Yak-derived bacillus subtilis and application thereof in drug sensitivity and antibacterial activity

Abstract

The invention discloses yak-derived bacillus subtilis and application of the yak-derived bacillus subtilis in drug sensitivity and antibacterial activity, and relates to the technical field of probiotics. The yak source bacillus subtilis disclosed by the invention is bacillus subtilis H4, and the preservation number of the yak source bacillus subtilis is CGMCC (China General Microbiological Culture Collection Center) No.31580. The invention also provides a microbial inoculum prepared from the bacillus subtilis H4. The invention also provides application of the bacillus subtilis H4 in preparation of a probiotic preparation for improving drug sensitivity and antibacterial activity. The invention also provides application of the bacillus subtilis H4 in preparation of a probiotic preparation for improving cholate resistance and high temperature resistance. The bacillus subtilis H4 disclosed by the invention accords with basic characteristics of bacillus, has relatively strong bacterial strain bacteriostatic activity and antibiotic sensitivity, has good biological characteristics, also has relatively good cholate resistance and high temperature resistance, can be used as a candidate bacterial strain for development of a yak-source microecological preparation, and has a wide application prospect. And an excellent probiotic strain is provided for further development of a special yak microbial preparation.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, in particular to a yak-derived Bacillus subtilis and applications of the same in terms of drug sensitivity and bacteriostasis. Background Art

[0002] In recent years, probiotic preparations have received much attention and have been widely developed and applied to avoid the hazards of DNA contamination, bacterial drug sensitivity, and even super bacteria caused by the use of antibiotics. Probiotics can improve the balance of animal gastrointestinal tract, promote animal growth, increase feed utilization, enhance animal health, and improve the quality of livestock products and breeding environment.

[0003] Bacillus subtilis is a spore-forming bacterium. Spores are a form of bacteria that resists harsh environments and can survive under adverse conditions such as high temperature, ultraviolet rays, dryness, and chemical disinfectants. Therefore, Bacillus subtilis has diverse physiological characteristics and good tolerance and adaptability, disease resistance, and stress resistance. It is widely used in livestock and poultry farming, and usually has the effects of improving livestock and poultry growth performance, improving intestinal flora balance, improving immune levels, and improving feeding environments. Therefore, it is often added to livestock and poultry feed as a microorganism with probiotic functions, becoming an important intestinal probiotic. As one of the two Bacillus species in feed additives, Bacillus subtilis has broad development prospects and huge ecological and social benefits. Since probiotics for yaks have certain requirements for drug sensitivity and antibacterial properties, and they also need to have good bile salt tolerance and high temperature resistance, the candidate strains that meet the conditions are limited. Therefore, we propose a yak-derived Bacillus subtilis and its drug sensitivity and antibacterial properties to solve the above-mentioned problems. Summary of the invention

[0004] The purpose of the present invention is to provide a yak-derived Bacillus subtilis and its drug sensitivity and antibacterial application to solve the problems in the current market raised by the above background technology.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a yak-derived Bacillus subtilis, wherein the yak-derived Bacillus subtilis is Bacillus subtilis H4, which is separated from healthy yak feces. The yak-derived Bacillus subtilis is named Bacillus subtilis H4, with a preservation number of CGMCC No.31580, a preservation date of August 14, 2024, a preservation classification name of Bacillus subtilis, a preservation unit of the General Microbiology Center of the China Microbiological Culture Collection Administration, and an address of the preservation unit of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a classification name of Bacillus subtilis.

[0006] The present invention also provides a bacterial agent prepared by the above-mentioned Bacillus subtilis H4.

[0007] The present invention also provides the use of the above-mentioned Bacillus subtilis H4 in the preparation of a probiotic preparation with improved drug sensitivity and antibacterial properties.

[0008] The probiotic preparation is in the form of powder or tablet.

[0009] The present invention also provides the use of the above-mentioned Bacillus subtilis H4 in the preparation of a probiotic preparation having improved bile salt tolerance and high temperature resistance.

[0010] The probiotic preparation is in the form of powder or tablet.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The Bacillus subtilis H4 of the present invention meets the basic characteristics of Bacillus, has strong antibacterial activity and antibiotic sensitivity, has good biological properties, and has good bile resistance and high temperature resistance. It can be used as a candidate strain for the development of yak-derived microecological preparations, and provides an excellent probiotic strain for further developing yak-specific microbial preparations.

[0013] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of morphological identification of Bacillus subtilis; A. MRS agar culture medium colony morphology; B. Gram staining (10×100);

[0015] Figure 2 The figure is a schematic diagram of the genomic DNA agarose gel detection results; where M is DNA Marker DL 5000; 1 is H3; 2 is H4;

[0016] Figure 3 Schematic diagram of the phylogenetic tree of isolate H4. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The invention provides a yak-derived Bacillus subtilis H4 separated from healthy yak feces.

[0019] Sample source: The isolated samples of the present invention were collected from a yak herd in Ganzi Prefecture. Thirty portions of fresh feces of healthy yaks were randomly collected using sterile sampling bags, placed in ice bags, brought back, and stored at -80°C.

[0020] Rough sample processing: Take 5g of fecal sample and add it to 45mL of nutrient broth culture medium, culture it at constant temperature and shaking for 15min, then treat it in an 80℃ water bath for 15min, and then centrifuge it in a centrifuge at 4000r / min for 15min, aspirate the upper liquid, inoculate it into nutrient broth culture medium, culture it at constant temperature and shaking for 2-3h again, and finally transfer it to a 37℃ constant temperature incubator and culture it for 24h.

[0021] Isolation and culture of Bacillus subtilis: Use an inoculation loop to dip the culture and inoculate it into soluble starch medium, MRS agar medium, and LB agar medium respectively, and perform streak separation and culture for 24 hours. Select single colonies that are white, dry, and have raised and wrinkled surfaces and are suspected to be Bacillus subtilis growing on the surface of the culture medium, and perform staining and microscopic examination. After multiple isolation, purification and culture, it was found that the suspected Bacillus subtilis grew best on MRS agar medium. Pick a single colony from the culture medium and inoculate it on MRS culture medium for isolation, purification and culture.

[0022] The Bacillus subtilis isolated from the feces of a yak herd in Ganzi Prefecture was identified by comprehensive identification including bacterial morphological observation, biochemical identification and 16S rRNA sequence analysis. Ten strains of Bacillus subtilis were isolated and identified from the samples, and the two strains with the best morphological identification were selected and named H3 and H4.

[0023] 1. Morphological identification: The isolated strains were streaked and inoculated on MRS solid medium, cultured at 37°C for 24 hours, and the morphological characteristics of the colonies were observed. Secondly, bacterial slides were prepared, and a thin layer of the isolated strains was smeared on a glass slide using the Gram staining method and fixed. After four steps of crystal violet primary staining, iodine solution mordanting, ethanol decolorization, and safranin counterstaining, the smear was dried and placed under a microscope for observation.

[0024] Results: The colony morphology is round or irregular (such as Figure 1 A), dry, moist and opaque, raised and wrinkled, white or yellow in color; short rod-shaped under a microscope, purple in Gram stain (such as Figure 1 B), indicating that the isolated strain is a Gram-positive bacterium.

[0025] 2. Biochemical identification: Follow the instructions for use of the Bacillus subtilis biochemical identification tube. Physiological and biochemical characteristics including VP test, nitrate reduction test, propionate utilization test, citrate utilization test, D-xylose fermentation, L-arabinose fermentation, mannitol fermentation, 7% sodium chloride growth test, starch hydrolysis test and pH determination are all referred to "Lactic Acid Bacteria Classification and Test Methods" and "Common Bacteria System Identification Manual".

[0026] Table 1. Results of biochemical identification of 2 isolates

[0027]

[0028] Results: As shown in Table 1, the results of VP test tube, nitrate test tube, starch hydrolysis tube, citrate utilization tube, D-xylose fermentation tube, L-arabinose fermentation tube, mannitol fermentation tube, 7% sodium chloride growth tube and pH5.7 growth tube were positive; the result of propionate utilization tube was negative, and aerobic growth was required. According to the staining characteristics, biochemical tests and colony morphology of the isolated bacteria, the identification results were consistent with the characteristics of Bacillus subtilis.

[0029] 3. Molecular identification:

[0030] (1) According to the instructions of the Bacteria Genomic DNA Kit (centrifugal column type), genomic DNA of the isolated strain was extracted.

[0031] (2) PCR amplification was performed using the extracted genomic DNA. The 16S rRNA amplification primers of Bacillus subtilis in the literature "Xie Xiaopei, Wang Qi, Kong Xinping, et al. Isolation, culture and identification of Bacillus subtilis from Tibetan yaks [J]. Journal of Animal Ecology, 2021, 42(03): 26-31" were used. The upstream primer was 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, and the downstream primer was 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The PCR reaction system (25 μL) is shown in Table 2.

[0032] Table 2. PCR reaction system

[0033]

[0034] Add all reagents in sequence according to the above PCR reaction system, place in a gene amplification instrument and start amplification.

[0035] (3) After PCR, the amplified product was subjected to 1% agarose gel electrophoresis (electrophoresis conditions: 130 v, 20 min), and the results were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA sequencing. The BLAST program in NCBI was used to query strains in GenBank with similar sequencing results to isolate H4 for comparison, and finally a phylogenetic tree was constructed using MEGA11.

[0036] Results: DNA was extracted using the kit, and the genomic DNA agarose gel test results showed that the PCR band of H4 was around 1500 bp (such as Figure 2 According to the 16SrRNA sequencing results of Sangon Biotech (Shanghai) Co., Ltd., the sequence similarity between isolate H4 and the standard Bacillus subtilis sequence is as high as 99.4%, and the evolutionary tree is shown (as shown in Figure 3 Combined with the results of biochemical and molecular biological identification, the isolated strain was determined to be Bacillus subtilis.

[0037] The present invention also provides the use of the Bacillus subtilis H4 in the preparation of a probiotic preparation with improved drug sensitivity and antibacterial properties, wherein the probiotic preparation is a powder or a tablet.

[0038] 1. Drug sensitivity test

[0039] The KB paper diffusion method was used to select 10 kinds of drug paper discs, including kanamycin (K), ceftriaxone (CRO), chloramphenicol (C), norfloxacin (NOR), amikacin (AK), sulfamethoxazole (SXT), ofloxacin (OFX), streptomycin (STR), gentamicin (GM), and ciprofloxacin (CIP), for drug sensitivity test. The concentration of H3 strain and H4 strain was adjusted to 10 8 CFU / mL, dip a small amount of bacterial solution with sterile cotton and evenly apply it on LB agar medium. After it dries naturally, take the drug sensitivity sheet and stick it on the surface of the medium. After culturing at 37℃ for 24h, measure the diameter of the inhibition zone. Repeat the test 3 times and take the average value. Refer to the NCCLS2014 version standard to judge the results, and divide the test strains into sensitive (S), moderately sensitive (I), and resistant (R) according to their drug sensitivity.

[0040] Table 3. Drug diameter determination criteria (mm)

[0041]

[0042] Table 4.2 Sensitivity of 2 strains of Bacillus subtilis to the following 10 drugs

[0043]

[0044]

[0045] Results: As shown in Table 4, the H3 strain was moderately sensitive (I) to ceftriaxone (CRO), streptomycin (STR), and sulfamethoxazole (SXT), while the H4 strain was only moderately sensitive (I) to ceftriaxone (CRO) and was sensitive (S) to other drugs. The comparison showed that the drug sensitivity of the H4 strain was stronger than that of the H3 strain.

[0046] 2. In vitro antibacterial test

[0047] The Oxford cup antibacterial test was used. Before the test, the concentration of indicator bacteria Escherichia coli, Staphylococcus aureus, and Salmonella was raised to 10 8 CFU / mL, take 100μL of bacterial solution respectively, evenly spread it on LB agar medium, then use tweezers to take out the sterile Oxford cup and place it on the medium coated with indicator bacteria, and place 2 evenly on each medium. The bacteria to be tested are activated and cultured in LB broth for 12 hours, and 200μL of bacterial solution is taken and added to the Oxford cup respectively. After 24 hours in a constant temperature incubator at 37℃, the diameter of the inhibition zone is measured. The test is repeated 3 times and the average value is taken.

[0048] Table 5.2 Antibacterial Test Results of Bacillus subtilis (mm)

[0049]

[0050] Results: As shown in Table 5, the two strains of Bacillus subtilis had antibacterial effects on Escherichia coli, Salmonella and Staphylococcus aureus. Among them, the H4 strain had the best antibacterial effect on Escherichia coli, with an inhibition zone of 17.63 mm, which is of great significance for preventing bacterial diseases caused by Escherichia coli.

[0051] The present invention also provides the use of the above-mentioned Bacillus subtilis H4 in the preparation of a probiotic preparation having improved bile salt tolerance and high temperature resistance, wherein the probiotic preparation is a powder or a tablet.

[0052] 1. High temperature tolerance test

[0053] The Bacillus subtilis culture to be tested is divided into clean test tubes, each tube contains 10mL of culture, and each culture is divided into 4 test tubes. Take any 3 test tubes from the 4 test tubes, place them in a constant temperature water bath at 85℃, 90℃, 95℃, and 100℃ for 30 minutes, and keep 1 test tube at room temperature as a control group. After the water bath, use a pipette to draw 200μl of culture solution from each test tube and spread it on the LB nutrient agar medium. After the coating is completed, place the LB nutrient agar medium in a constant temperature incubator at 37℃ for 24h. Repeat the above experiment three times and calculate the survival rate of the Bacillus subtilis at 85℃, 90℃, 95℃, and 100℃ respectively.

[0054] Table 6. High temperature tolerance test results

[0055]

[0056] Results: As shown in Table 6, different Bacillus subtilis have different tolerance levels under the same temperature; the survival rate of the same Bacillus subtilis decreases with the increase of temperature. Among them, only the H4 strain still survived partially under the 100℃ experimental environment, and H3 could not survive under the 100℃ experimental environment. Among the experimental strains, H4 has a higher survival rate in a high temperature environment and better high temperature tolerance. The processing temperature of general microbial preparations is 70-80℃, and the temperature of the bovine rumen is 37-40℃. In the experiment, Bacillus subtilis H4 can still maintain a survival rate of more than 80% after high temperature treatment at 85℃ for 30 minutes. It seems that Bacillus subtilis H4 can be added to yak feed as a feed probiotic.

[0057] 2. Pig bile salt test

[0058] Use pig bile salt solution to prepare 50 mL of selective culture medium with concentrations of 0.03 g / 100 mL, 0.1 g / 100 mL, 0.2 g / 100 mL and 0.3 g / 100 mL, respectively, and dispense 3 selective culture media. After high-temperature and high-pressure steam sterilization, inoculate the Bacillus subtilis culture to be tested, place it in a 37°C constant temperature incubator and culture it for 24 hours. Calculate the survival rate of the Bacillus subtilis in the pig bile salt solution environment with concentrations of 0.03 g / 100 mL, 0.1 g / 100 mL, 0.2 g / 100 mL and 0.3 g / 100 mL.

[0059] Table 7. Results of pig bile salt experiment

[0060]

[0061] Results: As shown in Table 7, with the increase of pig bile salt concentration, the survival rate of Bacillus subtilis decreased, but the experimental strains had a good overall tolerance to pig bile salts. In the experimental environment of 0.3g / 100mL pig bile salt concentration culture medium, the survival rate was above 50%.

[0062] In summary, the Bacillus subtilis H4 isolated and cultured in the present invention meets the basic characteristics of Bacillus, has strong antibacterial activity and antibiotic sensitivity, has good biological properties, and has good bile resistance and high temperature resistance. It can be used as a candidate strain for the development of yak-derived microecological preparations, and provides an excellent probiotic strain for further developing yak-specific microbial preparations.

[0063] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0064] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A yak-derived Bacillus subtilis, characterized in that: The yak-derived Bacillus subtilis is named Bacillus subtilis H4, the preservation number is CGMCC No.31580, the preservation date is August 14, 2024, the preservation classification is named Bacillus subtilis, the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, and the preservation unit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The bacterial agent prepared by Bacillus subtilis H4 as claimed in claim 1.

3. Use of the Bacillus subtilis H4 as claimed in claim 1 in the preparation of a probiotic preparation with improved drug sensitivity and antibacterial properties.

4. The use according to claim 3, characterized in that: The probiotic preparation is in the form of powder or tablet.

5. Use of the Bacillus subtilis H4 according to claim 1 in the preparation of a probiotic preparation having improved bile salt tolerance and high temperature resistance.

6. The use according to claim 5, characterized in that: The probiotic preparation is in the form of powder or tablet.

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