution is not less than 10 7CFU / mL.

[0022] As a further improvement, the tobacco products include cigar tobacco leaves, cigarettes and tobacco sheets.

[0023] In order to achieve the above object, the technical solution of a tobacco product mildew inhibitor in the present invention is:

[0024] A tobacco product mildew preventer, wherein the active ingredient of the tobacco product mildew preventer is the bacterial suspension of Bacillus Velezii, fermentation supernatant or volatile substances produced by fermentation.

[0025] The beneficial effect of the above technical scheme is that the bacterial liquid, fermentation supernatant and volatile substances produced by the Bacillus Velez subtilis of the present invention can inhibit the growth of Penicillium chrysogenum, Aspergillus niger and Aspergillus fumigatus, and Bacillus Velez subtilis is a microorganism with high biological safety, so the auxiliary materials commonly used in the field can be added as a mildew inhibitor for tobacco products.

[0026] As a further improvement, the concentration of Bacillus Velezii in the bacterial suspension is not less than 10 7 CFU / mL.

[0027] As a further improvement, the tobacco products include cigar tobacco leaves, cigarettes and tobacco sheets. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a colony morphology diagram of strain DY-4 in Example 1 of the present invention on solid LB medium;

[0029] Figure 2 The phylogenetic tree of strain DY-4 in Example 1 of the present invention constructed based on the 16S rDNA gene sequence;

[0030] Figure 3 This is a diagram showing the inhibitory effect of the fermentation supernatant of Bacillus Velez subtilis DY-4 and DY-7 on mold in Example 2 of the present invention;

[0031] Figure 4 This is a comparison of the inhibitory effects of the volatile metabolites of Bacillus Velez DY-4 and DY-7 on mold in Example 2 of the present invention;

[0032] Figure 5 This is a diagram showing the antibacterial effect of the volatile metabolites of Bacillus Velez subtilis DY-4 in Example 4 of the present invention. DETAILED DESCRIPTION

[0033] Among the prevention and control measures for the mildew of tobacco products in the prior art, the traditional chemical and physical mildew prevention and control means are difficult to exert the ideal effect, and the biological prevention and control method has the advantages of full environmental protection, high efficiency and pollution-free, and is increasingly valued. However, the currently disclosed tobacco product mildew-proof strains are all inoculated on tobacco leaves for fermentation treatment when applied, but for some cigars that cannot be inoculated and fermented with exogenous strains, this inoculation and fermentation method is obviously not applicable. Based on this, the present invention provides a kind of Bacillus velezensis DY-4. The Bacillus velezensis can not only prevent and control molds by inoculation and fermentation, but also prevent and control molds by fumigation, providing a new and available microbial resource for the mildew prevention and control of tobacco products.

[0034] 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.

[0035] The culture medium formula and preparation process used in the embodiments of the present invention are as follows:

[0036] Preparation of LB solid culture medium: LB agar medium (Cat. No. 028330) from Guangdong Huankai Microbiological Technology Co., Ltd., prepared at 36 g / L, sterilized at 121°C for 15 min.

[0037] Preparation of LB liquid culture medium: LB broth (catalog number: 028320) from Guangdong Huankai Microbiological Technology Co., Ltd., prepared at 21 g / L, sterilized at 121°C for 15 min.

[0038] Preparation of PDA medium: Select the potato dextrose agar (PDA) medium (Cat. No. 021050) from Guangdong Huankai Microbiological Technology Co., Ltd., configure it at 40.1 g / L, and sterilize it at 115°C for 30 min.

[0039] Biomaterials used in the embodiments of the present invention:

[0040] Strains: Penicillium chrysogenum, Aspergillus niger and Aspergillus fumigatus were isolated from the surface of cigar leaves of Sichuan China Tobacco, Hubei China Tobacco and Yunnan production areas respectively.

[0041] 1. A specific embodiment of a Bacillus Velezii of the present invention:

[0042] The Bacillus velezensis of the present invention is Bacillus velezensis DY-4, with a deposit number of CCTCC NO: M 20232640; a deposit date of December 22, 2023; a deposit unit of the China Center for Type Culture Collection; and a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The specific implementation operations are as follows:

[0043] Example 1 Isolation and Identification of Bacillus Velezii DY-4

[0044] In this example, a strain of Bacillus Velezii DY-4 was isolated from cigar tobacco leaves. The specific implementation steps are as follows:

[0045] 1. Isolation of strain DY-4

[0046] Take 2.0g of cigar tobacco leaves and add 20mL of sterile PBS buffer, shake at 37℃, 180rpm for 30min, and let stand at room temperature for 1min to obtain the bacterial suspension stock solution. Place the bacterial suspension in an 80℃ water bath for 30min to obtain the coating stock solution, and dilute it 10 times to obtain a dilution of 10 -1 , 10 -2 The yeast coating liquid was taken, and 100 μL of the coating liquid of different dilutions was taken to the LB solid culture medium plate. After culturing at 37°C to obtain the strain to be identified, the single strain to be identified was inoculated into the LB test tube culture medium again, and cultured at 37°C, 180rpm to obtain the seed liquid. The seed liquid of the strain to be screened was spread on the LB culture medium to obtain strain DY-4.

[0047] 2. Identification of strain DY-4

[0048] 1) Morphological identification

[0049] The Bacillus DY-4 obtained by the above screening was inoculated onto LB solid culture medium plates and cultured at 37°C for 12 hours before observing the colony morphology. Figure 1 As shown, the surface of the colony of strain DY-4 is smooth, opaque, relatively round, slightly convex, and has a viscous texture.

[0050] 2) Molecular biological identification

[0051] The strain DY-4 was sent to BGI for 16S sequencing, and the 16S rDNA nucleotide sequence of the strain DY-4 was measured as shown in SEQ ID No. 1. The sequencing results were submitted to the NCBI database for comparison and strain identification. The identification results showed that the 16S rDNA sequence of the strain DY-4 was 95% similar to the 16S sequence of Bacillus velezensis (MT 184820.1), and the phylogenetic tree was constructed using MEGA 7.0 software. The phylogenetic tree is shown in Figure 2 As shown. Figure 2 It can be seen that strain DY-4 belongs to the Bacillus velezensis clade.

[0052] Combined with morphological characteristics and 16S rDNA sequence analysis, the strain DY-4 was identified as Bacillus velezensis and named DY-4. It was deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with the accession number CCTCC NO: M 20232640. The storage address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0053] 2. Specific examples of the use of Bacillus Velezii of the present invention in inhibiting fungal growth and / or mildew of tobacco products:

[0054] Example 2 Analysis of antibacterial activity of Bacillus velez DY-4

[0055] In this example, the Oxford cup method was used to analyze the antagonistic ability of Bacillus Velezii DY-4 against Aspergillus fumigatus, Aspergillus niger and Penicillium chrysogenum. The specific implementation operation is as follows:

[0056] 1. Antibacterial activity analysis of fermentation supernatant of Bacillus velez DY-4

[0057] A single colony of Bacillus Velez subtilis DY-4 was inoculated into LB liquid culture medium and cultured in a horizontal shaker at 37°C and 180rpm for 12 hours to obtain a seed solution. Subsequently, the culture was transferred to LB liquid culture medium at a 5% inoculation rate and cultured for 12 hours. After centrifugation, the supernatant was filtered through a 0.22μm filter membrane to obtain the fermentation supernatant of Bacillus Velez subtilis DY-4, and then the fermentation supernatant was used for antibacterial experiments, with a blank LB liquid culture medium as a control. The diameter d after mold antagonism was measured and compared with the control diameter D to obtain the inhibition rate, and the inhibition rate (%) = (Dd) / D. The results are shown in Figure 3 And as shown in Table 1.

[0058] Table 1 Inhibition results of fermentation supernatant of Bacillus Velez DY-4 on different molds in cigars

[0059] Fungal species DY-4 inhibition rate against mold % DY-7 inhibition rate against mold % Penicillium chrysogenum 28.12 25.42 Aspergillus fumigatus 23.36 18.33 Aspergillus niger 18.15 14.21

[0060] 2. Analysis of the antibacterial activity of volatile substances of Bacillus velezinii DY-4 and its comparison with DY-7

[0061] Single colonies of Bacillus velez DY-4 and DY-7 (a strain of Bacillus velez disclosed in a Chinese invention patent application with a publication date of May 16, 2023 and publication number CN116121104A) were inoculated into LB liquid culture medium and cultured in a horizontal shaker at 37°C and 180rpm for 12 hours to obtain seed liquid. Subsequently, the inoculation amount was transferred to LB liquid culture medium at 5% and cultured for 12 hours. Diluted with sterile water to obtain OD 600=0.8 bacterial suspension. Take 100 μL of bacterial suspension and spread it on LB solid medium plate, and take another 1 μL 1×10 6 CFU / mL mold seed liquid was spotted onto a PDA solid culture medium plate, and the inoculated LB solid culture medium plate and the PDA fixed culture medium plate were "buttoned" and sealed, and cultured at 28°C for 4 days. The diameter d of the mold after antagonism was measured and compared with the control diameter D to obtain the inhibition rate, and the inhibition rate (%) = (Dd) / D. Among them, the blank YPD solid culture medium plate was used as the control. The results are shown in Figure 4 And as shown in Table 2.

[0062] Table 2 Inhibition results of volatile metabolites of Bacillus velez DY-4 on different molds in cigar tobacco

[0063] Fungal species DY-4 inhibition rate against mold % DY-7 inhibition rate against mold % Penicillium chrysogenum 51.47 21.08 Aspergillus fumigatus 51.52 28.90 Aspergillus niger 12.27 15.17

[0064] 3. Experimental results

[0065] It can be seen from Table 1 that the fermentation supernatant of Bacillus Velez DY-4 has an inhibition rate of 28% on Penicillium chrysogenum in cigar tobacco, 23% on Aspergillus fumigatus, and 18% on Aspergillus niger.

[0066] It can be seen from Table 2 that the volatile metabolites of Bacillus Velez DY-4 have an inhibitory effect on the growth of molds such as Penicillium chrysogenum, Aspergillus fumigatus, and Aspergillus niger. Among them, the volatile substances produced by Bacillus Velez DY-4 have an inhibition rate of 51.47% on Penicillium chrysogenum, an inhibition rate of 51.52% on Aspergillus fumigatus, and an inhibition rate of 12.27% on Aspergillus niger, and the inhibitory effect of the volatile substances on Aspergillus fumigatus of Penicillium chrysogenum is significantly better than the inhibitory effect of DY-7 reported. It can be seen that the Bacillus Velez DY-4 provided by the present invention has a certain inhibitory effect on molds in cigars, and has great application potential for biological control of cigar mildew.

[0067] Example 3 Application of Bacillus Velezii DY-4 strain in inhibiting mildew of tobacco products

[0068] A single colony of Bacillus Velezii DY-4 was inoculated into LB liquid medium and cultured in a horizontal shaker at 37°C and 180rpm for 12h to obtain seed liquid. Sterile water was used for gradient dilution to obtain DY-4 bacterial suspensions of different concentrations. 3.5mL DY-4 bacterial suspension was sprayed on the surface of 5g cigar tobacco leaves (cigar tobacco leaves from Sichuan production area, i.e., the same batch of cigar tobacco leaves from which yellow mold was isolated) to make the moisture content of the tobacco leaves reach 50%, and the control group was sprayed with an equal amount of sterile PBS buffer; among them, the concentrations of DY-4 bacterial suspensions were 1.0×10 6 CFU / mL, 1.0×10 7 CFU / mL, 1.0×108 The treated group and the control group were placed in static culture at 28°C for 7 days, and the condition of the cigar tobacco leaves after 7 days of culture of strain DY-4 was recorded to compare the mildew of the cigar tobacco leaves.

[0069] By comparing the experimental treatments of spraying and not spraying DY-4 strain, it can be seen that the cigar tobacco leaves of the control group without spraying were obviously moldy and smelled moldy; while the spraying concentration of 1.0×10 6 CFU / mL of DY-4 suspension, the cigar tobacco leaves turned dark and smelled musty, and could not play a role in preventing and controlling mildew; the spraying concentration was 1.0×10 7 CFU / mL and 1.0×10 8 In the DY-4 bacterial suspension group with 100 CFU / mL, no moldy smell was detected, and compared with the control group without spraying, the tobacco leaves were bright and bright, and the mildew could be obviously inhibited. Therefore, the Bacillus Velezii DY-4 provided in the embodiment of the present invention can effectively inhibit the mildew of cigar tobacco leaves.

[0070] Example 4 Application of volatiles of Bacillus Velezii DY-4 strain in inhibiting mildew of tobacco products

[0071] A single colony of Bacillus Velez DY-4 was streaked onto LB solid medium, and then the inoculated plate was placed in a glass desiccator. At the same time, normal cigar tobacco leaves (Sichuan production area) were added to the desiccator. The control group was a blank plate without inoculation of the strain. The desiccator was then sealed and placed in a 28°C incubator for 4 days to observe the inhibition of tobacco leaf mildew. The results are as follows Figure 5 shown.

[0072] Comparing the experimental treatment results of fumigated and unfumigated DY-4 strains, it can be seen that the cigar tobacco leaves in the unfumigated control group were obviously moldy and smelled moldy; while in the fumigated DY-4 strain experimental group, the cigar tobacco leaves were bright and did not smell moldy. Therefore, the Bacillus Velez DY-4 provided in the embodiment of the present invention can effectively inhibit the mold of cigar tobacco leaves.

[0073] 3. Specific embodiments of a tobacco product mildew inhibitor of the present invention:

[0074] Example 5 A tobacco product mildew inhibitor

[0075] The active ingredient of the tobacco product mildew inhibitor of this embodiment is a bacterial suspension of Bacillus Velezii with CCTCC NO: M 20232640, and the concentration of Bacillus Velezii in the bacterial suspension is ≥ 1.0×10 7 CFU / mL.

[0076] 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: M20232640.

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

3. The use of Bacillus Velezii in inhibiting fungal growth and / or mildew of tobacco products according to claim 2, characterized in that: The fungus is Penicillium chrysogenum, Aspergillus fumigatus and / or Aspergillus niger.

4. The use of Bacillus Velezii according to claim 2 or 3 in inhibiting fungal growth and / or mildew of tobacco products, characterized in that: The tobacco products are fumigated by using the Bacillus Velez.

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

6. The use of the Bacillus Velezii in inhibiting fungal growth and / or mildew of tobacco products according to claim 5, characterized in that: The concentration of Bacillus Velez in 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 mildew of tobacco products, characterized in that: The tobacco products include cigar tobacco leaves, cigarettes and strip tobacco.

8. A tobacco product mildew inhibitor, characterized in that: The active ingredient of the tobacco product mildew inhibitor is the bacterial suspension, fermentation supernatant or volatile substances produced by fermentation of the Bacillus Velezii according to claim 1.

9. The tobacco product mildew inhibitor according to claim 8, characterized in that: The concentration of Bacillus Velez in the bacterial suspension is not less than 10 7 CFU / mL.

10. The tobacco product mildew inhibitor according to claim 8 or 9, characterized in that: The tobacco products include cigar tobacco leaves, cigarettes and strip tobacco.

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