Bacillus subtilis XNW-KCYB1, microecological preparation as well as preparation method and application of microecological preparation

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis XNW-KCYB1, a microecological preparation as well as a preparation method and application of the microecological preparation. The invention provides a strain of bacillus subtilis XNW-KCYB1, and the preservation number of the bacillus subtilis is CCTCC (China Center For Type Culture Collection) M 20241806. The bacillus subtilis XNW-KCYB1 disclosed by the invention is separated from intestinal tracts of healthy piglets, the strain can survive at high temperature, and the bacillus subtilis XNW-KCYB1 can be used for inhibiting reproductive and respiratory syndrome viruses and inhibiting the activity of streptococcus suis, actinobacillus pleuropneumoniae, pasteurella multocida, clostridium perfringens and escherichia coli. The bacillus subtilis XNW-KCYB1 disclosed by the invention is a bacillus subtilis strain with application potential, and has extremely important significance on prevention and control of porcine reproductive and respiratory syndrome, livestock breeding and development of feed industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus subtilis XNW-KCYB1, a microecological preparation, and a preparation method and application thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an enveloped positive-strand RNA virus belonging to the genus Porrartevirus of the family Arteriviridae. It can cause Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease. Blue ear disease is a contact infectious disease that causes severe reproductive disorders in sows and respiratory diseases in piglets. It is currently one of the most serious infectious diseases affecting the world's pig farming industry. The typical characteristics of PRRS are anorexia and fever, abortion in late pregnancy of sows, birth of stillbirths and mummies, and severe respiratory symptoms in piglets during the lactation period, which will die within a short period of time. The morbidity and mortality of PRRS are high, and the economic losses caused by it are incalculable.

[0003] Vaccines are widely used to prevent and control animal infectious diseases. Currently, a variety of commercial PRRSV vaccines have been approved for use in my country. Although vaccines play an important role in the prevention and control of PRRSV, it cannot be ignored that current vaccines cannot completely prevent the spread of PRRSV within and between pig herds. In addition, there are many types of PRRSV strains and they are constantly mutating and recombining. In addition, they have biological characteristics such as antibody-dependent enhancement (ADE), persistent infection, secondary bacterial infection and immunosuppression, making the prevention, control and eradication of PRRSV a difficult problem facing the pig industry worldwide.

[0004] In addition, some bacteria can also cause diseases in pigs, bringing huge economic losses to the pig farming industry. For example, bacteria that cause pig pneumonia and can cause lesions on their own can be classified as primary pathogens, including Actinobacillus pleuropneumoniae and Pasteurella multocida; Streptococcus suis can cause acute sepsis and meningitis in pigs; diseases caused by Clostridium perfringens mainly include clostridial enteritis in piglets and sudden death syndrome in pigs; Escherichia coli can cause three major diseases in pigs: yellow diarrhea in piglets, white diarrhea in piglets and edema in piglets. These bacterial diseases are more harmful to pigs.

[0005] Bacillus subtilis is widely present in nature, is harmless to humans and animals, and can produce a variety of secondary metabolites. In addition, Bacillus subtilis is a type of Bacillus that can be used as a feed additive. At present, research on Bacillus subtilis is mainly focused on aquatic health care, plant pesticides, sewage treatment and environmental pollution. There are no reports on the use of Bacillus subtilis in the animal husbandry industry to simultaneously prevent and control PRRS and bacteria. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a strain of Bacillus subtilis XNW-KCYB1, a microecological preparation, and a preparation method and application thereof. The Bacillus subtilis XNW-KCYB1 or the microecological preparation of the present invention has the function of inhibiting porcine reproductive and respiratory syndrome virus and Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli bacteria.

[0007] In order to solve the above technical problems, the following technical solutions are proposed:

[0008] The invention provides a Bacillus subtilis XNW-KCYB1 strain, whose deposit number is CCTCC NO: M 20241806.

[0009] The present invention provides a microecological preparation, comprising the Bacillus subtilis XNW-KCYB1 of the above technical solution.

[0010] Preferably, the microecological preparation includes a liquid preparation or a solid preparation; the liquid preparation includes a fermentation broth; the bacterial concentration of Bacillus subtilis XNW-KCYB1 in the liquid preparation is ≥1×10 10 CFU / mL; the bacterial concentration of Bacillus subtilis XNW-KCYB1 in the solid preparation is ≥1×10 10 CFU / g.

[0011] The invention provides a method for preparing a fermentation liquid, comprising: inoculating the Bacillus subtilis XNW-KCYB1 of the technical solution into a Landy medium for fermentation culture to obtain the Bacillus subtilis XNW-KCYB1 fermentation liquid.

[0012] Preferably, the temperature of the fermentation culture is 26-30° C., and the time is 40-48 hours; and the dissolved oxygen concentration of the fermentation broth of the fermentation culture is 53%-57%.

[0013] The present invention provides a method for preparing a solid preparation of a microecological preparation, comprising: drying the Bacillus subtilis XNW-KCYB1 fermentation liquid obtained by the preparation method described in the above technical scheme to obtain a solid preparation.

[0014] Preferably, the drying method includes spray drying; the conditions of the spray drying are: inlet temperature of 150-180°C, outlet air temperature of 75-95°C, feed rate of 300-600L / h, air inlet velocity of 10-25m / h, and air inlet temperature of 150-180°C. 3 / h and spray pressure 0.1~0.2MPa.

[0015] Preferably, before the drying, the Bacillus subtilis XNW-KCYB1 fermentation liquid is mixed with a protective agent; the protective agent includes one or more of maltodextrin, trehalose and skim milk powder.

[0016] The present invention provides the use of the Bacillus subtilis XNW-KCYB1 described in the above technical solution, the microecological preparation described in the above technical solution, the fermentation liquid prepared by the preparation method described in the above technical solution, or the solid preparation prepared by the preparation method described in the above technical solution in the preparation of antiviral and / or antibacterial products.

[0017] Preferably, the virus includes porcine reproductive and respiratory syndrome virus; the bacteria include one or more of Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli.

[0018] Beneficial effects of the present invention: The present invention provides a strain of Bacillus subtilis XNW-KCYB1, the deposit number of the Bacillus subtilis is CCTCC NO: M 20241806. The Bacillus subtilis XNW-KCYB1 of the present invention is isolated from the intestine of a healthy piglet, and the strain can survive at high temperatures. Bacillus subtilis XNW-KCYB1 can inhibit the activity of reproductive and respiratory syndrome virus and bacteria such as Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli.

[0019] Bacillus subtilis XNW-KCYB1 is a strain of Bacillus subtilis with application potential. It is of great significance to the prevention and control of porcine reproductive and respiratory syndrome, animal husbandry and the development of feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a bacterial morphology diagram of Bacillus subtilis XNW-KCYB1 of the present invention;

[0021] Figure 2 The electrophoresis result of 16S rDNA PCR product of Bacillus subtilis XNW-KCYB1 of the present invention;

[0022] Figure 3 It is a schematic diagram of the 16srDNA evolutionary tree of Bacillus subtilis XNW-KCYB1 of the present invention; Figure 3The numbers "1, 9, 11, 12" represent the relationship between the two strains. The larger the number, the closer the relationship.

[0023] Biological Deposit Description

[0024] Bacillus subtilis XNW-KCYB1 was deposited in the China Center for Type Culture Collection (CCTCC) on August 16, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20241806. DETAILED DESCRIPTION

[0025] The present invention provides a strain of Bacillus subtilis XNW-KCYB1, with a deposit number of CCTCC NO: M 20241806. The 16srDNA gene sequence of the Bacillus subtilis XNW-KCYB1 of the present invention is shown in SEQ ID NO.3.

[0026] The present invention provides a microecological preparation, comprising the Bacillus subtilis XNW-KCYB1 of the above technical solution.

[0027] The present invention uses the intestinal contents of healthy weaned piglets as raw materials to isolate Bacillus subtilis XNW-KCYB1 which has both antiviral and antibacterial effects. The present invention uses the 16s rDNA sequence of Bacillus subtilis XNW-KCYB1 to perform BLAST homologous sequence search in NCBI, and constructs an evolutionary tree with sequences with high homology, as shown in FIG. Figure 3 Combined with the bacterial morphological characteristics, the strain XNW-KCYB1 was determined to be Bacillus subtilis.

[0028] The bacterium colony of the Bacillus subtilis XNW-KCYB1 of the present invention on the LB culture medium plate has a crater and a serrated edge. The Bacillus subtilis XNW-KCYB1 provided by the present invention is not resistant to each antibiotic, will not cause abuse of strains, has high safety, and the Bacillus subtilis XNW-KCYB1 does not have a drug-resistant phenotype and does not carry drug-resistant genes, and will not cause horizontal transmission of drug-resistant genes during use. After the formation of spores, the Bacillus subtilis XNW-KCYB1 has good heat resistance, can resist bacteria such as Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli, and resists reproductive and respiratory syndrome virus, has good antiviral and antibacterial effects, and can use Bacillus subtilis XNW-KCYB1 to prepare safe, efficient, green natural antiviral and antibacterial products, which are suitable for animal husbandry.

[0029] As an optional embodiment, the microecological preparation of the present invention includes a liquid preparation or a solid preparation. As an optional embodiment, the liquid preparation of the present invention includes a fermentation broth. As an optional embodiment, the bacterial concentration of Bacillus subtilis XNW-KCYB1 in the liquid preparation of the present invention is ≥1×10 10 CFU / mL, or 1×10 10 CFU / mL; the bacterial concentration of Bacillus subtilis XNW-KCYB1 in the solid preparation of the present invention is ≥1×10 10 CFU / g, or 1×10 10 CFU / g.

[0030] The present invention provides a method for preparing a fermentation broth, comprising:

[0031] The Bacillus subtilis XNW-KCYB1 described in the above technical solution is inoculated into Landy medium for fermentation to obtain Bacillus subtilis XNW-KCYB1 fermentation liquid.

[0032] As an optional implementation mode, the present invention inoculates the seed liquid of the Bacillus subtilis XNW-KCYB1 into Landy medium for fermentation culture. As an optional implementation mode, the preparation method of the seed liquid of the present invention comprises: streaking the Bacillus subtilis XNW-KCYB1 on a solid culture medium plate, activating the culture to obtain a single colony; inoculating the single colony into a liquid culture medium for seed culture to obtain a seed liquid. As an optional implementation mode, the temperature of the activation culture is 33-37°C, and can also be 35-36°C; in a specific embodiment of the present invention, the temperature of the activation culture is 33, 34, 35, 36 or 37°C. As an optional implementation mode, the activation culture time is 17-20h, and can also be 18-19h. In a specific embodiment of the present invention, the activation culture time is 17, 18, 19 or 20h. As an optional implementation mode, the solid culture medium includes LA solid culture medium. The composition of each 1L of LA solid culture medium includes: 5g yeast extract, 10g tryptone, 10g sodium chloride, 15g agar powder and the rest of distilled water, and the pH is 7.0. As an optional embodiment, a single colony is obtained, and the present invention inoculates the single colony into a liquid culture medium for seed culture to obtain a seed liquid. The temperature of the seed culture of the present invention is 33-37°C, and can also be 35-36°C; in a specific embodiment of the present invention, the temperature of the seed culture is 33, 34, 35, 36 or 37°C; the time of the seed culture is 17-20h, and can also be 18-19h. In a specific embodiment of the present invention, the time of the seed culture is 17, 18, 19 or 20h. As an optional embodiment, the liquid culture medium includes LB liquid culture medium. As an optional embodiment, the rotation speed of the seed culture is 170-200rpm. In a specific embodiment of the present invention, the rotation speed of the seed culture is 170, 180, 190, or 200rpm.

[0033] As an optional embodiment, the present invention inoculates the seed liquid into Landy medium for fermentation culture to obtain a fermentation liquid. The inoculation amount of the seed liquid of the present invention is 8% to 12%, or 10% to 11%. The temperature of the fermentation culture of the present invention is 26 to 30°C, or 27 to 29°C; in a specific embodiment of the present invention, the temperature of the first fermentation culture is 26, 27, 28, 29 or 30°C. The fermentation culture time of the present invention is 46 to 50h, or 47 to 49h. In a specific embodiment of the present invention, the second fermentation culture time is 46, 47, 48, 49 or 50h. As an optional embodiment, the dissolved oxygen concentration of the fermentation liquid of the second fermentation culture is 53% to 57%, or 54% to 55%. In a specific embodiment of the present invention, the dissolved oxygen concentration of the fermentation liquid of the second fermentation culture is 53%, 54%, 55%, 56% or 57%. As an optional embodiment, the bacterial concentration of the fermentation liquid is ≥1×10 10 CFU / mL, or 5×10 10 CFU / mL. As an optional embodiment, a defoaming agent is added during the fermentation culture. The present invention does not specifically limit the type and addition method of the defoaming agent, and a conventional method can be used. As an optional embodiment, the rotation speed of the fermentation culture is 180 to 220 rpm. In a specific embodiment of the present invention, the rotation speed of the fermentation culture is 180, 190, 200, 210 or 220 rpm. As an optional embodiment, the equipment used for fermentation culture is a fermentor. The fermentation culture of the present invention can obtain a large amount of fermentation liquid through the large volume advantage of the fermenter, reduce time cost and increase the number of Bacillus in the fermentation liquid.

[0034] As an optional embodiment, the composition of each 1L of Landy medium includes: 30g glucose, 5g L-glutamic acid, 0.5g MgSO4, 0.5g KCl, 1gKH2PO4, 0.15mg FeSO4, 5mg MnSO4, 0.16mg CuSO4·5H2O and 1000mL deionized water. The Landy medium is used for culturing in order to control the bacterial growth of Bacillus subtilis XNW-KCYB1 and promote it to produce more metabolites.

[0035] The fermentation liquid prepared by the invention has a high content of metabolites of Bacillus subtilis XNW-KCYB1.

[0036] The present invention provides a method for preparing a solid preparation of a probiotic preparation, comprising: drying the Bacillus subtilis XNW-KCYB1 fermentation broth obtained by the preparation method of the above technical solution to obtain a solid preparation. As an optional embodiment, the drying of the present invention includes spray drying. As an optional embodiment, the inlet temperature of the spray drying of the present invention is 150-180°C, or 160-170°C; the outlet air temperature of the spray drying is 75-95°C, or 80-90°C; the feed rate of the spray drying is 300-600L / h, or 400-500L / h; the air inlet rate of the spray drying is 10-25m / h. 3 / h, or 15~23m 3 / h; 10~25m 3 / h means 10~25m per hour 3 The air is added to the fermentation tank. The spray pressure of the spray drying of the present invention is 0.1-0.2MPa, and can also be 0.13-0.18MPa. The advantages of the spray drying of the present invention are that the product is guaranteed to be active, the moisture content of the generated dry powder meets the requirements, and the powder output rate is fast.

[0037] As an optional embodiment, the present invention mixes the Bacillus subtilis XNW-KCYB1 fermentation broth with a protective agent before the drying. As an optional embodiment, the protective agent includes one or more of maltodextrin, trehalose and skim milk powder. As an optional embodiment, the mass volume ratio of the protective agent to the Bacillus subtilis XNW-KCYB1 fermentation broth of the present invention is (8-12) g: 100 mL; the addition of the protective agent of the present invention can reduce the mortality rate of Bacillus subtilis XNW-KCYB1 during the drying process, increase the number of viable bacteria and protect the activity of the dry powder.

[0038] As an optional embodiment, the viable bacterial count of the solid preparation of the microecological preparation prepared by the present invention is ≥1×10 10 CFU / g, or 1×10 10 CFU / g, the content of viable bacteria in solid preparations is high.

[0039] The present invention provides the use of the Bacillus subtilis XNW-KCYB1 described in the above technical solution, the microecological preparation described in the above technical solution, the fermentation liquid prepared by the preparation method described in the above technical solution, or the solid preparation prepared by the preparation method described in the above technical solution in the preparation of antiviral and / or antibacterial products.

[0040] As an optional embodiment, the virus includes reproductive and respiratory syndrome virus; the bacteria include one or more of Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli. The Bacillus subtilis XNW-KCYB1 of the present invention can inhibit Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli. In a specific embodiment of the present invention, the Escherichia coli is verified by Escherichia coli ATCC29212; the Actinobacillus pleuropneumoniae is verified by Actinobacillus pleuropneumoniae ATCC 43336; the Pasteurella multocida is verified by Pasteurella multocida ATCC 43137; the Clostridium perfringens is verified by Clostridium perfringens CVCC2030; the Streptococcus suis is verified by Streptococcus suis SC19. Among them, Streptococcus suis SC19 is publicly published and used in the prior art (see "Zou G, Zhou J, Xiao R, Zhang L, Cheng Y, Jin H, Li L, Zhang L, Wu B, Qian P, Li S, Ren L, Wang J, Oshota O, Hernandez-Garcia J, Wileman TM, Bentley S, Weinert L, Maskell DJ, Tucker AWD, Zhou R. Effects of Environmental and Management-Associated Factors on Prevalence and Diversity of Streptococcus suis in Clinically Healthy Pig Herds in China and the United Kingdom. Appl Environ Microbiol. 2018 Apr 2; 84 (8): e02590-17."; DOI: 10.1128 / AEM.02590-17), and other strains are standard strains that can be purchased conventionally. As an optional embodiment, the product includes a feed additive or a drug. The present invention has no special limitation on the preparation method of the product, and a conventional method can be used. The dosage form of the product of the present invention can be a conventional dosage form.

[0041] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] 1. The reagents, strains and equipment used in the present invention are all commercially available unless otherwise specified.

[0043] 2. Culture medium composition

[0044] (1) The composition of LB liquid culture medium is: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride and 1000 mL of deionized water. The preparation method is: after mixing and dissolving the above components, adjust the pH to 7.0±0.2, sterilize with high pressure steam at 121°C for 20 min, and then place at room temperature for use.

[0045] (2) The composition of LB solid culture medium is: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder and 1000 mL of deionized water; the preparation method is: after mixing and dissolving the above components, adjust the pH to 7.0±0.2, sterilize with high-pressure steam at 121°C for 20 min, and then prepare a solid plate when the temperature drops to about 55°C.

[0046] (3) The composition of Landy medium is as follows: 30 g glucose, 5 g L-glutamic acid, 0.5 g MgSO4, 0.5 g KCl, 1 g KH2PO4, 0.15 mg FeSO4, 5 mg MnSO4, 0.16 mg CuSO4.5H2O and 1000 mL deionized water; the preparation method is as follows: after mixing and dissolving the above components, the pH is adjusted to 7.0±0.2, and sterilized by high-pressure steam at 121°C for 20 min, and then placed at room temperature for use.

[0047] (4) TSA medium is composed of 15 g of casein pancreatic digest, 5 g of soy flour papain digest, 5 g of sodium chloride, 15 g of agar powder and 1000 mL of deionized water. The preparation method is as follows: the aforementioned components are mixed and dissolved, the pH is adjusted to 7.0 ± 0.2, and the medium is sterilized by high-pressure steam at 121°C for 20 min and then placed at room temperature for use.

[0048] (5) TSB medium is composed of 15 g of casein pancreatic digest, 5 g of soy flour papain digest, 5 g of sodium chloride, and 1000 mL of deionized water. The preparation method is as follows: the aforementioned components are mixed and dissolved, and the pH is adjusted to 7.0 ± 0.2. After high-pressure steam sterilization at 121°C for 20 min, a solid plate is prepared when the temperature drops to about 55°C.

[0049] (6) The composition of the semi-solid culture medium is as follows: TSA culture medium and TSB culture medium are mixed in a mass ratio of 5:3 and dissolved in 100 mL of distilled water; the preparation method is as follows: 2.5 g of TSA culture medium and 1.5 g of TSB culture medium are mixed and dissolved in 100 mL of distilled water, the pH is adjusted to 7.0±0.2, and the mixture is sterilized by high-pressure steam at 121°C for 20 min and then used when the temperature drops to about 55°C.

[0050] (7) The composition of LA solid medium per 1 L is: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 15 g agar powder, and the volume is made up to 1000 mL with distilled water. The pH is 7.0.

[0051] Example 1 Isolation and Identification of Bacillus subtilis XNW-KCYB1

[0052] 1. Strain Isolation and Purification

[0053] Take the intestinal contents of healthy weaned piglets and put them in sterile saline, where the mass percentage of NaCl in the sterile saline is 0.9%. Use a vortex instrument to fully vortex and mix, then place in a water bath at 85℃ for 10 minutes, dilute 10 times, spread on LB agar medium plate, and culture at 37℃ for 12 hours. Pick a single colony with a crater and a serrated edge and streak it on the LB agar medium. After streaking, subculture at 37℃ for 12 hours. Repeat the streaking and culturing operations 5 times until the colony morphology in the LB agar medium is single, and a purified single colony is obtained. The colony morphology is shown in Figure 1 , recorded as strain XNW-KCYB1.

[0054] 2. 16S rDNA sequencing and analysis of strain XNW-KCYB1

[0055] According to the instructions of the MicroElute Genomic DNA Kit, the genome of strain XNW-KCYB1 was extracted, and then PCR amplification was performed using bacterial 16S rDNA universal primers: upstream primer, 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and downstream primer: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO.2).

[0056] PCR product electrophoresis results, see Figure 2 The band size is about 1550 bp. The 16SrDNA sequence of strain XNW-KCYB1 is shown in SEQ ID NO.3.

[0057]

[0058] The 16S rDNA sequence of strain XNW-KCYB1 was compared with the known sequences in NCBI by BLAST. The 16S rDNA sequence of this strain (SEQ ID NO.3) had the highest homology with Bacillus subtilis WES3 (NCBIID: MN960117.1), reaching 99.81%. A phylogenetic tree was constructed, see Figure 3 ; Combined with the strain morphology and other characteristics, the strain XNW-KCYB1 was identified as Bacillus subtilis.

[0059] Example 2 Experiment on temperature tolerance of spores of Bacillus subtilis XNW-KCYB1 strain

[0060] 1. Composition of DSM medium: 8g yeast powder, 0.1g MgSO4·7H2O, 1g KCl, 0.02g NaOH, 1.25mg MnCl2 and 1000mL distilled water; Preparation method: Mix and dissolve the above components and sterilize them with high pressure steam at 121℃ for 20min. Before inoculating XNW-KCYB1 bacterial solution into DSM medium, add 1mL of 1mol / LCaCl2 and 1mmol / LFeSO4 per liter of medium.

[0061] 2. Strain activation and spore preparation: The Bacillus subtilis XNW-KCYB1 obtained in Example 1 was streaked on an LB agar medium plate, cultured in a constant temperature incubator at 37°C for 12 h, and a single colony was picked and transferred to an LB liquid medium, and cultured at 37°C and 200 rpm to the logarithmic phase to obtain a bacterial solution; then the bacterial solution was transferred to a DSM medium at a volume ratio of 1:100, and cultured at 37°C and 180 rpm for 48 h to obtain a fermentation broth of Bacillus subtilis XNW-KCYB1, and the spore concentration in the fermentation broth was 1×10 9 CFU / mL.

[0062] 3. Strain spore collection: The fermentation liquid of Bacillus subtilis XNW-KCYB1 in step 2 was centrifuged at 4°C and 8000 rpm for 5 min to collect the bacteria, and the bacteria were washed 3 times with sterile saline to obtain a bacterial suspension; the bacterial suspension was then water-bathed at 85°C for 10 min to obtain a water-bathed bacterial suspension, and the water-bathed bacterial suspension was centrifuged at 4°C and 8000 rpm for 5 min to collect spores, and the obtained spores were washed 3 times with sterile saline to obtain a Bacillus subtilis XNW-KCYB1 spore liquid, and the spore concentration in the spore liquid was about 1×10 9 CFU / mL.

[0063] 4. Determination of temperature tolerance of spores of strains

[0064] Treatment 1: Take 1 mL of the spore solution prepared in step 3, place it in a water bath at 75°C for 3 min, and set up 3 parallel experiments.

[0065] Treatment 2: Take 1 mL of the spore solution prepared in step 3, place it in a 75°C water bath for 5 min, and set up 3 parallel experiments.

[0066] Treatment 3: Take 1 mL of the spore solution prepared in step 3, place it in a 75°C water bath for 10 min, and set up 3 parallel experiments.

[0067] Treatment 4: Take 1 mL of the spore solution prepared in step 3, place it in a water bath at 80°C for 3 min, and set up 3 parallel experiments.

[0068] Treatment 5: Take 1 mL of the spore solution prepared in step 3, place it in a water bath at 80°C for 5 min, and set up 3 parallel experiments.

[0069] Treatment 6: Take 1 mL of the spore solution prepared in step 3, place it in a water bath at 80°C for 10 min, and set up 3 parallel experiments.

[0070] Treatment 7: Take 1 mL of the spore solution prepared in step 3, incubate in a water bath at 85°C for 3 min, and set up 3 parallel experiments.

[0071] Treatment 8: Take 1 mL of the spore solution prepared in step 3, incubate in a water bath at 85°C for 5 min, and set up 3 parallel experiments.

[0072] Treatment 9: Take 1 mL of the spore solution prepared in step 3, incubate in a water bath at 85°C for 10 min, and set up three parallel experiments.

[0073] Control group: Take 1 mL of the spore solution prepared in step 3 without water bathing, and set up 3 parallel experiments.

[0074] After the experiment, the spore solution of treatments 1 to 9 and the control group was diluted, spread on LB agar plates, cultured overnight at 37°C, and the bacteria were counted to determine the survival rate of Bacillus subtilis XNW-KCYB1. Survival rate (%) = (number of bacteria in the treatment group / number of bacteria in the control group) × 100%. The results are shown in Table 1. It can be seen that Bacillus subtilis XNW-KCYB1 has good heat resistance after forming spores.

[0075] Table 1 Survival rate of Bacillus subtilis XNW-KCYB1 spores at different temperatures

[0076]

[0077] Example 3 Determination of antibiotic sensitivity of Bacillus subtilis XNW-KCYB1 strain

[0078] 1. Activation and concentration adjustment of Bacillus subtilis XNW-KCYB1: streak Bacillus subtilis XNW-KCYB1 on LB agar medium, culture in a constant temperature incubator at 37°C for 12 hours, pick a single colony and transfer it to LB liquid medium, culture it at 37°C and 200rpm overnight, then transfer the bacterial solution obtained from overnight culture to fresh LB liquid medium, culture it at 37°C and 200rpm for about 2.5 hours to reach the logarithmic growth phase, collect the cells by centrifugation, resuspend them with sterile physiological saline, and adjust the concentration of Bacillus subtilis XNW-KCYB1 in the bacterial suspension to 1×10 5 CFU / mL.

[0079] 2. Determination of antibiotic sensitivity of Bacillus subtilis XNW-KCYB1 strain: Use commercial drug sensitivity tablets to evaluate the sensitivity of the strain to tetracycline, piperacillin, meropenem, gentamicin, chloramphenicol, sulfisoxazole, erythromycin, rifampicin and nitrofurantoin. Spread the above-prepared Bacillus subtilis XNW-KCYB1 bacterial suspension on LB agar medium and dry it. Stick different types of drug sensitivity tablets on the medium coated with Bacillus subtilis XNW-KCYB1, invert it, and culture it at 37°C for 16 to 18 hours.

[0080] 3. Criteria for determining antibiotic sensitivity of Bacillus subtilis XNW-KCYB1 strain: Referring to the National Committee for Clinical Laboratory Standards (NCCLS) drug sensitivity test standard, the diameter of the inhibition zone is above 20 mm for extremely sensitive, 15 mm to 20 mm for highly sensitive, 10 mm to 14 mm for moderately sensitive, and below 10 mm for low sensitivity.

[0081] 4. The antibiotic sensitivity results of Bacillus subtilis XNW-KCYB1 strain are shown in Table 2.

[0082] Table 2 Antibiotic sensitivity of strains

[0083]

[0084]

[0085] Note: “+” means sensitive, “-” means insensitive

[0086] From the results in Table 2, it can be seen that the Bacillus subtilis XNW-KCYB1 strain is sensitive to all antibiotics and is not resistant, which means that the strain does not have a resistant phenotype and does not carry resistant genes, and will not cause horizontal transmission of resistant genes during use.

[0087] Example 4 Preparation method of Bacillus subtilis XNW-KCYB1 microecological preparation

[0088] The composition of 1L of LA medium is: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar powder and 1000mL of deionized water.

[0089] 1. Preparation of seed solution: Bacillus subtilis XNW-KCYB1 was streaked on LA medium plates and activated for 18 h to obtain a single colony. A sterile inoculation loop was used to pick a single colony and inoculated it into LB liquid medium. Seed culture was carried out at 37 ° C and 200 rpm. After 18 h of culture, XNW-KCYB1 seed solution was obtained. The bacterial concentration of the seed solution was 1×10 9 CFU / mL.

[0090] 2. Preparation of Fermentation Broth

[0091] Fermentation culture: The seed solution obtained above was inoculated into a Landy medium fermenter at an inoculation rate of 10% of the volume of the culture medium to obtain a fermentation liquid. The number of viable Bacillus subtilis XNW-KCYB1 in the fermentation liquid was 1×10 10 CFU / mL, the volume of the fermenter is 5000 liters, and the volume of Landy medium in the fermenter is 50% of the volume of the fermenter. The culture conditions are: the dissolved oxygen concentration during the culture process is controlled to be 55%, the temperature is 28°C, the culture is 48 hours, and the defoamer is added online in real time. There is no requirement for the amount and type of the defoamer, as long as the defoaming effect is achieved.

[0092] 3. The fermentation broth of Bacillus subtilis XNW-KCYB1 obtained in step 3 was added with 10% maltodextrin by volume, with maltodextrin as a carrier, and then added to a spray drying device for spray drying. The spray drying conditions were as follows: the inlet air temperature of the spray drying device was 175°C, the outlet air temperature was 80°C, the feed rate was 500L / h, and the inlet air velocity was 25m 3 / h, spray pressure 0.2MPa. After spray drying, the Bacillus subtilis XNW-KCYB1 microecological preparation was obtained.

[0093] The viable bacterial count of the Bacillus subtilis XNW-KCYB1 microecological preparation prepared in this example was 1×10 10 CFU / g.

[0094] Example 5 Inhibitory effect of Bacillus subtilis XNW-KCYB1 probiotic preparation on cells infected with porcine reproductive and respiratory syndrome virus (PRRSV)

[0095] Preparation group: The Bacillus subtilis XNW-KCYB1 microecological preparation obtained in Example 4 was dissolved in physiological saline to prepare solutions of different concentrations. The specific concentrations of the solutions are shown in Table 3. The maximum concentration of the solution was 50 g / L. The specific experimental conditions of the solutions of different concentrations are as follows:

[0096] Preparation of PPRSV virus liquid: PRRSV with MOI=1 was inoculated into a cell bottle, and the cell bottle was full of Mac-145 cells. Then the cell bottle was placed in an incubator with a volume concentration of 5% CO2 and cultured at 37°C for 48 hours. After the 48-hour culture, the cell bottle was repeatedly frozen and thawed three times at -80°C to promote the release of the virus from the cells, and a 0.22μm filter was used to remove cell debris to obtain the PRRSV virus liquid.

[0097] Treatment 1: The concentration of the solution was 50 g / L and the titer of the PPRSV virus solution was 1000 TCID 50 , 50 μL of the lysate and 50 μL of the PPRSV virus solution were added to a 95-well cell culture plate full of Mac-145 cells, and cultured in a 5% CO2 incubator at 37°C. The cell lesions were observed and recorded at 48 hours, and the OD value of each well was determined by the MTT method. Three parallel experiments were set up;

[0098] Treatment 2: Same as treatment 1, the only difference is that the concentration of the solution is 25 g / L, and three parallel experiments are set;

[0099] Treatment 3: Same as treatment 1, the only difference is that the concentration of the dissolving solution is 12.5 g / L, and three parallel experiments are set;

[0100] Treatment 4: Same as treatment 1, the only difference is that the concentration of the solution is 6.25 g / L, and three parallel experiments are set;

[0101] Treatment 5: Same as treatment 1, the only difference is that the concentration of the solution is 3.13 g / L, and three parallel experiments are set;

[0102] Treatment 6: Same as treatment 1, the only difference is that the concentration of the solution is 1.56 g / L, and three parallel experiments are set;

[0103] Treatment 7: Same as treatment 1, the only difference is that the concentration of the dissolving solution is 0.78 g / L, and three parallel experiments are set;

[0104] Treatment 8: Same as treatment 1, the only difference is that the concentration of the dissolving solution is 0.40 g / L. Set up 3 parallel experiments.

[0105] Cell control group: only 100 μL of cell maintenance solution was added. 100 μL of cell maintenance solution was added to a 95-well cell culture plate full of Mac-145 cells, and then cultured in a 5% CO2 incubator at 37°C. The cell lesions were observed and recorded at 48 hours, and the OD value of each well was determined by MTT method. Three parallel experiments were set up. The cell maintenance solution was DMEM culture medium, the content of penicillin in DMEM culture medium was 100 U / mL, the concentration of streptomycin was 100 μg / mL, and the volume concentration of fetal bovine serum was 2%.

[0106] Virus control group: only 1000 TCID 50 100 μL of virus solution was added and cultured in a 5% CO2 incubator at 37°C. The cytopathic effect was observed and recorded at 48 h, and the OD value of each well was determined by MTT method. Three parallel experiments were set up.

[0107] The inhibition rate of different concentrations of microecological preparations was calculated according to the following formula:

[0108] Probiotics inhibition rate = (OD value of the preparation group - OD value of the virus control group) / (OD value of the cell control group - OD value of the virus control group) × 100%;

[0109] The results are shown in Table 3. The results show that the half effective dose of the microecological preparation in Example 4 for the inhibitory effect on virus-infected cells is 1.56 g / L, the Bacillus subtilis XNW-KCYB1 microecological preparation has an anti-PRRSV effect, and the microecological preparation can be used for the subsequent preparation of anti-PRRSV drugs.

[0110] Table 3 The half effective dose of probiotics to inhibit PRRSV infection of cells

[0111] Group Probiotic concentration (g / L) Inhibition rate (%) Process 1 50 98.5 Process 2 25 94.6 Process 3 12.5 92.5 Process 4 6.25 90.0 Process 5 3.13 85.5 Process 6 1.56 78.0 Process 7 0.78 48.3 Process 8 0.40 22.8 Cell control group 0 0 Virus control group 0 0

[0112] Example 6

[0113] An LB agar plate was used as the basic culture medium, and a sterile Oxford cup was placed at a fixed position therein. The diameter of the Oxford cup was 7 mm.

[0114] The preparation of LB agar basal medium 1 is as follows: the semi-solid medium is cooled to 55°C, 100 μL of indicator bacteria Streptococcus suis SC19 liquid is added, and it is immediately mixed by inversion, and poured into the LB agar basal medium in the Oxford cup, and cooled to room temperature until solidified, and the Oxford cup is gently pulled out with sterile hemostatic forceps to obtain LB agar basal medium 1.

[0115] The preparation of LB agar base medium 2 is the same as that of LB agar base medium 1, with the only difference that 100 μL of indicator bacteria Streptococcus suis SC19 liquid is replaced with 100 μL of Actinobacillus pleuropneumoniae ATCC 43336 liquid.

[0116] The preparation of LB agar base medium 3 is the same as that of LB agar base medium 1, with the only difference that 100 μL of indicator bacteria Streptococcus suis SC19 bacterial solution is replaced with 100 μL of Pasteurella multocida ATCC 43137 bacterial solution.

[0117] The preparation of LB agar base medium 4 is the same as that of LB agar base medium 1, with the only difference being that 100 μL of indicator bacteria Streptococcus suis SC19 solution is replaced with 100 μL of Clostridium perfringens CVCC2030 solution.

[0118] The preparation of LB agar base medium 5 is the same as that of LB agar base medium 1, with the only difference being that 100 μL of indicator bacteria Streptococcus suis SC19 solution is replaced with 100 μL of Escherichia coli ATCC29212 solution.

[0119] The fermentation broth obtained by fermentation culture of Bacillus subtilis XNW-KCYB1 prepared in Example 4 was centrifuged at 10000 r / min at 4° C. for 5 min to obtain a supernatant;

[0120] The obtained supernatant was added to wells 1 to 5 of LB agar basal medium at 100 μL / well, and then cultured upright in a 37°C constant temperature incubator for 16 h, and the diameter of the inhibition zone was measured.

[0121] The results are shown in Table 4.

[0122] Table 4 Inhibition results of Bacillus subtilis XNW-KCYB1 against common pathogenic bacteria

[0123] Pathogen name Diameter of inhibition zone (mm) Streptococcus suis SC19 18±1.0 Actinobacillus pleuropneumoniae ATCC43336 12±1.0 Pasteurella multocida ATCC43137 10±1.0 Clostridium perfringens CVCC2030 17±1.0 Escherichia coli ATCC29212 14±1.0

[0124] As shown in Table 4, Bacillus subtilis XNW-KCYB1 has strong antibacterial activity against a variety of porcine bacteria and has the potential to prevent and control bacterial diseases in animal husbandry.

[0125] In summary, the Bacillus subtilis XNW-KCYB1 provided by the present invention can resist PRRSV and Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli, and has good application prospects.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis XNW-KCYB1, characterized in that: The deposit number is CCTCCNO: M 20241806.

2. A microecological preparation, characterized in that: The invention comprises the Bacillus subtilis XNW-KCYB1 of claim 1.

3. The microecological preparation according to claim 2, characterized in that: The microecological preparation includes a liquid preparation or a solid preparation; the liquid preparation includes a fermentation broth; the bacterial concentration of Bacillus subtilis XNW-KCYB1 in the liquid preparation is ≥1×10 10 CFU / mL; the bacterial concentration of Bacillus subtilis XNW-KCYB1 in the solid preparation is ≥1×10 10 CFU / g.

4. A method for preparing a fermentation broth, characterized in that: include: The Bacillus subtilis XNW-KCYB1 of claim 1 is inoculated into Landy medium for fermentation to obtain a fermentation liquid of the Bacillus subtilis XNW-KCYB1.

5. The preparation method according to claim 4, characterized in that: The temperature of the fermentation culture is 26-30° C., and the time is 46-50 hours; the dissolved oxygen concentration of the fermentation liquid of the fermentation culture is 53%-57%.

6. A method for preparing a solid preparation of a probiotic preparation, characterized in that: include: The Bacillus subtilis XNW-KCYB1 fermentation liquid obtained by the preparation method according to claim 4 or 5 is dried to obtain a solid preparation.

7. The preparation method according to claim 6, characterized in that: The drying method includes spray drying; the conditions of the spray drying are: inlet temperature of 150-180°C, outlet air temperature of 75-95°C, feed rate of 300-600L / h, air inlet speed of 10-25m 3 / h and spray pressure 0.1~0.2MPa.

8. The preparation method according to claim 6 or 7, characterized in that: Before the drying, the fermentation liquid of Bacillus subtilis XNW-KCYB1 is mixed with a protective agent; the protective agent comprises one or more of maltodextrin, trehalose and skim milk powder.

9. Use of the Bacillus subtilis XNW-KCYB1 according to claim 1, the probiotic preparation according to claim 2 or 3, the fermentation broth prepared by the preparation method according to claim 4 or 5, or the solid preparation prepared by the preparation method according to any one of claims 6 to 8 in the preparation of antiviral and / or antibacterial products.

10. The use according to claim 9, characterized in that: The virus includes porcine reproductive and respiratory syndrome virus; the bacteria include one or more than two of Streptococcus suis, Actinobacillus pleuropneumoniae, Pasteurella multocida, Clostridium perfringens and Escherichia coli.

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