Bacillus altitudinis strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus altitudinis strain and application thereof, the bacillus altitudinis strain is a bacillus altitudinis strain H50 and has been preserved in China General Microbiological Culture Collection Center (CGMCC) on December 16, 2024, and the preservation number is CGMCC No.33070. The bacillus altitudinis strain is a bacillus altitudinis strain H50 which has been preserved in China General Microbiological Culture Collection Center (CGMCC). The bacillus altitudinis strain H50 disclosed by the invention is a strain with broad-spectrum antibacterial activity, and the strain particularly has an obvious antagonistic effect on tritirachium album, so that effective treatment on tritirachium album and escherichia coli infection is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bacillus subtilis strain and application thereof. Background Art

[0002] Candida albicans, also known as white Candida, is widely found in nature and in normal human skin, oral cavity, upper respiratory tract, intestine and vagina. It is an opportunistic fungal pathogen and an important foodborne pathogen. In recent years, the incidence of Candida albicans infection has been increasing year by year. Due to its own characteristics, it is easy to form biofilm on the surface of biomaterials and is highly resistant to most existing antifungal drugs, which brings great difficulties to clinical treatment. Therefore, it is particularly important to find new strategies to treat this systemic fungal disease.

[0003] Biological control is considered to be a more friendly and safe method. As a complex, open and changing biological and organic system, the ocean has a wide distribution of bacteria and many types. The active substances produced are diverse and have novel and unique molecular structures. They are widely used in the development of new drugs. Among them, Bacillus is a dominant microbial flora with many types and wide distribution in nature. Most of them are non-pathogenic and harmless to humans and animals, and can produce a variety of antibacterial substances, such as lipopeptides, peptides, phospholipids, bacteriocins, etc. These substances have the effect of inhibiting the growth of fungal hyphae and spore germination. Therefore, people focus on screening strains with antagonistic effects on pathogenic microorganisms in marine microorganisms for biological control. In recent years, through the screening and research of marine microbial resources, many excellent strains of marine microorganisms with antibacterial activity have been discovered. For example, the GM-1-1 strain of Bacillus amyloliquefaciens has been shown to have a strong inhibitory effect on a variety of pathogens. Studies have shown that Bacillus amyloliquefaciens MR14M3 can produce metabolites with antifungal activity. These metabolites act on Candida albicans by diffusion, significantly reducing its cell size and even causing cell rupture. The study also evaluated the cytotoxicity of MR14M3 culture fluid to human fibroblasts (HFF-1) through the MTT experiment. The results showed no significant cytotoxicity, indicating that MR14M3 has high safety in biological control applications. Therefore, the application of biological control strategies will reduce the drug resistance of Candida albicans and promote the development of new antifungal drugs. At present, the main technologies used for plant control by Bacillus are as follows:

[0004] The Chinese invention patent publication number is CN114921358 A, and its name is a method for preparing a biocontrol agent for preventing and treating tea anthracnose, which discloses the following steps: fermenting the Bacillus altitudinis GS-16 to obtain a fermentation product, that is, a biocontrol agent. The endogenous Bacillus altitudinis provided by the present invention has a strong inhibitory effect on Colletotrichum gloeosporioides of tea leaves, and its antibacterial mechanism is mainly to destroy the mycelial growth of the pathogenic bacteria of Colletotrichum gloeosporioides, and to affect the integrity of the cell membrane, protein synthesis, energy metabolism, and damage to the cell structure of Colletotrichum gloeosporioides. At the same time, it has an inhibitory effect on Colletotrichum japonicum, Fusarium oxysporum, Fusarium graminearum, Alternaria alternata, Fusarium monoseptum, Cucumis sativus, White capsule rake tooth and Alternaria alternata, and has a broad spectrum of antibacterial activity.

[0005] The Chinese invention patent publication number is CN112608868A, which discloses that Bacillus subtilis 181-7 exhibits antibacterial effects on rice bacterial leaf blight and rice bacterial leaf streak, and also has antibacterial effects on banana bacterial wilt, bean wilt, soybean scab, and some pathogenic fungi such as gray mold, phytophthora, and graminearum fusarium. The results of the present invention show that the strain has a broad-spectrum antibacterial ability, provides a good biocontrol resource for the prevention and control of rice bacterial leaf blight and bacterial leaf streak, and lays a foundation for the exploration of new microbial agents.

[0006] At present, there is no application of Bacillus for inhibiting or treating infections or diseases caused by Candida albicans. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a strain of Bacillus subtilis and its application, which can prevent and treat Candida albicans by using microbial agents. The technical solution used to achieve the purpose of the present invention is:

[0008] The first object of the present invention is to provide a Bacillus altitudinis strain, which is the Bacillus altitudinis strain H50, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 16, 2024, with a deposit number of CGMCC No.33070, and a classification name of: Bacillus altitudinis.

[0009] The 16S rDNA sequence of Bacillus altitudinis strain H50 is 100% similar to that of Bacillus altitudinis41KF2b in the literature. The evolutionary tree of the bacterium is as follows: Figure 1 shown.

[0010] In one embodiment of the present invention, the suitable growth temperature of the Bacillus subtilis strain H50 is 35-37°C.

[0011] The second object of the present invention is to provide a microbial agent containing the above-mentioned Bacillus subtilis and / or the fermentation broth of the above-mentioned Bacillus subtilis.

[0012] The third object of the present invention is to provide a method for preparing a microbial agent, comprising the following steps:

[0013] The highland Bacillus strain H50 was inoculated into a liquid culture medium, and cultured by shaking at a temperature of 35-37° C. for 24-48 hours to obtain a culture solution.

[0014] In one embodiment of the invention, the liquid culture medium is any one of YPD liquid culture medium, Sabouraud liquid culture medium or LB liquid culture medium.

[0015] The fourth object of the present invention is to provide a use of the microbial agent as described above in preventing Candida albicans.

[0016] The present invention further provides a use of the above-mentioned microbial agent in preventing Escherichia coli.

[0017] In particular, the present invention provides a use of the above-mentioned Bacillus subtilis strain H50 in a biological control agent.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention uses the highland Bacillus strain H50 to antagonize Candida albicans, achieving effective treatment of Candida albicans infection, which is an application of biological control strategy, will reduce the drug resistance of Candida albicans, thereby promoting the development of new antifungal drugs. And experiments have verified that the highland Bacillus strain H50 also has an antibacterial effect on Escherichia coli.

[0020] The conditions required for the preparation of the microbial agent containing the bacteria are simple and easy to obtain, the method is simple and easy, and the application of biological control strategies will reduce the drug resistance of Candida albicans and Escherichia coli, thereby promoting the development of new antifungal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the phylogenetic tree of the 16srDNA sequence of Bacillus subtilis strain H50.

[0022] Figure 2 This is the colony morphology of Bacillus subtilis strain H50 on YPD solid medium.

[0023] Figure 3 This is a diagram showing the confrontation effect of Bacillus subtilis strain H50 against Candida albicans (double-layer plate method). DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental method of the present invention adopts the general experimental method of this area, and the reagents or instruments of the present invention are conventional reagents that can be obtained commercially without indicating the manufacturer. It should be understood that the following description is only for explaining the present invention and is not intended to limit its content.

[0025] A Bacillus subtilis strain, the Bacillus subtilis strain is Bacillus subtilis strain H50, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 14, 2024, with the deposit number CGMCC No.33070.

[0026] The 16S rDNA sequence of Bacillus altitudinis strain H50 is 100% similar to that of Bacillus altitudinis41KF2b in the literature. The evolutionary tree of the bacterium is as follows: Figure 1 shown.

[0027] In one embodiment, the suitable growth temperature of the Bacillus subtilis strain H50 is 35-37°C.

[0028] The present invention also provides a microbial agent containing the above-mentioned Bacillus subtilis and / or the fermentation liquid of the above-mentioned Bacillus subtilis.

[0029] In one embodiment, the preparation method of the microbial agent comprises the following steps:

[0030] The highland Bacillus strain H50 was inoculated into a liquid culture medium, and cultured by shaking at a temperature of 35-37° C. for 24-48 hours to obtain a culture solution.

[0031] In one embodiment, the liquid culture medium is any one of YPD liquid culture medium, Sabouraud liquid culture medium or LB liquid culture medium.

[0032] The present invention also provides a use of the microbial agent as described above in preventing Candida albicans.

[0033] The present invention further provides a use of the above-mentioned microbial agent in preventing Escherichia coli.

[0034] In particular, the present invention provides a use of the above-mentioned Bacillus subtilis strain H50 in a biological control agent.

[0035] Application Examples

[0036] The specific steps of using Bacillus subtilis strains in biological control against Candida albicans are:

[0037] The culture medium formulations described in the following examples are as follows:

[0038]

[0039]

[0040] Isolation and purification of strains:

[0041] The purified strain H50 was obtained by diluting the seawater samples collected from Shijiao Mangrove Nature Reserve (21°36'56"N, 108°13'55"E) in Fangchenggang City, Guangxi Zhuang Autonomous Region using the dilution plate method and the plate streak method. The genomic DNA was extracted using the FastPure Bacteria DNA Isolation Mini Kit, and the 16S rRNA gene was amplified by PCR using the universal bacterial identification primers 27F and 1492R. The strain was then sent to BGI for sequencing. NCBI BLAST confirmed that the strain H50 was Bacillus altaica. Its phylogenetic tree is shown in the following figure. Figure 1 shown.

[0042] Strain H50 was inoculated on a YPD solid medium plate and incubated at 37°C for 24 h. The morphological characteristics of the colonies were observed. Figure 2 .

[0043] Antibacterial phenotype:

[0044] Double-layer plate method: Streak Bacillus subtilis H50 on a YPD plate and culture at 37°C overnight. Pick a single colony of Candida albicans and shake it, then culture it at 37°C overnight. After 24 hours, adjust the OD600 of Candida albicans to 0.5, prepare 100 ml YPD solid medium, add 200 μl of Candida albicans with OD600=0.5, pour double layers, and culture at 37°C for 24 hours. Observe the results, such as Figure 3 .

[0045] Antibacterial rate:

[0046] 1. Test tube method

[0047] 1. Shake the bacteria: Bacillus subtilis H50 and Candida albicans 2310223029, 2310223003 were all grown in YPD liquid medium and cultured at 37°C for 24 hours.

[0048] 2. Transfer: After 24 hours, transfer the bacterial solution to the new culture medium at a ratio of 1:100 and culture the bacteria at 37℃ with shaking for 24 hours.

[0049] ①Experimental group: Bacillus subtilis H50+Candida albicans 2310223029, Bacillus subtilis H50+Candida albicans 2310223003.

[0050] ②Control group: Candida albicans 2310223029, 2310223003.

[0051] 3. Coating: Dilute the bacterial solution of the experimental group and the control group with PBS to 10 -1 ~10 -6 , the experimental group took 10 -3 ~10 -5 100 μl of the gradient bacterial solution was coated on Sabouraud plates, and 10 -5 ~10 -6 100 μl of the gradient bacterial solution was spread on Sabouraud plates, repeated 3 times, and cultured in a 37°C incubator. The colonies were counted after 24 hours.

[0052] 4. Colony count: The culture of Candida albicans alone is CK, and the co-culture of Bacillus subtilis H50 and Candida albicans is T. The antibacterial rate is calculated as follows:

[0053]

[0054] The results are as follows:

[0055] Indicator bacteria Antibacterial rate (%) Candida albicans 2310223029 61.13 Candida albicans 2310223003 46.12

[0056] The antibacterial rate of Bacillus subtilis H50 against Candida albicans was verified by repeating the test tube method with 10 strains of Candida albicans from different sources. The results are as follows:

[0057]

[0058]

[0059] Test tube method to measure the antibacterial rate of Bacillus subtilis H50 against clinically important pathogens:

[0060] 1. Shaking bacteria: Antagonistic bacteria: Bacillus subtilis H50 and indicator bacteria: Klebsiella pneumoniae 47434, Acinetobacter baumannii 17978, Escherichia coli 73552, all were shaken in LB liquid culture medium and cultured at 37°C for 24 hours.

[0061] 2. Transfer: After 24 hours, transfer the bacterial solution to the new culture medium at a ratio of 1:100 and culture the bacteria at 37℃ with shaking for 24 hours.

[0062] ①Experimental group: H50+47434, H50+17978, H50+73552.

[0063] ②Control group: 47434, 17978, 73552.

[0064] 3. Coating: Dilute the bacterial solution of the experimental group and the control group with PBS to 10-1 ~10 -6 , H50+47434, H50+17978, H50+73552 are 10 respectively -3 ~10 -5 100 μl of the gradient bacterial solution was coated on MEM = 4 μg / ml, CH1 = 25 μg / ml, GEN = 32 μg / ml resistance plates, and 10 μl of each of the control groups 47434, 17978, and 73552 were taken. -5 ~10 -6 100 μl of the gradient bacterial solution was coated on MEM=4 μg / ml, CH1=25 μg / ml, and GEN=32 μg / ml resistance plates respectively, repeated 3 times, placed in a 37°C incubator for culture, and colonies were counted after 24 hours.

[0065] 4. Colony count: The indicator bacteria cultured alone is CK, and the antagonistic bacteria and indicator bacteria cultured together is T. The antibacterial rate is calculated as follows:

[0066]

[0067]

[0068]

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention.

Claims

1. A strain of Bacillus subtilis, characterized in that The Bacillus subtilis strain is the Bacillus subtilis strain H50, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 16, 2024, with the deposit number CGMCC No.33070.

2. A Bacillus subtilis strain according to claim 1, characterized in that The suitable growth temperature of the Bacillus subtilis strain H50 is 35-37°C.

3. A microbial agent, characterized in that: A fermentation broth containing the Bacillus subtilis according to any one of claims 1 to 2 and / or the Bacillus subtilis according to any one of claims 1 to 2.

4. The microbial agent according to claim 3, characterized in that The preparation method of the microbial agent comprises the following steps: The highland Bacillus strain H50 was inoculated into a liquid culture medium, and cultured by shaking at a temperature of 35-37° C. for 24-48 hours to obtain a culture solution.

5. The microbial agent according to claim 4, characterized in that: The liquid culture medium is any one of YPD liquid culture medium, Sabouraud liquid culture medium or LB liquid culture medium.

6. Use of the microbial agent as described in any one of claims 3 to 5 in preventing Candida albicans.

7. Use of the microbial agent as described in any one of claims 3 to 5 in preventing Escherichia coli.

8. Use of the Bacillus subtilis strain H50 according to any one of claims 1 to 3 in a biological control agent.

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