Bacillus velezensis and application thereof

Abstract

The invention relates to bacillus velezensis and application thereof, and belongs to the technical field of microorganisms. The bacillus velezensis disclosed by the invention is ZD122, and the preservation number of the bacillus velezensis is CCTCC (China Center For Type Culture Collection) NO: M 20241480. According to the present invention, a strain capable of efficiently antagonizing a variety of molds is separated from cigar tobacco leaves, and physiological, biochemical and molecular biological identification results show that the strain is Bacillus velezensis, is named as ZD122, and has an antagonistic effect on Aspergillus versicolor, Aspergillus mongolicus, Aspergillus pseudoglaucus and Penicillium chrysogenum with strong mildewing ability. Furthermore, the bacillus velezensis is a strain with relatively high biological safety, and is bacillus capable of being added into 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 important economic crop that mainly harvests tobacco leaves. The natural aging or artificial fermentation of tobacco leaves is one of the important links to improve the quality and availability of tobacco leaves. The natural aging storage period of tobacco leaves is long. Since tobacco leaves have strong hygroscopicity and contain various nutrients required for the growth of microorganisms (such as various molds), once the conditions are suitable, tobacco leaves will breed various microorganisms and become moldy during storage. This will cause the tissue structure of tobacco leaves to be destroyed, and the appearance of moldy and rancid odors will appear. In addition, some molds can produce toxins, which seriously affect the appearance quality and aroma quality of tobacco leaves and reduce the use value of tobacco leaves. Therefore, the control of tobacco leaf mildew is an important research direction during the storage period of tobacco leaves. Solving the mildew and mildew prevention problems during the storage period of tobacco leaves has become a top priority.

[0003] There are many reasons for tobacco leaf mildew, mainly including the following: ① The influence of pathogenic microorganisms. At present, the pathogens isolated from moldy tobacco leaves mainly include Aspergillus and Penicillium, as well as a small amount of Rhizopus and Muor. In some places, Bacillus and Lactobacillus are also isolated; ② The influence of temperature and humidity. Tobacco leaves must contain a certain amount of moisture so that they will not break during storage and avoid the loss of broken tobacco. However, if the moisture content is high and exceeds the moisture standard for safe storage, pathogenic microorganisms such as mold can grow and multiply, which is also one of the main reasons for tobacco leaf mildew; ③ Tobacco leaves contain nutrients for the growth of microorganisms; ④ The influence of tobacco leaf maturity and packaging materials.

[0004] Since tobacco leaf mildew requires certain temperature, humidity and nutrients necessary for mold growth, the main measures to prevent tobacco leaf mildew should start from inhibiting mold growth conditions, take comprehensive prevention measures, focus on prevention, strictly control the moisture content of tobacco leaves, master and adjust the changes in temperature and humidity in the storage environment, and cooperate with exogenous physical mildew prevention, chemical mildew prevention or biological mildew prevention to achieve the purpose of inhibiting mold growth. Among them, physical mildew prevention mainly includes irradiation mildew prevention and microwave mildew prevention, but these two methods may affect the quality of tobacco leaves, and there are problems such as poor effect, high energy consumption and high cost, so they have not been promoted; chemical mildew prevention usually uses mildew inhibitors such as sorbic acid (salt), benzoic acid (salt) and hydroxybenzoic acid (salt) to intervene in the growth of mold, but these mildew inhibitors are easy to have adverse effects on the sensory quality of tobacco leaves, so their application is limited; and biological mildew prevention mainly uses microorganisms or microbial metabolites to inhibit or kill mold, thereby preventing the occurrence of tobacco leaf mildew. This method has the advantages of safety, environmental protection, high efficiency and no pollution, and has been increasingly valued.

[0005] However, there are currently very few reports on mildew-resistant strains in cigar tobacco leaves, and the mildew-resistant ability of the reported strains still needs to be strengthened. Therefore, screening strains with high antibacterial activity is of great significance for tobacco biological mildew prevention. Summary of the invention

[0006] The first object of the present invention is to provide a Bacillus velezensis that can effectively antagonize a variety of molds.

[0007] The second purpose of the present invention is to provide an application of Bacillus Velez in preventing and controlling mold contamination of tobacco products, and to provide a method for preventing mold of tobacco products with good prevention and 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 Bacillus Velez subtilis is ZD122, and the deposit number is CCTCC NO: M20241480.

[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 multiple molds from cigar tobacco leaves. After physiological, biochemical and molecular biological identification, the strain is Bacillus velezensis, named ZD122, which has antagonistic effects on Aspergillus versicolor, Aspergillus monteritus, Aspergillus pseudoglaucoides and Penicillium chrysogenum, which have strong mildew ability.

[0011] Furthermore, the strain screened by the present invention that can effectively antagonize multiple molds is Bacillus Velezii, which is a strain with high biological safety and is a Bacillus that can be added to tobacco products.

[0012] In order to achieve the above object, the technical solution of the application of Bacillus Velezii in preventing and controlling mold contamination of tobacco products in the present invention is:

[0013] The invention discloses an application of Bacillus Velezii in preventing and controlling mold contamination of tobacco products, wherein the molds are Aspergillus versicolor, Aspergillus monteridium, Aspergillus pseudoglaucus and / or Penicillium chrysogenum.

[0014] The beneficial effect of the above technical scheme is that: the present invention isolates a strain ZD122 from cigar tobacco leaves, which is identified as Bacillus Velez through morphology and molecular biology, and then through the Oxford Cup antibacterial experiment and the cigar tobacco mildew prevention experiment, it is found that it can effectively antagonize Aspergillus versicolor, Aspergillus monteridium, Aspergillus pseudogrey green and Penicillium chrysogenum. Aspergillus and Penicillium are the main mildew-causing bacteria in tobacco products at present, therefore, the Bacillus Velez ZD122 provided by the present invention can be used as a biological mildew-preventing agent for preventing and controlling mildew contamination of tobacco products. The present invention provides a kind of mold that can effectively antagonize multiple mildew-causing molds with strong mildew-causing ability, and provides a new microbial resource for mildew prevention and control of tobacco products.

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

[0016] As a further improvement, the concentration of Bacillus Velezii in the bacterial solution is not less than 10 7 CFU / mL.

[0017] As a further improvement, the tobacco products include cigar tobacco leaves, cigarettes and tobacco sheets. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a colony morphology diagram of strain ZD122 in solid LB medium in Example 2 of the present invention;

[0019] Figure 2 This is a microscopic morphology of strain ZD122 in Example 2 of the present invention;

[0020] Figure 3 The phylogenetic tree constructed based on the 16S rDNA gene sequence in Example 2 of the present invention;

[0021] Figure 4 This is a graph showing the antibacterial results of strain ZD122 in Example 3 of the present invention;

[0022] Figure 5This is a comparison chart of the antibacterial results of strains DY-7 and ZD122 in Example 3 of the present invention;

[0023] Figure 6 This is a diagram showing the anti-mildew effect of strain ZD122 in Example 4 of the present invention in cigar tobacco leaves;

[0024] Figure 7 This is a diagram showing the anti-mildew effect of strain DY-7 in Example 4 of the present invention in cigar tobacco leaves. DETAILED DESCRIPTION

[0025] Among the existing tobacco product mildew prevention and control measures, traditional chemical and physical mildew prevention and control methods are difficult to achieve ideal results, while biological prevention and control methods are increasingly valued due to their advantages of being fully environmentally friendly, highly efficient and pollution-free. However, there are currently very few tobacco product mildew-proof strains that have been disclosed, and the mildew-proof ability of the mildew-proof strains that have been reported still needs to be strengthened. Based on this, the present invention provides a Bacillus Velezii.

[0026] The present invention separates a strain that can effectively antagonize multiple molds from cigar tobacco leaves. After physiological, biochemical and molecular biological identification, the strain is Bacillus velezensis, named ZD122, and has antagonistic effects on Aspergillus versicolor, Aspergillus monteritus, Aspergillus pseudogrey green and Penicillium chrysogenum, which have strong mildew resistance. Moreover, the strain that can effectively antagonize multiple molds selected by the present invention is Bacillus velezensis, which is a strain with high biological safety and is a Bacillus that can be added to tobacco products.

[0027] 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.

[0028] 1. A specific embodiment of a Bacillus Velezii of the present invention:

[0029] The Bacillus velezensis of the present invention is Bacillus velezensis ZD122, with a deposit number of CCTCC NO: M 20241480; a deposit date of July 4, 2024; a deposit unit of the China Center for Type Culture Collection; and a deposit address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The specific implementation operation is as follows:

[0030] Example 1 Isolation and screening of Bacillus velez ZD122

[0031] In this embodiment, a strain that can effectively antagonize multiple molds is isolated from cigar-filled tobacco leaves. The specific implementation operation is as follows:

[0032] Take 1g of cigar tobacco leaves from different regions, add them into a sterilized test tube containing 9mL of sterile water, seal it with a cotton plug, shake it for 2min, and draw 0.1mL of the supernatant to dilute it 10 -1 ~10 -4 , 10 -2 , 10 -4 The gradient dilutions were spread on LB plates and cultured at 37°C for 12 hours. The single colonies on the plates were picked and purified to obtain further purified single colonies, and the obtained strain colonies were numbered for further study. The Oxford cup method was used to explore the antagonistic ability of the numbered strains to different molds, and the diameter of the inhibition zone around the Oxford cup was measured to select strains with large diameters and broad-spectrum antifungal ability. That is, the strain ZD122 that can effectively antagonize Aspergillus versicolor, Aspergillus monteridium, Aspergillus pseudogrey green and Penicillium chrysogenum was obtained.

[0033] Example 2 Identification of strain ZD122

[0034] In this example, the strain ZD122 isolated and obtained in the example was identified by morphology and molecular biology, which is Bacillus velez. The specific implementation operation is as follows:

[0035] 1. Morphological identification

[0036] The strain ZD122 is short rod-shaped, Gram-positive, with spores that are approximately round and lateral. On LB solid medium, the colonies are flat, rough, with irregular edges, and the surface of the bacterial lawn is dull, opaque, and spreads. Figure 1 and Figure 2 (eyepiece 10 times, objective lens 100 times oil lens) The optimum growth temperature is 30-37℃, and the suitable pH is 6.8-7.2.

[0037] 2. Molecular Biology Identification

[0038] The 16S rDNA gene fragment of strain ZD122 was amplified by PCR, purified and recovered. After DNA sequencing, the measured sequence was analyzed for sequence similarity using the Blastn program of NCBI. The 16S rDNA gene sequences of representative strains of similar species were selected from the Genbank database and the ribosome database. Phylogenetic analysis was performed using MEGA6.0 software. A phylogenetic tree based on the 16S rDNA gene sequence was constructed using the Neighbor Joining method. The number of repeated samplings used to test the bootstrap support rate was 1000 times. The purified product was sequenced, and the 16S rDNA gene sequence of strain ZD122 obtained by sequencing had 1367 bases. The homology analysis results showed that the similarity between the 16S rDNA gene sequence of strain ZD122 and the 16S rDNA of Bacillus velezensis FZB42 (NR075005.2) was 99.93%, and the phylogenetic tree was constructed by MEGA6.0 software. Figure 3 As shown, it was shown that strain ZD122 belonged to Bacillus velez.

[0039] Combined with morphological characteristics and 16S rDNA gene sequence analysis, the strain was identified as Bacillus velezensis and named Bacillus velezensis ZD122. It was deposited in the China Center for Type Culture Collection on July 4, 2024, with the deposit number CCTCC NO: M 20241480.

[0040] Example 3 Analysis of antibacterial activity of Bacillus velez ZD122

[0041] This example uses the Oxford cup antibacterial test to analyze the antibacterial activity of Bacillus velez ZD122, and the specific implementation operation is as follows:

[0042] 1. Preparation of mold spore suspension

[0043] Pick colonies from the Sabouraud slant of the mold stored in a 4℃ refrigerator and transfer them to a 100mL conical flask containing 50mL Sabouraud solid culture medium. Keep it at a constant temperature of 28℃ for 6 days, and when the surface is covered with colonies, add 12-15mL of non-distilled water to the conical flask, and use a sterile inoculation loop to gently scrape the culture medium and the growth surface of the bacteria. Then pour the liquid containing hyphae and spores into a disposable syringe plugged with sterile cotton wool in advance, slowly push and collect the spore suspension drops without hyphae, collect the spore suspension drops into a 10mL sterile centrifuge tube, draw about 10μL of the spore suspension and drop it on the blood cell counting plate, count and observe under a biological microscope and adjust to the laboratory required 10 4The spore 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.

[0044] 2. Oxford cup antibacterial experiment

[0045] A single colony of Bacillus velez ZD122 was inoculated into LB seed medium and cultured at 37°C and 180 rpm for about 12 h until the bacterial concentration reached 10 8 CFU / mL. Mix 10 mL of Sabouraud medium with 100 μL of fungal spore suspension (10 4 CFU / mL) was mixed and added to a disposable culture dish with a diameter of 9 mm as the first layer, and then the sterile Oxford cup was picked up with sterilized tweezers to place the layer, and the Oxford cup was removed after the Sabouraud poured in again solidified. 8 100 μL of Bacillus Velezii liquid with CFU / mL was added to each agar well. After culturing at 28°C for 5 days, the size of the inhibition zone was observed.

[0046] The strains with antibacterial activity previously stored in the laboratory were used as control bacteria. The sizes of the inhibition zones are shown in Table 1 and Figure 4 As shown. Compared with the control bacteria, the inhibitory activity of Bacillus velez ZD122 against Aspergillus versicolor, Aspergillus monteridium, Aspergillus pseudoglaucus, and Penicillium chrysogenum was significantly improved. Bacillus velez has the advantages of heat resistance and desiccation resistance. In actual production applications, it can enhance product stability and reduce production costs, making it have broad application potential.

[0047] Table 1 Diameters of inhibition zones of strain ZD122 against different molds (cm)

[0048]

[0049] The reported antibacterial Bacillus velezensis DY-7 was used as the control bacteria. The sizes of the inhibition zones are shown in Table 2 and Figure 5 As shown in the figure. Compared with the control bacteria, the inhibitory activity of Bacillus velez ZD122 against Aspergillus versicolor, Aspergillus monteritus, Aspergillus pseudoglaucus, and Penicillium chrysogenum was significantly improved. This shows that strain ZD122 has stronger antibacterial activity and has better potential in antifungal applications.

[0050] Table 2 Diameters of inhibition zones of strains ZD122 and DY-7 against different molds (cm)

[0051]

[0052] 2. Specific examples of the use of Bacillus Velezii in preventing and controlling mold contamination of tobacco products according to the present invention:

[0053] The present invention isolates a strain ZD122 that can antagonize multiple molds from cigar tobacco leaves, which is identified as Bacillus Velezii. Spraying its fermented bacterial liquid into tobacco products can effectively prevent and control mold pollution. The specific implementation operation is as follows:

[0054] Example 4: Application of Bacillus Velezii ZD122 in the prevention of mildew in cigar tobacco leaves

[0055] 1. Bacteria activation

[0056] The frozen and stored strains were inoculated into LB solid medium (containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH 7.0), and the bacterial moss was scraped and inoculated into LB liquid medium and cultured at 37°C, 180 rpm for 12 h; a certain volume of bacterial liquid was pipetted and stored in a glycerol preservation tube, with the final glycerol volume in the tube being 20%; and frozen and stored at -80°C.

[0057] 2. Seed liquid culture

[0058] The frozen and stored strains were transferred to LB solid medium plates and cultured at 37°C for 24-36 hours to activate them. The activated seeds were transferred to liquid seed medium in a triangular flask (containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and cultured at 37°C, 180 rpm for 14-16 hours until the mid-logarithmic growth period. The optimum concentration was 10 8 CFU / mL, and the bacterial liquid of Bacillus Velez ZD122 was obtained.

[0059] 3. Anti-mildew experiment in cigar tobacco leaves

[0060] Use a sterile pipette tip to pierce the surface of the cigar tobacco leaf and inject 20 μL of 1×10 4 CFU / mL Aspergillus versicolor spore suspension was inoculated into each wound. After natural air drying for 2 h, 20 μL of different concentrations of Bacillus Velezii ZD122 suspension (10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL) was inoculated into each wound, and 20 μL of sterile water was added to the control group. The cells were placed in a 28°C constant temperature incubator for 4 days to observe the anti-mildew effect.

[0061] The anti-mold effect of Bacillus Velez ZD122 in cigar tobacco leaves Figure 6 As shown in the figure, the diameter of cigar tobacco leaf mold infection decreased with the increase of Bacillus Velez ZD122 concentration. Compared with the control, 10 7CFU / mL of Bacillus velezensis ZD122 can reduce the lesion diameter from 14 mm to 0.3 mm (a reduction of 97.85%). 5 CFU / mL and 10 6 CFU / mL) of Bacillus Velezii ZD122 had a weak effect on tobacco leaf mildew prevention, while high concentration (10 7 CFU / mL) of Bacillus Velezii ZD122 has the strongest mildew-proof effect on tobacco leaves. The reported mildew-proof effect of Bacillus Velezii DY-7 with antibacterial activity in cigar tobacco leaves is as follows Figure 7 As shown, compared with the control, the best anti-mildew effect is 10 7 CFU / mL high concentration group, 10 7 CFU / mL of Velez Bacillus DY-7 can reduce the lesion diameter from 13mm to 5.3mm (reduced by 59.23%). Under the condition of the same concentration of antagonistic strains, the anti-mildew effect of strain ZD122 is stronger, which is about 30% higher than that of Velez Bacillus DY-7.

[0062] 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 Bacillus Velez is ZD122, and its deposit number is CCTCCNO: M 20241480.

2. A use of the Bacillus Velezii as claimed in claim 1 in preventing and controlling mold contamination of tobacco products, characterized in that: The mold is Aspergillus versicolor, Aspergillus monteridium, Aspergillus pseudoglaucus and / or Penicillium chrysogenum.

3. The use of Bacillus Velezii in preventing and controlling mold contamination of tobacco products according to claim 2, characterized in that: The bacterial liquid of Bacillus Velez is sprayed into tobacco products.

4. The use of Bacillus Velezii in preventing and controlling mold contamination of tobacco products according to claim 3, characterized in that: The concentration of Bacillus Velez in the bacterial solution is not less than 10 7 CFU / mL.

5. The use of Bacillus Velezii according to any one of claims 2 to 4 in preventing and controlling mold contamination of tobacco products, characterized in that: The tobacco products include cigar tobacco leaves, cigarettes and strip tobacco.

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