Bacillus amyloliquefaciens with broad-spectrum bacteriostasis from pig and application thereof

Abstract

The present application relates to the technical field of Bacillus amyloliquefaciens, and discloses a pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens and application thereof; the Bacillus amyloliquefaciens provided by the present application is Bacillus amyloliquefaciens Y106-A1-3. The bacterium is isolated from the excrement of healthy pigs, and the gastrointestinal tolerance test proves that the bacterium has strong gastrointestinal tolerance; and experiments prove that the fermentation supernatant of the Bacillus amyloliquefaciens has a bacteriostatic effect and a wide bacteriostatic spectrum, and can be used for regulating the intestinal microecological balance of animals, preventing diseases and improving the growth and production performance of animals. The microbial preparation containing the Bacillus amyloliquefaciens and the related components of the fermentation liquor has very important significance for reducing the use of antibiotics, improving the quality of agricultural and livestock products and protecting the environment, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Bacillus amyloliquefaciens, in particular to a pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens and application thereof. BACKGROUND

[0002] With the use of antibiotics in animal husbandry, the animal growth performance is improved in a short time, the animal disease probability is reduced, and the economic benefit of animal husbandry is improved. However, with the passage of time, problems such as antibiotic residues, drug-resistant bacteria, and animal excrement pollution of the environment are increasingly prominent. In order to ensure that the animal production benefit in animal husbandry is not greatly affected, and to reduce the use of antibiotics, it is urgent to find effective substances that can replace or reduce the use of antibiotics. At the same time, people have increasingly high requirements for the quality of animal products, and the use of safe, environmentally friendly, and efficient substitutes to replace antibiotics has become a trend. At this time, Bacillus in probiotics has attracted the attention of researchers and animal husbandry producers due to its high stress resistance, non-toxicity, non-harmfulness, significant effect, and low cost.

[0003] Bacillus amyloliquefaciens is a gram-positive bacillus that can produce spores and widely exists in environmental soil, plant surfaces, and animal gastrointestinal tracts. The survival environment of Bacillus amyloliquefaciens is very diverse. Bacillus amyloliquefaciens can colonize in the animal body, form a biofilm, produce various bacteriostatic substances, prevent and treat animal diseases, improve the ecological environment of the animal gastrointestinal tract, improve the growth performance of animals, improve the quality of animal products, degrade animal waste and improve the environment, and has many advantages such as the ability to form spores, resistance to storage, high temperature, acid and alkali, strong stress resistance, and friendliness to humans, animals, and the environment. Bacillus amyloliquefaciens is widely used in agriculture and animal husbandry as a probiotic. Although Bacillus amyloliquefaciens has strong probiotic potential, due to the different survival environments and sources of Bacillus amyloliquefaciens, not all strains have ideal ability and potential to be applied in animal husbandry production. It is usually necessary to screen a large number of strains with good probiotic properties as probiotics. Therefore, screening strains with good probiotic properties and suitable for animal production is of great significance to animal husbandry production and environmental protection. SUMMARY

[0004] The present application aims to provide a pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens and application thereof, so as to solve the problems in the background art.

[0005] In order to solve the above technical problems, the present application provides the following technical solutions:

[0006] A pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens, the DNA sequence of the Bacillus amyloliquefaciens is shown as SEQ ID NO. 1.

[0007] Further, the Bacillus amyloliquefaciens is a pig-derived Bacillus amyloliquefaciens Y106-A1-3, the preservation date is February 7, 2023, the preservation number is CGMCC No. 26510, the preservation center name is China General Microbiological Culture Collection Center, the address is No. 1, Yihuangyuan 3rd Road, Beijing, China, and the classification name is Bacillus amyloliquefaciens.

[0008] Further, the pig-derived Bacillus amyloliquefaciens Y106-A1-3 is prepared by the following method:

[0009] S1: Sterilely collect a healthy pig fecal sample, take the pig feces into a sterile centrifuge tube, add sterile PBS to the fecal sample and mix thoroughly, and use a sterilized inoculation loop to streak on LB solid medium; after inoculation, incubate in a 36.5-37°C constant temperature incubator for 12-24 hours; pick single colonies suspected to be Bacillus subtilis and continue to streak on LB plates, and incubate at 36.5-37°C; repeat the streaking 1-3 times until uniform single colonies grow on the plates, and obtain pure culture;

[0010] S2: Take the above-mentioned pure culture and evenly spread it on a glass slide to prepare a smear, observe the bacterial morphology under a microscope after Gram staining; select strains that are positive for Gram staining and can produce spores, and perform 16S rDNA sequencing identification to obtain the pig-derived Bacillus amyloliquefaciens Y106-A1-3.

[0011] Further, the application of the one strain of pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens is the preparation, fermentation broth, metabolic broth, bacterial suspension or culture broth prepared from Bacillus amyloliquefaciens for preparing an antibacterial or bacteriostatic product.

[0012] Further, the antibacterial or bacteriostatic bacteria are any one or more of Enterococcus, Staphylococcus, Clostridium perfringens, Clostridium difficile, Salmonella, Burkholderia cepacia and Escherichia coli.

[0013] Further, the application of the one strain of pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens is the preparation, fermentation broth, metabolic broth, bacterial suspension or culture broth prepared from Bacillus amyloliquefaciens for preparing a product for regulating the microecological balance of animals.

[0014] Further, the application of the one strain of pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens is the preparation, fermentation broth, metabolic broth, bacterial suspension or culture broth prepared from Bacillus amyloliquefaciens for preparing a product for preventing animal diseases or reducing the probability of animal diseases.

[0015] Further, the application of the strain of pig-derived broad-spectrum bacteriostatic Bacillus amyloliquefaciens is that the preparation, fermentation liquor, metabolic liquor, bacterial suspension or culture liquor prepared from the Bacillus amyloliquefaciens is used in the preparation of a product for improving the growth performance of an animal body.

[0016] Further, the product prepared from the pig-derived Bacillus amyloliquefaciens has any one or more of the following functions:

[0017] (1) antibacterial and / or bacteriostatic;

[0018] (2) regulating the microecological balance of an animal body;

[0019] (3) preventing animal diseases;

[0020] (4) reducing the disease probability of an animal;

[0021] (5) improving the growth performance of an animal body.

[0022] Compared with the prior art, the application has the beneficial effects that the application provides the Bacillus amyloliquefaciens Y106-A1-3. The Bacillus amyloliquefaciens Y106-A1-3 has the inhibitory activity to Enterococcus (HC-5, HC-28, HC-25, HC-32), Staphylococcus (AC-13 white, AC-39 white, GC-50, MC-50-1, PC-10-2, QC-1, SC-6-white, SC-23-white, TC-22-white, TC-41-white, TC-34-white, TC-11-white, ATCC25923, ATCC29213), Clostridium perfringens (ATCC13124, X27, V25, X41, X39), Clostridium difficile (ATCC43593), Salmonella (SL1344, SL7207, SP, CVCC514), Burkholderia cepacia (ATCC25416), and Escherichia coli (ATCC25922), and can be used for regulating the microecological balance of an animal body, preventing animal diseases, reducing the disease probability of an animal, and improving the growth performance of an animal body. Not only does it have very important significance for reducing the use of antibiotics, improving the quality of agricultural and pastoral products and protecting the environment, but also has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, illustrate the application, and are used together with the embodiments to explain the application, and do not constitute a limitation on the application. In the drawings:

[0024] Figure 1is a colony morphology diagram of Bacillus amyloliquefaciens Y106-A1-3 on LB solid culture medium in the embodiment of the present application;

[0025] Figure 2 is a morphology diagram of vegetative cell and spore of Bacillus amyloliquefaciens Y106-A1-3 observed under an oil microscope in the embodiment of the present application. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below. Apparently, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0027] The materials, reagents and the like used in the following embodiments can be obtained from commercial channels unless otherwise specified. In the quantitative test in the embodiments, three repeated experiments are set, and the average value is taken as the result.

[0028] The following strains in the examples indicate Clostridium perfringens ATCC13124 (from human gas gangrene isolates), Staphylococcus aureus ATCC25923, ATCC29213, Salmonella typhimurium ATCC13311, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Burkholderia cepacia ATCC25416, Candida albicans ATCC14053, Serratia marcescens ATCC13880, Clostridium difficile ATCC43593, Klebsiella oxytoca ATCC43086 purchased from American Type Culture Collection (ATCC), Clostridium perfringens V25, X27, X39, X41 are clinical isolates from the feces of piglets with diarrhea. Salmonella abortus equi CVCC514 is purchased from China Institute for Control of Veterinary Drug Administration, Pichia pastoris X33, Salmonella SL1344, SL7207, SP, SE, other Enterococcus (AC-41-1, AC-36-1, GC-12, GC-46, GC-38, HC-5, HC-28, HC-25, HC-32, MC-26, MC-50, MC-23), Staphylococcus (AC-13 white, AC-39 white, GC-50, MC-50-1, PC-10-2, QC-1, SC-6-white, SC-23-white, TC-22-white, TC-41-white, TC-34-white, TC-11-white), Escherichia coli (K88, OE-9, OE-6, OE-5, PE-13, PE-20, PE-21, PE-22, PE-23, DFE-8, XGE-17, LGE-26, LGE-21, CXE-22, CXE-16), Klebsiella pneumoniae (K7, KPP36, KPP41, KPP27, KPP7, KPL) strains are clinical isolates, all of which are preserved in the experiment.

[0029] Example

[0030] S1: Sterile collection of healthy pig feces samples, take about 0.1 g of pig feces in a sterile centrifuge tube, add 1 mL of sterile PBS and mix well with the fecal sample, take 100 μL and evenly coat on LB solid plate, or use sterilized inoculation ring to dip a small amount on LB solid medium and streak. After inoculation, incubate in a 37°C constant temperature incubator for 12-24 h. Pick single colonies suspected of being Bacillus subtilis and continue to streak on LB plates at 37°C. Streak for 3 times in succession until uniform single colonies are grown on the plates;

[0031] S2: Take the above pure culture and evenly coat on a glass slide to prepare a smear, observe the bacterial morphology under a microscope after Gram staining. Select strains that are positive for Gram staining and can produce spores, and perform 16S rDNA sequencing identification to obtain the pig-derived Bacillus amyloliquefaciens Y106-A1-3.

[0032] The Bacillus amyloliquefaciens Y106-A1-3 sequence is as follows:

[0033] This sequence is a 16S rDNA sequence:

[0034]

[0035] Test:

[0036] Pig-derived Bacillus amyloliquefaciens colonies that have formed wrinkles on solid culture medium for 1-2 days were dissolved in 1 mL of PBS and mixed well using a vortex shaker. This was the spore suspension of pig-derived Bacillus amyloliquefaciens. It was used for the tolerance test of artificial gastric juice, artificial intestinal juice, and bile salt solution.

[0037] Artificial gastric juice tolerance test

[0038] Preparation of artificial gastric juice: 16.4 mL of 9.5%-10.5% dilute hydrochloric acid was taken and adjusted to pH 2.0 with distilled water. 0.33 g of pepsin was dissolved in 100 mL of the above solution with pH 2.0, mixed well, and filtered using a 0.22 μm sterile filter membrane. It was stored at 4°C for standby use and prepared fresh every time.

[0039] 100 μL of the spore suspension was added to 900 μL of artificial gastric juice and mixed well, serving as the experimental group. 100 μL of the spore suspension was added to 900 μL of PBS, serving as the control group. The experimental group and the control group were simultaneously placed in a 37°C water bath and incubated for 3 h. After removal, they were diluted by 2-fold, 100 μL was spread on LB plates, and incubated at 37°C overnight. The number of colonies was counted. The experiment was repeated three times.

[0040] Survival rate (%) = number of colonies after 3 h treatment of the experimental group / number of colonies after 3 h treatment of the control group x 100%.

[0041] The results showed that the survival rate of Bacillus amyloliquefaciens Y106-A1-3 was 79.34 ± 0.47%.

[0042] Artificial intestinal juice tolerance test

[0043] Preparation of artificial intestinal juice: 6.8 g of potassium dihydrogen phosphate was weighed and added to 500 mL of distilled water to dissolve completely. The pH value was adjusted to 6.8 with 0.4% sodium hydroxide, and diluted with water to 1000 mL. 0.21 g of trypsin was added to 100 mL of the above solution and dissolved and mixed well. It was filtered using a 0.22 μm sterile filter membrane and stored for standby use. The blank artificial intestinal juice was artificial intestinal juice without trypsin, which was filtered using a 0.22 μm sterile filter membrane and stored for standby use. The above solutions were prepared fresh every time.

[0044] Take 100 μL spore suspension to add 900 μL artificial intestinal fluid, mix, as experimental group; take 100 μL spore suspension to add 900 μL blank artificial intestinal fluid, as control group. The experimental group and the control group are placed in 37℃ at the same time, water bath incubation 3h, take out after dilution, take 100 μL on LB plate, 37℃ incubator overnight culture, count the number of bacteria. Experimental triplicate.

[0045] Survival rate (%) = the number of bacteria after 3h treatment of experimental group / the number of bacteria after 3h treatment of control group x 100%.

[0046] The results show that: Bacillus amyloliquefaciens Y106-A1-3 survival rate can reach 89.13±7.94%.

[0047] Bile salt tolerance test

[0048] Preparation of artificial bile salt solution: use PBS to prepare 0.5% pig bile salt solution 100mL, sterilized after high pressure (121℃, 20min) for standby.

[0049] Take 100 μL spore suspension to add 900 μL artificial bile salt solution, mix, as experimental group; take 100 μL spore suspension to add 900 μL PBS, as control group. The experimental group and the control group are placed in 37℃ at the same time, incubation 3h, take out after dilution, take 100 μL on LB plate, 37℃ incubator overnight culture, count the number of bacteria. Experimental triplicate.

[0050] Survival rate (%) = the number of bacteria after 3h treatment of experimental group / the number of bacteria after 3h treatment of control group x 100%.

[0051] The results show that: Bacillus amyloliquefaciens Y106-A1-3 survival rate can reach 98.45±1.43%.

[0052] Drug sensitivity test

[0053] Take 100 μL Bacillus amyloliquefaciens culture to OD600 value of 0.6 on LB plate; respectively take tetracycline, ampicillin, penicillin, kanamycin, lincomycin, erythromycin, polymyxin B, vancomycin, ceftriaxone, gentamicin, streptomycin, doxycycline drug sensitive paper to the above LB plate, 37℃ incubator culture overnight, measure and record the diameter of inhibition zone.

[0054] The results showed that the most sensitive antibiotic for Bacillus amyloliquefaciens Y106-A1-3 was erythromycin, with an inhibition zone diameter of 29.67±0.47 mm, while lincomycin showed no obvious inhibition zone for Y106-A1-3; the inhibition zone diameter of Y106-A1-3 to polymyxin B was 8.33±0.47 mm. It was shown that Y106-A1-3 had certain resistance to lincomycin and polymyxin B. The specific inhibition zone diameter is shown in Table 1.

[0055] Table 1 Drug sensitivity test

[0056]

[0057] Note: "N" means no inhibition activity

[0058] Determination of the inhibition effect of Bacillus amyloliquefaciens

[0059] Culture of the isolated strain and preparation of cell-free supernatant (CFS):

[0060] The glycerol stock of Bacillus amyloliquefaciens isolated strain stored at -80°C was streaked on LB solid medium and cultured at 37°C overnight. A single colony was picked and inoculated in LB liquid medium and cultured at 37°C, 180 rpm for 12 h. Then 3% (V / V) of the culture was transferred into 100 mL of LB liquid medium and cultured at 37°C, 180 rpm for 36 h.

[0061] After the culture was completed, the sample was centrifuged at 8000 rpm, 4°C for 15 min, and the precipitate was discarded. The supernatant was filtered with a 0.22 μm filter to obtain the cell-free supernatant (CFS). The CFS was stored at 4°C for later use.

[0062] Culture of the indicator bacteria:

[0063] The strain stored at -80°C was streaked twice on solid medium plates, and a single colony was picked and inoculated in liquid medium suitable for the growth of the strain. The culture was incubated under suitable conditions for each strain until the OD600 value was about 0.6, and then the culture was stored at 4°C for later use.

[0064] The inhibition test used the punch method: 100 μL of the indicator bacteria liquid was evenly spread on the LB solid medium, and several small holes were punched on the spreaded plate, with a hole diameter of 8-9 mm. The CFS of Bacillus subtilis and Bacillus amyloliquefaciens prepared above was added to the small holes, and the culture dishes were incubated at 37°C for 24 h. The inhibition zone diameter was measured. Each group had three repeated tests, and the inhibition zone diameter was used to evaluate the inhibition effect of each test strain.

[0065] Results show that: 66 strains of indicator bacteria are used for antibacterial experiment. The CFS of Bacillus amyloliquefaciens Y106-A1-3 shows antibacterial effect on 30 strains of indicator bacteria, and the CFS of Bacillus amyloliquefaciens Y106-A1-3 does not show obvious antibacterial effect on the remaining strains. The CFS of Bacillus amyloliquefaciens Y106-A1-3 shows antibacterial effect on Enterococcus (HC-5, HC-28, HC-25, HC-32), Staphylococcus (AC-13 white, AC-39 white, GC-50, MC-50-1, PC-10-2, QC-1, SC-6-white, SC-23-white, TC-22-white, TC-41-white, TC-34-white, TC-11-white, ATCC25923, ATCC29213), Clostridium perfringens (ATCC13124, X27, V25, X41, X39), Clostridium difficile (ATCC43593), Salmonella (SL1344, SL7207, SP, CVCC514), Burkholderia cepacia (ATCC25416), Escherichia coli (ATCC25922). Compared with other genera of indicator bacteria, the inhibitory effect of the CFS of Bacillus amyloliquefaciens Y106-A1-3 on Clostridium perfringens is obviously stronger.

[0066] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the foregoing detailed description of the present application is made with reference to the foregoing embodiments, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included in the protection scope of the present application.

Claims

1. A broad-spectrum antibacterial Bacillus amyloliquefaciens strain derived from pigs, characterized by: The Bacillus amyloliquefaciens mentioned is porcine Bacillus amyloliquefaciens Y106-A1-3, with the preservation number CGMCCNo.26510.

2. The application of the porcine-derived broad-spectrum antibacterial Bacillus amyloliquefaciens strain according to claim 1, characterized in that: Bacterial suspensions or culture media prepared from Bacillus amyloliquefaciens are used to prepare antibacterial or bacteriostatic products; The antibacterial or bacteriostatic bacteria are any one or more of the following: Enterococcus, Staphylococcus, Clostridium perfringens, Clostridium difficile, Salmonella, Burkholderia cepacia, and Escherichia coli.

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