A strain of Bacillus AK Bac-1, culture, fermentation supernatant, bacteriocin, preparation method and application thereof

Abstract

The present invention relates to the field of microbial technology, and relates to a strain of Bacillus AK Bac‑1, a culture, a fermentation supernatant bacteriocin, a preparation method and an application thereof, wherein the strain deposit number of the Bacillus AK Bac‑1 is CCTCC NO: M 20232684, and the deposit date is January 2, 2024. The culture, the fermentation supernatant and the extracted bacteriocin obtained from the screened Bacillus AK Bac‑1 can effectively inhibit the growth of multidrug-resistant Staphylococcus aureus, and provides a new idea for solving the problem of antibiotic resistance.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and relates to a strain of Bacillus AK Bac-1, culture, fermentation supernatant, bacteriocin, and a preparation method and application thereof. Background Art

[0002] In the livestock industry, Staphylococcus aureus can not only cause various diseases, such as mastitis, arthritis, endometritis, urinary tract infection and exudative dermatitis in piglets, but also its drug resistance is gradually increasing, making treatment more difficult. This problem has brought huge economic losses to the livestock industry. Therefore, many researchers and people engaged in the livestock industry are committed to finding new solutions.

[0003] The current solutions to the problem of bacterial resistance are: (1) Rational selection of drugs. After infection occurs, conduct drug sensitivity tests and select sensitive antimicrobial drugs for treatment based on the results. Avoid blindly using broad-spectrum antibiotics to reduce the emergence of drug-resistant strains. Advantages: Ensure that the infection is effectively treated and reduce the probability of the emergence of drug-resistant strains. Disadvantages: Drug sensitivity tests are required, which may delay treatment. (2) Combination therapy. For severe infections or situations where a single antimicrobial drug is ineffective, a combination therapy can be considered, using two or more antimicrobial drugs for simultaneous treatment to improve efficacy and reduce the emergence of drug resistance. Advantages: Improve treatment efficacy and reduce the emergence of drug resistance. Disadvantages: May cause drug interactions, increase treatment costs and complexity. (3) Develop new antimicrobial drugs. Actively develop new antimicrobial drugs, improve drug structure and treatment efficacy to respond to the emergence of drug-resistant strains. At the same time, simplify the drug approval process and accelerate the application of new antimicrobial drugs. Advantages: Response to the emergence of drug-resistant strains and provide new treatment options. Disadvantages: Long R&D cycle, high cost, and cumbersome approval process. (4) Establish a drug resistance monitoring system. Establish a complete drug resistance monitoring system to regularly monitor and analyze the development trend and spread of bacterial drug resistance to provide a basis for formulating effective prevention and control strategies. Advantages: Regularly monitor and analyze the development trend of drug resistance to provide a basis for formulating prevention and control strategies. Disadvantages: The establishment and maintenance costs of the monitoring system are high and require continuous investment. (5) Alternative treatment strategies. Develop Chinese herbal preparations to extract bioactive substances with antibacterial and antiviral effects to replace traditional antibiotic treatments. These substances have the characteristics of low toxicity and side effects and no drug resistance, and can be used as an alternative strategy to solve the problem of bacterial resistance. Advantages: Low toxicity and side effects, no drug resistance, and can replace traditional antibiotic treatment. Disadvantages: There are many types of Chinese herbal medicines, a wide screening range, and the antibacterial effect on pathogens is not specific and targeted.

[0004] Therefore, it is very important to find an efficient, low-cost microorganism that has an inhibitory effect on drug-resistant Staphylococcus aureus to solve the problem of increased drug resistance of Staphylococcus aureus faced in the aquaculture industry. Summary of the invention

[0005] The invention provides a strain of Bacillus AK Bac-1, a culture, a fermentation supernatant, a bacteriocin, a preparation method and an application thereof for screening out a strain capable of inhibiting the growth of Staphylococcus aureus. The Bacillus AK Bac-1 isolated by the invention has a very obvious antibacterial effect on multidrug-resistant Staphylococcus aureus.

[0006] The invention provides Bacillus altitudinis AKBac-1. The strain preservation number of the Bacillus altitudinis AKBac-1 is CCTCC NO: M 20232684, and the preservation date is January 2, 2024.

[0007] The present invention also provides an AKBac-1 strain culture prepared by the Bacillus subtilis AKBac-1.

[0008] The present invention also provides a method for preparing the AK Bac-1 strain culture, comprising the following steps: inoculating the Bacillus subtilis AK Bac-1 strain into a culture medium and culturing at 35-40° C. to obtain the culture of the Bacillus subtilis AK Bac-1 strain.

[0009] The present invention also provides a fermentation supernatant prepared by the Bacillus subtilis AKBac-1, wherein the fermentation supernatant contains bacteriocin.

[0010] The present invention also provides a method for preparing the fermentation supernatant, comprising the following steps: inoculating the Bacillus subtilis AK Bac-1 strain into a culture medium, culturing at 35-40°C, centrifuging at 7000-8000rpm for 10-15min, removing the bacteria, and retaining the supernatant, which is the fermentation supernatant of the Bacillus subtilis AK Bac-1 strain.

[0011] Furthermore, the culture medium is selected from any one of nutrient broth medium, LB liquid medium, NB liquid medium, and PDA medium.

[0012] The present invention also provides a bacteriocin produced by the Bacillus subtilis AKBac-1.

[0013] The present invention also provides a method for preparing the bacteriocin, comprising the following steps: first preparing a fermentation supernatant, adjusting the pH value of the fermentation supernatant to 1.8-2.2, keeping it at a low temperature overnight to produce a precipitate, then centrifuging at 10000-12000 rpm for 12-15 min, discarding the supernatant, and dissolving the precipitate with 2-5 mL PBS to obtain a crude bacteriocin.

[0014] The present invention also provides an application of the Bacillus subtilis AK Bac-1, the Bacillus subtilis AKBac-1 strain culture, the fermentation supernatant or the bacteriocin in inhibiting pathogenic bacteria.

[0015] Furthermore, the use of the Bacillus subtilis AK Bac-1, Bacillus subtilis AKBac-1 strain culture, fermentation supernatant or bacteriocin in inhibiting pathogenic bacteria is multidrug-resistant Staphylococcus aureus.

[0016] The present invention also provides a product comprising the culture of the Bacillus subtilis AK Bac-1 strain, which is a wettable powder, a water-dispersible granule, a suspension, a suspoemulsion, an aqueous emulsion or a microemulsion.

[0017] Furthermore, the product is prepared by fermenting the culture of the Bacillus subtilis AKBac-1 strain through conventional liquid fermentation, and then adding a surfactant or an adsorption carrier and mixing them in a certain proportion.

[0018] Furthermore, the surfactant is any one or more of a dispersant, a stabilizer, a wetting agent, a binder, a defoaming agent, a disintegrant, an antifreeze agent, and the like.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. In the present invention, a strain of Bacillus subtilis AKBac-1 was isolated and identified, and it was found that its culture, fermentation supernatant and crude bacteriocin had obvious antibacterial effects on multidrug-resistant Staphylococcus aureus, among which the crude bacteriocin had the best antibacterial effect, with an inhibition zone diameter of 18 mm.

[0021] 2. The pathogenic Staphylococcus aureus tested in the present invention is a multi-drug resistant bacterium. A bacterial drug sensitivity test was performed on it, and it was found to be multi-drug resistant to erythromycin, tetracycline, ciprofloxacin, norfloxacin and clindamycin.

[0022] 3. The current multi-drug resistance problem of pathogenic Staphylococcus aureus is relatively serious in the breeding industry. Therefore, the present invention has significant therapeutic effects in the prevention and treatment of the pathogenicity of multi-drug resistant Staphylococcus aureus, and has broad application prospects in the future, such as being used as a feed additive to regulate the intestinal flora of animals, being non-toxic, harmless, and having no residue, and having an obvious antibacterial effect, which can significantly improve the resistance of animals to pathogenic Staphylococcus aureus, thereby reducing the use of traditional antibacterial drugs in veterinary clinics, reducing economic losses, and reducing environmental pollution.

[0023] Information on the deposit of biological materials

[0024] AK Bac-1, referred to as Bacillus altitudinis AK Bac-1 in this application, has been deposited in the China Center for Type Culture Collection on January 2, 2024, with a deposit number of CCTCC NO: M 20232684. The address of the depository is: Wuhan University, Wuhan, China, Postal Code: 430072, and the classification name is Bacillus altitudinis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The common nutrient agar culture characteristics of Bacillus subtilis AKBac-1 in the present invention;

[0027] Figure 2 The blood agar culture characteristics of Bacillus subtilis AKBac-1 in the present invention;

[0028] Figure 3 The TSA agar culture characteristics of Bacillus subtilis AKBac-1 in the present invention;

[0029] Figure 4 The results of Gram staining microscopy of Bacillus subtilis AKBac-1 in the present invention;

[0030] Figure 5 The biochemical test results of Bacillus AK Bac-1 of the present invention are shown, wherein from right to left, 2 tubes of maltose, 2 tubes of lactose, 2 tubes of sucrose and 2 tubes of glucose are shown respectively;

[0031] Figure 6 The biochemical test results of Bacillus subtilis AK Bac-1 of the present invention are shown from right to left, respectively representing the indole formation test, methyl red test, and diacetyl test;

[0032] Figure 7 This is a genetic evolution tree constructed based on the AKBac-116S rRNA gene sequence of Bacillus subtilis in the present invention; X-1 in the figure represents the AKBac-1 strain sequence in the present invention, and other strains are represented by GenBank accession numbers.

[0033] Figure 8The results are as follows: the inhibition of Bacillus subtilis AK Bac-1 against multi-drug resistant Staphylococcus aureus, wherein the plate markers represent: 1-crude bacteriocin, 2-fermentation supernatant, 3-culture.

[0034] Fig. 9 The results of the drug sensitivity test of the tested bacteria (multi-drug resistant Staphylococcus aureus) in the present invention are: 1-erythromycin, 2-tetracycline, 3-ciprofloxacin, 4-norfloxacin, and 5-clindamycin. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0036] Example 1: Isolation and screening of a strain of Bacillus subtilis AK Bac-1 and its application.

[0037] The present invention is a bacterium accidentally discovered on a nutrient agar medium for culturing multi-drug resistant Staphylococcus aureus in a laboratory. The bacterium is characterized by neat edges, moist surface, slight bulges, medium-sized milky white round colonies on the surface of the culture medium, and no Staphylococcus aureus grows around the colonies. The present invention isolates, cultures and purifies the strain of bacteria, and then conducts studies such as bacterial Gram staining microscopy, biochemical tests, PCR molecular biological identification, and antimicrobial drug sensitivity tests.

[0038] 1. Isolation and identification of strains

[0039] 1. Isolation, culture and purification

[0040] The bacteria to be tested can grow when inoculated on ordinary nutrient agar, blood agar, tryptone soy agar (TSA) and other media. The colonies are white, medium-sized, with irregular edges, dry surface, wrinkled surface, and opaque. The results are shown in Figure 1 , Figure 2 , Figure 3 The growth of the culture medium after culturing in nutrient broth medium is manifested by turbidity of the culture medium and flocculent precipitation at the bottom.

[0041] Nutrient broth medium formula: 5 g beef extract, 10 g peptone, 1 g dipotassium hydrogen phosphate, 5 g sodium chloride, 1000 mL distilled water.

[0042] 2. Smear staining and microscopy

[0043] A single colony was picked up after purification to prepare a bacterial smear, and then Gram staining was performed under a microscope. It was found that the strain was rod-shaped, most of which were scattered in a single arrangement, and a few were arranged in chains. It was able to form spores. After Gram staining, the bacteria were blue-purple, and it was a Gram-positive Bacillus. The results are shown in Figure 4 .

[0044] 3. Biochemical test

[0045] Glucose, lactose, maltose, sucrose fermentation tests, methyl red test, diacetyl test and indole formation test were carried out respectively. Use an inoculation loop or inoculation needle to inoculate the bacteria to be tested into peptone water and glucose peptone water, as well as sugar fermentation tubes (glucose, lactose, maltose, sucrose), and culture at 37°C for 24 hours. The results showed that it can decompose glucose and sucrose, but not lactose and maltose. It only produces acid for glucose, and the color changes from purple to yellow, and no bubbles are produced. The methyl red test and indole formation test were both negative, with no color change and still light yellow; the diacetyl test was positive, and the color changed from yellow to red. The results of the sucrose, lactose, glucose and maltose fermentation tests are shown in Figure 5 , the results of methyl red test, diacetyl test and indole formation test are shown in Figure 6 .

[0046] 4. Molecular identification

[0047] The universal primers 27F and 1492R of bacterial 16S rRNA gene were used for PCR molecular biological identification;

[0048] (1) Nucleic acid extraction: Use an inoculation loop to pick a single colony of the strain and inoculate it into broth culture medium, and culture it at 37°C and 180 rpm for 24 hours. Then use the boiling method to extract nucleic acid: take out 1 mL of bacterial solution and put it into a 1.5 mL centrifuge tube, centrifuge it at 5000 rpm for 5 minutes, remove the supernatant, dissolve the bacterial precipitate with 200 μL of sterile water, boil it at 100°C for 10 minutes, ice bath it for 5 minutes, and finally centrifuge it at 12000 rpm for 5 minutes. The supernatant after centrifugation is the DNA template for PCR reaction.

[0049] (2) PCR amplification and sequencing analysis of 16S rRNA: The PCR reaction system was 50 μL: PCR MasterMix 25 μL, upstream primer 2 μL, downstream primer 2 μL, DNA template 5 μL, sterile water 16 μL. PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 32 cycles; 72°C for 10 min. The PCR product was detected by agarose gel electrophoresis and sent to Shanghai Sangon Biotechnology Technology Service Co., Ltd. for sequencing.

[0050] (3) The gene sequencing results were compared with the nucleic acid sequences published in GenBank for homology analysis and phylogenetic tree analysis. It was found that it was located in the same branch as the 16S rRNA gene of Bacillus subtilis IRHB1-80 strain (GenBank accession number: OP364558.1), with the closest relationship and the highest homology. The results are shown in Figure 7 .

[0051] Table 1 Detailed information of reference strains of Bacillus subtilis

[0052] GenBank Accession Number Strain name source KT003254.1 HN-8 Spacecraft surface OP364558.1 IRHB1-80 Soybean root system MZ713012.1 IRHB1-80 Gray tea looper intestine OP104190.1 EMJ43 Agaricus bisporus cap and stipe OR462724.1 h217 - MZ895387.1 HY-6 ocean KC172039.1 SH91 soil OL989110.1 D5-13 - MN511801.1 JYM50 - MK070089.1 D30102 - OR594194.1 SE14 - ON514240.1 GTB22 spider MW025980.1 AS13 Rhizosphere of Paris polyphylla

[0053] 2. Antibacterial Test

[0054] 1. Preparation of AK Bac-1 strain culture

[0055] The Bacillus subtilis AKBac-1 strain was inoculated into a test tube containing 5 mL of nutrient broth medium, and cultured at 37° C. and 180 rpm for 24 hours to obtain a culture of the Bacillus subtilis AKBac-1 strain.

[0056] 2. Preparation of fermentation supernatant

[0057] The Bacillus subtilis AK Bac-1 strain was inoculated into a 250 mL conical flask containing 100 mL nutrient broth medium, cultured at 37° C., 180 rpm shaking for 24 h, centrifuged at 7000 rpm for 15 min, the bacteria were removed, and the supernatant was retained as the fermentation supernatant of the Bacillus subtilis AK Bac-1 strain.

[0058] 3. Crude extraction of bacteriocin by acid precipitation

[0059] The pH value of the fermentation supernatant was slowly adjusted to 2.0 with concentrated hydrochloric acid, and precipitated in a refrigerator at 4°C overnight. The supernatant was then centrifuged at 12,000 rpm for 15 min, and the supernatant was discarded. The precipitate was dissolved with 2 mL of PBS to obtain the crude bacteriocin, which was stored at -20°C for later use.

[0060] 4. Determination of antibacterial effect by drug sensitive paper method

[0061] ① Preparation of drug sensitivity sheets: Soak blank drug sensitivity sheets (without drugs) in 2 mL cryovials containing 1 mL of Bacillus subtilis AKBac-1 strain culture, crude bacteriocin, and fermentation supernatant, respectively. Soak 10 round paper sheets in each tube. Take them out after soaking for 40 minutes, dry them naturally in a clean bench after ultraviolet irradiation, and set them aside.

[0062] ② Cultivation of pathogenic bacteria: Inoculate the multidrug-resistant Staphylococcus aureus preserved in this laboratory (recorded in the article "Diagnosis and Treatment of Mixed Intestinal Infection of Salmonella and Staphylococci in Geese. Lu Zhenxiang et al. Chinese Veterinary Journal", the strain name in the article is "Strain 1") into 5 mL of nutrient broth culture medium, and culture at 37°C and 180 rpm for 24 hours.

[0063] ③ Antibacterial test: Take out 50μL of multidrug-resistant Staphylococcus aureus liquid and drop it onto ordinary nutrient agar plates. Use a sterilized spreading stick to evenly spread the liquid. After slightly drying, place the round paper pieces soaked with Bacillus subtilis AKBac-1 strain culture, bacteriocin, and fermentation supernatant on the agar plates and mark them. After culturing at 37℃ for 24h, the inhibition zone was determined. The results showed that the AK Bac-1 strain culture, fermentation supernatant, and crude bacteriocin had a very obvious antibacterial effect on Staphylococcus aureus, with the diameters of the inhibition zones being 15mm, 17mm, and 18mm, respectively. See details. Figure 8 .

[0064] ④ Drug resistance test of the test bacteria: Take out 50μL of the test bacteria solution (multi-drug resistant Staphylococcus aureus) and spread it evenly on the ordinary agar medium with a glass-spreading rod. Use tweezers to pick up 5 kinds of antimicrobial drug sensitive tablets and place them on the ordinary agar medium coated with bacteria, and mark them. After culturing at 37℃ for 12h, the inhibition zone is determined. The results show that the test bacteria are resistant to 1-erythromycin, 2-tetracycline, 3-ciprofloxacin, 4-norfloxacin, and 5-clindamycin, and no inhibition zone appears. For details, see Fig. 9 .

[0065] At present, the problem of multidrug resistance of Staphylococcus aureus has become a serious problem both in human life and in the animal breeding industry. Therefore, the present invention has a good effect on preventing diseases caused by multidrug-resistant Staphylococcus aureus. The Bacillus subtilis AKBac-1 strain of the present invention and the bacteriocin secreted and produced can be used as a feed additive to regulate the intestinal flora of animals. It is non-toxic, harmless, and has no residue, and has an obvious antibacterial effect. It can significantly improve the resistance of animals to pathogenic Staphylococcus aureus, thereby reducing the use of traditional antibiotics in veterinary clinics. In addition, further improving the extraction and purification methods and coating methods of bacteriocin can greatly enhance the antibacterial effect of bacteriocin. Therefore, the present invention can be applied to the research and development of new feed additives, and also lays the foundation for the research and development of new antibiotics, thereby ensuring the health and production benefits of farmed animals.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Bacillus subtilis ( Bacillus altitudinis ) AK Bac-1, characterized in that The strain preservation number of the Bacillus subtilis AK Bac-1 is: CCTCC NO: M 20232684, and the preservation date is January 2, 2024.

2. A culture of AK Bac-1 strain prepared by the Bacillus subtilis AK Bac-1 according to claim 1, characterized in that: The method for preparing the AK Bac-1 strain culture comprises the following steps: inoculating the Bacillus subtilis AK Bac-1 strain into a culture medium and culturing at 35-40° C. to obtain the culture of the Bacillus subtilis AK Bac-1 strain.

3. A fermentation supernatant prepared by the Bacillus AK Bac-1 according to claim 1, characterized in that: The fermentation supernatant contains bacteriocin.

4. A method for preparing the fermentation supernatant according to claim 3, characterized in that: The method comprises the following steps: inoculating the Bacillus subtilis AK Bac-1 strain into a culture medium, culturing at 35-40°C, centrifuging at 7000-8000 rpm for 10-15 min, removing the bacteria, and retaining the supernatant as the fermentation supernatant of the Bacillus subtilis AK Bac-1 strain.

5. The method for preparing the AK Bac-1 strain culture according to claim 2 or the method for preparing the fermentation supernatant according to claim 4, characterized in that: The culture medium is selected from any one of nutrient broth medium, LB liquid medium, NB liquid medium, and PDA medium.

6. A method for preparing bacteriocin produced by Bacillus subtilis AK Bac-1 according to claim 1, characterized in that: The method specifically comprises the following steps: firstly preparing a fermentation supernatant, adjusting the pH value of the fermentation supernatant to 1.8-2.2, keeping the supernatant at low temperature overnight to produce a precipitate, then centrifuging at 10000-12000 rpm for 12-15 min, discarding the supernatant, dissolving the precipitate with 2-5 mL PBS, and obtaining a crude bacteriocin.

7. Use of the Bacillus AK Bac-1 according to claim 1, the Bacillus AK Bac-1 strain culture according to claim 2, the fermentation supernatant according to claim 3 or the bacteriocin according to claim 6 in inhibiting pathogenic bacteria, characterized in that: The pathogen is multidrug-resistant Staphylococcus aureus ( Staphylococcus aureus ).

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