阅读说明：本技术 一种具有抑制多种致病菌微生物生长的民猪源枯草芽孢杆菌 (Min swine bacillus subtilis capable of inhibiting growth of various pathogenic bacteria microorganisms ) 是由 石宝明 王晓旭 赵轩 郭志强 于 2021-06-16 设计创作，主要内容包括：本发明公开一种具有抑制多种致病菌微生物生长的民猪源枯草芽孢杆菌。保藏在中国典型培养微生物保藏中心,保藏日期为2021年4月25日,保藏编号为CCTCC NO:M2021440,分类命名为枯草芽孢杆菌Bacillussubtilis JX673943.1。该菌有助于减轻致病菌微生物对动物的危害,从而提高动物对抗病性,是一株具有较高应用价值的猪源益生菌,其对畜牧业和饲料发酵工业的发展具有重要意义。(The invention discloses a Min swine bacillus subtilis capable of inhibiting the growth of various pathogenic bacteria microorganisms. The bacillus subtilis JX673943.1 is preserved in China center for type culture of microorganisms with the preservation date of 2021, 4 months and 25 days, the preservation number is CCTCC NO: M2021440, and the bacillus subtilis JX673943.1 is classified and named. The strain is beneficial to reducing the harm of pathogenic bacteria microorganisms to animals, thereby improving the disease resistance of the animals, is a pig source probiotic strain with higher application value, and has important significance for the development of animal husbandry and feed fermentation industry.)

1. The civilian swine Bacillus subtilis capable of inhibiting the growth of various pathogenic bacteria microorganisms is preserved in China center for type culture collection with the preservation date of 2021, 4 months and 25 days and the preservation number of M2021440, and is classified and named as Bacillus subtilis JX 673943.1.

2. The Bacillus subtilis of claim 1, wherein the strain has a 16S rDNA sequence as shown in SEQ ID No. 1.

3. The bacillus subtilis of claim 1 having biological properties comprising: the colonies were white and had irregular edges.

4. The bacillus subtilis of claim 1 having the ability to inhibit the growth of a plurality of pathogenic microorganisms, wherein: the growth conditions are that the temperature is 30-50 ℃, the pH value is 5.1-8, and the mass concentration of NaCl is 1-5%.

5. The Bacillus subtilis of claim 4 having the ability to inhibit the growth of a plurality of pathogenic microorganisms, wherein: the growth conditions were 37 ℃ temperature, pH 6, and NaCl concentration 2.4%.

6. According to the rightThe method for culturing the Min swine bacillus subtilis capable of inhibiting the growth of a plurality of pathogenic microorganisms according to claim 1, which comprises the following steps: taking OD_100nmInoculating 1 percent of the seed solution by volume into a culture medium seed, and performing shake culture on a shaker at the temperature of 37 ℃ and the speed of 220r/min, wherein the culture medium adopts an LB liquid culture medium.

7. The bacillus subtilis of civilian origin capable of inhibiting the growth of a plurality of pathogenic microorganisms according to claim 1, wherein the bacillus subtilis of civilian origin has bacteriostatic activity against escherichia coli 25922, escherichia coli K88, escherichia coli K99, escherichia coli 1799, escherichia coli 1515, staphylococcus aureus 43300, pseudomonas aeruginosa 27853, pseudomonas aeruginosa PA01, salmonella pullorum C7913, enterococcus faecalis 29212, aspergillus niger 2377, streptococcus pneumoniae 31001, staphylococcus epidermidis 12228, and salmonella murine nociceps C7731.

8. A probiotic formulation characterized by: the method for inhibiting the growth of pathogenic microorganisms comprises the bacillus subtilis of Minsui of claim 1.

Technical Field

The invention belongs to the field of agricultural and livestock application, and particularly relates to a Bacillus subtilis JX673943.1 of Min pig origin capable of inhibiting the growth of various pathogenic bacteria microorganisms.

Background

The obtained microorganism can reduce the damage of the disease-causing bacterium microorganism of Min pig origin to animal bodies. The bacillus subtilis is one of bacillus, is a probiotic which is approved and applied by the agricultural rural department in China, is nontoxic and harmless, has multiple functions of enhancing the immunity of organisms, improving the feed conversion rate, improving the microbial balance of animal intestinal tracts, improving the culture environment, inhibiting oxidative stress, inducing plant resistance, promoting plant growth and the like as metabolites, and is widely applied to livestock feed, food processing, medicine production and water purification.

In recent years, the economic losses due to infection with pathogenic microorganisms have increased in the pig industry. After the piglets are infected by pathogenic bacteria such as escherichia coli and salmonella, diarrhea and even death can be caused. Antibiotics are always the most main way for preventing diarrhea of weaned piglets, but long-term use of antibiotics has a plurality of hazards for treating piglet pathogenic bacteria infection, seriously pollutes the environment, causes bacterial drug resistance, kills pathogenic bacteria and a large amount of beneficial bacteria, further destroys intestinal flora structure, and has a plurality of adverse effects on human body. The excellent characteristics of the Min pig are necessarily closely related to the specific microbial flora in the digestive tract, and the abundant microorganisms in the digestive tract also provide abundant sources for the separation and screening of substances inhibiting pathogenic bacteria. The colon is used as a main part for bacteriostatic screening because the colon has low flowing speed, a large amount of substances are fermented, the microbial flora is most complex, and the types and the number of microorganisms are more. The screening of antagonistic pathogenic bacteria microorganisms in intestinal tracts of Min pigs can inhibit harmful bacteria by using beneficial bacteria and reduce the harm of the pathogenic bacteria to animals, so that the intestinal microorganisms obtained by animal source screening become a substitute of novel antibiotics. The probiotics has the functions of regulating the micro-ecological balance in the animal body, stimulating the immunologic function and the like, thereby being beneficial to recovering the normal physiological function of the animal body, preventing and treating diseases and promoting the health, and further improving the production performance of the cultured animals. The screening of the probiotic strains which can inhibit the growth of pathogenic bacteria microorganisms in the intestinal flora of the Min pig has practical significance.

Disclosure of Invention

Based on the practical problems and requirements in the livestock breeding production process, the invention aims to provide the Min swine source Bacillus subtilis JX673943.1 capable of inhibiting the growth of various pathogenic bacteria microorganisms, and the Min swine source Bacillus subtilis JX673943.1 is beneficial to improving the disease resistance of animals.

The purpose of the invention is realized by the following technical scheme: the Min swine-origin bacillus subtilis capable of inhibiting the growth of various pathogenic bacteria microorganisms is preserved in China center for type culture Collection at the address: china center for type culture Collection, one of Wuhan university in Wuchang district, Wuhan city, Hubei province, postal code: 430072, preservation date of 2021, 4 months and 25 days, preservation number of M2021440, and classified name of Bacillus subtilis JX 673943.1.

The invention also has the following technical characteristics:

1. the Min swine bacillus subtilis has the biological characteristics that: the colony is white, the edge is irregular, and the surface is dry.

2. The Min pig source bacillus subtilis has the growth conditions of 30-50 ℃ of temperature, 5.1-8 of pH and 1-5 mass percent of NaCl.

3. Further, the bacillus subtilis derived from min swine is grown under the conditions of 37 ℃ of temperature, 6 of pH and 2.4% of NaCl concentration.

4. The method for culturing the Min pig source bacillus subtilis comprises the following steps: inoculating the seed liquid into a culture medium according to the inoculation amount of 1% of the volume ratio, and performing shaking culture on a shaker at the temperature of 37 ℃ and at the speed of 220r/min, wherein the culture medium adopts an LB liquid culture medium.

5. The Min pig source bacillus subtilis has bacteriostatic activity on escherichia coli 25922, escherichia coli K88, escherichia coli K99, escherichia coli 1799, escherichia coli 1515, staphylococcus aureus 43300, pseudomonas aeruginosa 27853, pseudomonas aeruginosa PA01, salmonella pullorum C7913, enterococcus faecalis 29212, aspergillus niger 2377, streptococcus pneumoniae 31001, staphylococcus epidermidis 12228 and salmonella typhimurium C7731.

The invention has the advantages and beneficial effects that: the bacillus subtilis strain provided by the invention has bacteriostatic activity on escherichia coli 25922, escherichia coli K88, escherichia coli K99, escherichia coli 1799, escherichia coli 1515, staphylococcus aureus 43300, pseudomonas aeruginosa 27853, pseudomonas aeruginosa PA01, salmonella pullorum C7913, enterococcus faecalis 29212, aspergillus niger 2377, streptococcus pneumoniae 31001, staphylococcus epidermidis 12228 and salmonella typhimurium C7731. In conclusion, the civilian swine Bacillus subtilis JX673943.1 is a swine probiotic strain with high application value, and has important significance for the development of animal husbandry and feed fermentation industry.

Drawings

FIG. 1 is a diagram showing the morphology of cells according to the present invention;

FIG. 2 is a phylogenetic tree diagram of Min swine-origin Bacillus subtilis JX 673943.1;

FIG. 3 is a graph showing experimental results of growth ability of strains at different temperatures;

FIG. 4 is a graph showing the results of experiments on the growth ability of strains at different pH;

FIG. 5 is a graph showing the results of experiments on the growth ability of strains under different NaCl conditions;

FIG. 6 is a graph showing the results of growth performance tests under different bile salt concentrations.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.

Test materials: escherichia coli ATCC 25922, Escherichia coli K88, Escherichia coli K99, Escherichia coli 1799, Escherichia coli 1515, Staphylococcus aureus 43300, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa PA01, Salmonella pullorum C7913, enterococcus faecalis 29212, Aspergillus niger 2377, Streptococcus pneumoniae 31001, Staphylococcus epidermidis 12228, Salmonella typhimurium C7731, which are provided by the institute of animal nutrition of northeast university of agriculture; the civil pig manure is collected from the experimental base of the Ath city, northeast university of agriculture.

Example 1:

the screening and identification of the strain are specifically carried out according to the following steps:

1. preparation of a culture medium: in the experiment, an LB liquid culture medium is selected as a screening culture medium for determining the bacteriostatic activity. Nutrient agar medium: 15g of agar, 3g of yeast extract, 5g of tryptone, 5g of sodium chloride and 1000mL of water, adjusting the pH value to 7.4, and sterilizing at 121 ℃ for 20min under high temperature and high pressure; LB liquid medium: 3g of yeast extract, 5g of tryptone, 5g of sodium chloride and 1000mL of water, and sterilizing at 121 ℃ for 20min under high temperature and high pressure; LB solid medium: 15g of agar, 5g of tryptone, 3g of yeast extract, 5g of sodium chloride and 1000mL of water, and sterilizing at 121 ℃ for 20min under high temperature and high pressure.

2. Preparing an indication bacterial liquid: taking the frozen pathogenic bacteria as indicator bacteria, streaking the indicator bacteria into a nutrient agar culture medium, culturing at a constant temperature of 37 ℃, and activating a strain; selecting single colony, inoculating in LB liquid culture medium, culturing overnight at 37 deg.C at 220rpm, subculturing overnight in new liquid culture medium for 2-4 hr until indicator bacteria is in logarithmic phase, centrifuging to collect bacterial thallus, washing with sterile PBS, and adjusting concentration of obtained bacteria to 10⁵CFU/ml；

3. Collecting microbial metabolites: weighing 1.0g colon content of Min pig, dissolving in triangular flask containing 50mL sterilized deionized water, shaking in shaking table at room temperature for 30min to obtain uniform 10% colon content^-2Taking a proper amount of the diluent, and carrying out ten-fold gradient continuous dilution to obtain 10^-3-10^-5And (4) diluting the solution. 30 mu L of each dilution is evenly coated on a nutrient agar culture plate, the nutrient agar culture plate is placed in a constant temperature box at 37 ℃ for 24 hours, and a single colony is picked. The single colony is inoculated into LB liquid culture medium, shaking cultured at 37 ℃ and 220rpm for 24h, and then centrifuged at 12000rpm for 5 min. Sucking supernatant, and filtering with 0.22 μm water system filter;

4. screening for inhibition of growth of pathogenic microorganism strains: the primary screening was performed by the agar diffusion method. Selecting single colony of each preserved strain to be planted in an LB liquid culture medium,shaking-flask culture at 37 deg.C for 24h, centrifuging at 12000rpm for 5min, and adding 50 μ L supernatant dropwise into Oxford cup (1 × 10) containing primary sieve culture plate of indicator bacteria⁵CFU,8mm deep × 10mm outer diameter) wells, culturing at 37 ℃ for 48 hours at constant temperature, and recording the presence or absence of the zone of inhibition and the diameter (d) of each of the stored strains with a vernier caliper as a standard for evaluating the ability of the strains to antagonize the indicator bacteria. Selecting strains with larger diameter of the inhibition zone, performing shake flask culture and streak culture, repeatedly separating, purifying and culturing for 4-5 generations, and storing in 50% glycerol solution at-80 deg.C for use. Re-screening: preserving the antagonistic bacteria screened in the primary screening at-80 ℃ for a week, taking out again for activation culture, continuously observing the antibacterial activity of the antagonistic bacteria according to an agar diffusion method, and comparing the change of the antibacterial activity of the antagonistic bacteria in the preservation process of the strains to identify whether the antagonistic performance is stable.

The biological characteristics of the strains obtained by screening are as follows: as shown in FIG. 1, the colonies were white and had irregular edges.

The strain identification process and results are as follows: the screened strains are identified by a first-generation sequencing method. The extraction of bacterial genomic DNA was carried out according to the instructions of the bacterial DNA extraction kit (Omega Bio-Tek, USA), followed by PCR amplification with primers: an upstream primer, AGAGAGTTTGATCCTGGCTCAG and a downstream primer, GGTTACCTTGTTACGACTT. The sequences of the strains were then compared to the NCBI database using the blast program to determine the species, as shown in fig. 2, and phylogenetic trees of the strains were drawn.

Through morphological observation, physiological and biochemical reaction and 16S rDNA molecular identification, the strain with the capability of inhibiting the growth of pathogenic bacteria is determined to be bacillus subtilis JX 673943.1.

Example 2:

determination of bacterial strain bacteriostatic ability to pathogenic bacteria

1. Strain activation: streaking the frozen strains onto a nutrient agar culture medium, culturing activated strains at constant temperature of 37 ℃, selecting single colonies, inoculating the single colonies to an LB liquid culture medium, and culturing at the temperature of 37 ℃ for 24 hours at 220 rpm;

2. preparing an indication bacterial liquid: escherichia coli ATCC 25922, Escherichia coli K88, Escherichia coli K99, Escherichia coli 1799, Escherichia coli 1515, Staphylococcus aureus 43300, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa PA01, Salmonella pullorum C7913, enterococcus faecalis 29212, Aspergillus niger 2377, Streptococcus pneumoniae 31001, Staphylococcus epidermidis 12228 and Salmonella typhimurium C7731 are respectively used as indicator bacteria, and the preparation method is as the method for preparing the indicator bacterium liquid in the reference example 1;

3. collecting microbial metabolites: specific process reference example 1;

4. and (3) determining the antibacterial activity: culturing the bacterial liquid for 24h, centrifuging bacterial liquid 12000r, collecting supernatant, and filtering with 0.22 μm water system filter for 2 times for use. Taking 100 microliters of supernatant and an indicator bacterium solution, mixing the supernatant and the indicator bacterium solution in equal amount, adding the mixture into a 96-well plate, taking a mixture of the indicator bacterium solution and a sterilized PBS solution as a positive control group, taking an LB liquid culture medium as a negative control group, placing the mixture at the constant temperature of 37 ℃ for overnight culture, taking the clarification of the liquid culture medium as a standard, and measuring the absorbance by using an enzyme-labeling instrument;

5. the calculation formula of the bacteriostatic rate is as follows:

6. the result of the antibacterial activity of the strain is as follows: the bacteriostatic effect of the strain is shown in table 1, and the strain JX673943.1 has inhibitory effect on various pathogenic bacteria.

TABLE 1 bacteriostatic ability of the strains to pathogenic bacteria

Example 3

Determination of the growth Capacity of the Strain at different temperatures

1. Strain activation: inoculating stock solution of the strain to be frozen and preserved to LB liquid culture medium with the inoculation amount of 1% (50 mu l +5ml LB), and culturing at 37 ℃ overnight at 220rpm for 12 h;

2. growth assay of the strains at different temperatures: adjusting OD_100nmWhen the strain is 1, subculturing to LB liquid culture medium with 1% (50 μ l +5ml LB), at 20 deg.C, 30 deg.C, 37 deg.C,Culturing at 45 deg.C, 50 deg.C and 55 deg.C for 9 hr, and measuring OD600nm value. And then 100. mu.l of the dilution was added to the nutrient agar medium. Analyzing the growth conditions of the selected strains under different pH environmental conditions through absorbance values and viable count, and drawing a temperature growth curve;

3. as shown in FIG. 3, the results of the strain growth ability experiment are shown.

4. The experimental result of the growth ability of the strain shows that: the growth of the strain is most suitable at 37 ℃. The growth ability of the strain gradually increases with the temperature increase before 37 ℃; after exceeding 37 ℃, the growth ability of the strain gradually decreases with increasing temperature. The temperature range of 30-50 ℃ is more suitable for the growth of the strain.

Example 4

Determination of growth capacity of strain in different pH environments

1. Strain activation: the experimental procedure was as in example 3;

2. growth assay of the strains under the corresponding pH conditions: the pH of LB liquid medium was adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 with 1mol/L HCl and 1mol/L NaOH. The activated bacterial suspension was inoculated into LB medium adjusted to pH with an inoculum size of 1% (50. mu.l +5ml LB), and cultured under the same conditions for 6 hours. After completion of the culture, the OD of the culture was measured_600nmAnd inoculating 100. mu.L of the dilution solution onto a solid medium, and culturing at 37 ℃ for 12h to determine the growth of the selected strain;

3. as shown in FIG. 4, the results of the strain growth ability experiment.

4. The experimental result of the growth ability of the strain shows that: the growth of the strain is most suitable at pH 6, and the strain is viable at pH 4-9.

Example 5

Determination of growth capacity of strain in different NaCl environments

1. Strain activation: the experimental procedure was as in example 3;

2. growth assay of the strains under corresponding NaCl conditions: the activated bacterial suspension was inoculated in an LB liquid medium containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% NaCl in an inoculum size of 1% (50. mu.l +5ml LB), and simultaneously cultured in the same liquid medium without the bacterial suspension inoculatedThe medium was used as a control and cultured at 37 ℃ and 220rpm for 3 hours. After completion of the culture, the OD of the culture was measured_600nmAnd inoculating 100. mu.L of the dilution solution onto a solid medium, and culturing at 37 ℃ for 12h to determine the growth of the selected strain;

3. as shown in FIG. 5, the results of the strain growth ability experiment.

4. The experimental result of the growth ability of the strain shows that: the strain can survive under the conditions of different NaCl concentrations, but the growth capacity of the strain is reduced along with the increase of the NaCl concentration.

Example 6

Determination of growth ability of strain under different bile salt concentration conditions

1. Strain activation: the experimental procedure was as in example 3;

2. growth assay of strains under corresponding bile salt conditions: the activated bacterial suspension was inoculated in an LB liquid medium containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% bile salts in an inoculum size of 1% (50. mu.l +5ml LB), and the same liquid medium without the bacterial suspension was used as a control, and cultured at 37 ℃ and 220rpm for 3 hours. After completion of the culture, the OD of the culture was measured_600nmAnd inoculating 100. mu.L of the dilution solution onto a solid medium, and culturing at 37 ℃ for 12h to determine the growth of the selected strain;

3. as shown in FIG. 6, the results of the strain growth ability experiment.

4. The experimental result of the growth ability of the strain shows that: the strain grows better under the condition of no bile salt addition. After the bile salt was added, almost no growth occurred.

Sequence listing

<110> northeast university of agriculture

<120> Min swine-origin bacillus subtilis capable of inhibiting the growth of various pathogenic bacteria microorganisms

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1463

<212> DNA

<213> Bacillus subtilis

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acgaaagggc gtctataatg cagtcgagcg gacagatggg agcttgctcc ctgatgttag 60

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120

accggggcta ataccggatg gttgtttgaa ccgcatggtt caaacataaa aggtggcttc 180

ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240

aaggcaacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420

caagtaccgt tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctgg cagggcggtt attttcaagt gtgatttgaa tagcgcccac gctcaaccgc 600

ggagggtcat tgtaaagtgt gaaatgtgac tgcagagggg gggaacggaa ttacacgtgg 660

cgctcttaat tgcgtagcag atatggagga acactagtgt cgagggcgac cctctggtct 720

gtaatcgtcg gtgtggagtt gaaggcgtgg ggagcgaaca ggataagata cctcgttagt 780

ccacgccgta aaacgatgag tgctaagtgt taggggggtt tccgcccctt agtgctgcag 840

ctaacgcatt aagcactccg cctgggggag tacggtcgca agactgaaac tcaaaggaat 900

tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc 960

ttaccaggtc ttgacatcct ttgacaatcc tagagatagg acgtcccctt agggggcagc 1020

gtgacaggtg ggtgcatggg ttgtcgtcag ctcgtgtcgt gagatgtttg ggttagtccc 1080

cgcaacgagc gcaccctttg gatcttagtg accagcattc agtttgggca cctctaacgt 1140

gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1200

acctgggcta cacacgtgct acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa 1260

gccaatccca caaatctgtt ctcagttcgg atcgcagtct gcaactcgat tgcgtgaagc 1320

cggcattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg gcccttgtac 1380

acaccgcccg tcacaccacg agagtttgta acacccgacg tcggtgaggt aacctttacg 1440

agccagccgc tgaacgaccc agt 1463