A strain of Bacillus Velezii HBXN2020, microecological preparations and their applications

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Bacillus velezensis HBXN2020, a microecological preparation and an application thereof. The present invention provides a strain of Bacillus velezensis HBXN2020, and the deposit number of the Bacillus velezensis is CCTCCM20221720. The strain of the present invention has a broad-spectrum antibacterial property, can maintain normal intestinal flora of animals, and has no antibiotics and drug resistance.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus velez HBXN2020, a microecological preparation and applications thereof. Background Art

[0002] The period after weaning is the most difficult period in the life of suckling piglets, because environmental changes and different diet formulas force the digestive system and the composition of intestinal flora to undergo tremendous changes. In this case, the ecological balance of the intestinal flora will be severely damaged, and the gastrointestinal motility of piglets is weak, which makes piglets very susceptible to bacterial and viral infections, leading to diarrhea, growth stagnation, and even death. The early health of weaned piglets has a very important impact on their growth rate and feed utilization, which have a significant economic impact on breeding companies or farmers. However, maintaining the homeostasis of intestinal flora is the most effective way to reduce weaning-related damage. Intestinal flora has many functions in the host defense process, such as immune regulation, nutrient metabolism, energy supplementation, and the integrity of the gastrointestinal barrier. At present, due to the excessive use of antibiotics in the breeding industry, problems such as drug residues and bacterial resistance have emerged one after another, which have extremely adverse effects on human health and the ecological environment. For this reason, countries around the world have successively issued decrees prohibiting the addition of growth-promoting antibiotics in livestock and poultry feed.

[0003] In the past hundreds of years, probiotics have been mainly used in the food and health care industry. In recent years, more and more studies have found that probiotics have certain advantages in preventing and treating animal diarrhea, promoting animal growth, improving livestock and poultry immune function, and improving feed conversion rate. Their use as feed additives can also effectively avoid the emergence of antibiotic-resistant strains, antibiotic residues, environmental pollution and other problems caused by antibiotics.

[0004] Bacillus Velez is a newly discovered species of Bacillus. It is widely found in soil, rivers, oceans, feces and animal intestines in nature. Its strains have many excellent characteristics, such as heat stability, acid and alkali resistance, harmlessness to humans and animals, and the ability to produce a variety of secondary metabolites that antagonize pathogens. At present, research on Bacillus Velez is mainly focused on aquatic health care, plant pesticides, sewage treatment and environmental pollution. However, there are few reports on the application of Bacillus Velez in the animal breeding industry. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a strain of Bacillus Velezii HBXN2020, a microecological preparation and its application. The strain of the present invention has a broad-spectrum antibacterial property, can maintain normal intestinal flora of animals, and has no antibiotics and drug resistance.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a Bacillus velezensis HBXN2020, and the deposit number of the Bacillus velezensis is CCTCC M 20221720.

[0008] Preferably, the specific sequence of Bacillus velez is shown as SEQ ID No.4.

[0009] The present invention provides a probiotic preparation or an animal feed additive, wherein the effective ingredients of the probiotic preparation or the animal feed additive include the Bacillus Velez subtilis HBXN2020 described in the above technical solution, or the fermentation liquid of Bacillus Velez subtilis HBXN2020 or the metabolites of the Bacillus Velez subtilis HBXN2020.

[0010] Preferably, the bacterial activity of Bacillus Velezii HBXN2020 in the microecological preparation or animal feed additive is 1×10 10 CFU / g~3×10 10 CFU / g.

[0011] The invention provides a method for preparing a fermentation liquid of Bacillus Velez subtilis HBXN2020, comprising the following steps: inoculating Bacillus Velez subtilis HBXN2020 into a modified Landy medium for fermentation culture to obtain the fermentation liquid of Bacillus Velez subtilis HBXN2020.

[0012] The present invention provides the use of the Bacillus Velezii HBXN2020 described in the above technical solution or the microecological preparation or animal feed additive described in the above technical solution in preventing and controlling bacteria.

[0013] Preferably, the bacteria include one or more of Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Clostridium perfringens and Staphylococcus aureus.

[0014] The present invention provides the use of the Bacillus Velezii HBXN2020 described in the above technical scheme or the probiotic preparation or animal feed additive described in the above technical scheme in the preparation of a product for maintaining the balance of animal gastrointestinal flora.

[0015] The present invention provides the use of the Bacillus Velezii HBXN2020 described in the above technical scheme or the probiotic preparation or animal feed additive described in the above technical scheme in the preparation of a product for preventing animal diarrhea and / or improving animal disease resistance.

[0016] The present invention provides a breeding method for maintaining the balance of gastrointestinal flora of animals, comprising the following steps:

[0017] Feed animals with the microecological preparation or animal feed additive described in the above technical solution.

[0018] Beneficial effects: The present invention provides a strain of Bacillus velezensis HBXN2020, and the deposit number of the Bacillus velezensis is CCTCC M 20221720. The Bacillus velezensis HBXN2020 described in the present invention is isolated from the intestines of healthy piglets. The strain has a broad-spectrum antibacterial effect, can survive under high temperature and high pressure, strong acid and strong alkali, can produce cellulase, and has no antibiotics and drug resistance; the strain can also help maintain the balance of animal gastrointestinal flora, prevent animal diarrhea, and thus improve the disease resistance of animals. From the above, it can be seen that Bacillus velezensis HBXN2020 is a pig-derived probiotic spore-forming bacteria with huge application potential, which is of great significance to the development of animal health, animal husbandry and feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 This is the bacterial morphology of strain HBXN2020;

[0021] Figure 2 This is the electrophoresis of the PCR product of 16S rDNA of strain HBXN2020;

[0022] Figure 3 is the 16S rDNA phylogenetic tree of strain HBXN2020;

[0023] Figure 4 This is a Gram staining micrograph of strain HBXN2020;

[0024] Figure 5 This is a diagram showing the antagonistic effect of Bacillus Velez HBXN2020 on Escherichia coli;

[0025] Figure 6 This is a diagram showing the antagonistic effect of Bacillus Velez HBXN2020 on Salmonella Typhimurium and Salmonella Enteritidis SE006;

[0026] Figure 7 This is a diagram showing the antagonistic effect of Bacillus Velez HBXN2020 on Clostridium perfringens;

[0027] Figure 8 This is a diagram showing the antagonistic effect of Bacillus Velez HBXN2020 on Staphylococcus aureus;

[0028] Fig. 9 This is a qualitative result diagram of cellulase production by Bacillus Velez HBXN2020;

[0029] Fig.10 This is an electron micrograph of Bacillus velezensis HBXN2020.

[0030] Biological deposit information

[0031] Bacillus velezensis HBXN2020 is deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, Wuhan, Hubei. The deposit date is November 3, 2022, and the deposit number is CCTCC M 20221720. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the strains described in the present invention can be purchased routinely by those skilled in the art.

[0033] The present invention provides a strain of Bacillus velezensis HBXN2020, and the deposit number of the Bacillus velezensis is CCTCC M 20221720. The specific sequence of the Bacillus velezensis of the present invention is preferably shown as SEQ ID No.4, and the 16S rDNA sequence of the Bacillus velezensis is preferably shown as SEQ ID NO.3. The colony of the Bacillus velezensis HBXN2020 strain is milky white, with a smooth and transparent surface, neat edges, and wrinkles when dry; it is a Gram-positive bacterium that can form spores, is resistant to high temperatures, and is resistant to acid and alkali; the growth conditions are a temperature of 25°C to 42°C and a pH of 5.0 to 9.0. The nucleotide sequence of the 16S rDNA of the Bacillus velezensis HBXN2020 strain is shown as SEQ ID NO.3. This strain has antagonistic effects on multiple strains, especially Escherichia coli ATCC25922, Escherichia coli K88, Escherichia coli O157, Salmonella typhimurium ATCC14028, Salmonella Enteritidis SE006, Clostridium perfringens CVCC2030, Staphylococcus aureus ATCC29213 and Staphylococcus aureus ATCC43300.

[0034] The present invention provides a probiotic preparation or an animal feed additive, wherein the effective ingredients of the probiotic preparation or the animal feed additive include the Bacillus Velez subtilis HBXN2020 described in the above technical solution, or the fermentation liquid of Bacillus Velez subtilis HBXN2020 or the metabolites of the Bacillus Velez subtilis HBXN2020. The bacterial activity of Bacillus Velez subtilis HBXN2020 in the probiotic preparation or the animal feed additive of the present invention is preferably 1×10 10 CFU / g~3×10 10 CFU / g, more preferably 2×10 10 CFU / g~3×10 10The metabolites of Bacillus Velez HBXN2020 of the present invention preferably include the fermentation broth of Bacillus Velez HBXN2020 or the bacteriocin produced by Bacillus Velez HBXN2020, more preferably the fermentation broth of Bacillus Velez HBXN2020; the bacteriocin produced by Bacillus Velez HBXN2020 preferably includes the antimicrobial lipopeptide prepared by Bacillus Velez HBXN2020.

[0035] The present invention provides a method for preparing a fermentation broth of Bacillus Velez HBXN2020, comprising the following steps: inoculating Bacillus Velez HBXN2020 into a modified Landy medium for fermentation culture to obtain a fermentation broth of Bacillus Velez HBXN2020. The modified Landy medium of the present invention preferably comprises the following components: glucose 30 g / L, L-glutamic acid 5 g / L, MgSO4 0.5 g / L, KCl 0.5 g / L, KH2PO4 1 g / L, FeSO4 0.15 mg / L, MnSO4 5 mg / L and CuSO4.5H2O 0.16 mg / L. The temperature of the fermentation culture of the present invention is preferably 27-30°C, more preferably 28°C; the time is preferably 36-48 h, more preferably 48 h; the rotation speed is preferably 180-220 r / min, more preferably 200 r / min. The viable count of the fermentation broth of Bacillus Velezii HBXN2020 of the present invention is preferably 1×10 10 CFU / g~3×10 10 CFU / g, preferably 2×10 10 CFU / mL~3×10 10 CFU / mL, more preferably 3×10 10 CFU / mL. The inoculation amount of the Bacillus Velezii HBXN2020 of the present invention is 1% to 2% of the volume of the modified Landy medium, and more preferably 2%.

[0036] When inoculating the modified Landy with Bacillus velezii HBXN2020, the Bacillus velezii HBXN2020 is preferably inoculated in the form of a seed solution of Bacillus velezii HBXN2020. The method for preparing the seed solution of Bacillus velezii HBXN2020 of the present invention preferably comprises the following steps: inoculating Bacillus velezii HBXN2020 in an LB liquid culture medium to obtain a seed solution of Bacillus velezii HBXN2020. The inoculation amount of Bacillus velezii HBXN2020 of the present invention is one ring. The LB liquid culture medium of the present invention preferably comprises the following components at the following concentrations: 5g yeast extract, 10g tryptone, 10g sodium chloride, fixed to 1000mL with distilled water, pH 7.0; the culture time is preferably 12 to 16h, more preferably 16h; the temperature is preferably 30 to 37°C, more preferably 37°C; the rotation speed is preferably 160 to 220r / min, more preferably 180r / min. The viable bacterial count of the seed solution of Bacillus Velezii HBXN2020 of the present invention is preferably 5×10 8 CFU / mL~1×10 9 CFU / mL, more preferably 1×10 9 CFU / mL.

[0037] The present invention provides the use of Bacillus Velez HBXN2020 described in the above technical solution or the microecological preparation or animal feed additive described in the above technical solution in preventing and controlling bacteria. The bacteria described in the present invention preferably include one or more of Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Clostridium perfringens and Staphylococcus aureus. The Escherichia coli described in the present invention preferably includes one or more of Escherichia coli ATCC25922, Escherichia coli K88 and Escherichia coli O157; the Salmonella typhimurium preferably includes Salmonella typhimurium ATCC14028, the Salmonella enteritidis preferably includes Salmonella enteritidis SE006, the Clostridium perfringens preferably includes Clostridium perfringens CVCC2030, and the Staphylococcus aureus preferably includes Staphylococcus aureus ATCC29213 and / or Staphylococcus aureus ATCC43300. The Genbank number of the enteritidis Salmonella SE006 of the present invention is CP099973, which is disclosed in "Fitness Trade-Offs in Phage Cocktail-Resistant Salmonella enterica Serovar Enteritidis Results in Increased Antibiotic Susceptibility and Reduced Virulence", DOI: 10.1128 / spectrum.02914-22. The present invention has no special limitation on the source of the strain, and the strain can be purchased by a person skilled in the art.

[0038] The present invention provides the use of the Bacillus Velez HBXN2020 described in the above technical scheme or the probiotic preparation or animal feed additive described in the above technical scheme in the preparation of a product for maintaining the balance of gastrointestinal flora in animals, and also provides the use of the Bacillus Velez HBXN2020 described in the above technical scheme or the probiotic preparation or animal feed additive described in the above technical scheme in the preparation of a product for preventing animal diarrhea and / or improving animal disease resistance.

[0039] The present invention provides a breeding method for maintaining the balance of gastrointestinal flora of animals, comprising the following steps:

[0040] Feeding animals with the probiotic preparations or animal feed additives described in the above technical solution. The present invention preferably feeds animals with the probiotic preparations or animal feed additives described in the above technical solution after adding them to the feed. The amount of the probiotic preparation or animal feed additive added in the present invention is preferably 1kg / ton to 2kg / ton of feed, more preferably 1kg / ton of feed; the feeding frequency is 2 to 3 times / day, more preferably 1 time / day; the feeding cycle is 3 to 4 weeks, more preferably 3 weeks. The animals described in the present invention preferably include poultry and livestock, and some include pigs, cattle, sheep or chickens, more preferably weaned piglets. The present invention has described the probiotic preparations and animal feed additives in detail above, so they will not be repeated here.

[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] Medium preparation

[0043] The LB liquid medium formula is: peptone 10 g, yeast powder 5 g, NaCl 10 g, deionized water 1000 mL, pH 7.0;

[0044] The LB agar medium formula is: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride and 15 g agar powder, dilute to 1000 mL with distilled water, pH 7.0;

[0045] The modified Landy medium base formula is: glucose 30g, L-glutamic acid 5g, MgSO4 0.5g, KCl 0.5g, KH2PO4 1g, FeSO4 0.15mg, MnSO4 5mg, CuSO4.5H2O 0.16mg, double distilled water 1000mL, pH 7.0, sterilized at 115℃ for 15min;

[0046] The formula of TSA medium is: 15 g of casein tryptic digest, 5 g of soy flour papain digest, 5 g of sodium chloride and 15 g of agar, made up to 1000 mL with distilled water, pH 7.3 ± 0.2;

[0047] The formula of TSB culture medium is: 15wt.% tryptone, 5wt.% soy peptone, 5wt.% sodium chloride, and the volume is adjusted to 1000mL with distilled water, and the pH is 7.2±0.2.

[0048] Example 1 Isolation and identification of Bacillus velez strain HBXN2020

[0049] 1. Strain screening and identification

[0050] The intestinal contents of healthy weaned piglets were taken into sterile saline, wherein the mass percentage of NaCl in the sterile saline was 0.9%, and the mixture was thoroughly vortexed using a vortexer, placed in a water bath at 85°C for 10 min, and after 10-fold gradient dilution, spread on LB agar medium and cultured at 37°C for 12 to 14 h. A single colony with a crater and a serrated edge was picked and streaked on the LB agar medium for subculture at 37°C for 12 h. A single colony was picked again and streaked on the LB agar medium and cultured at 37°C for 12 h until the colony morphology in the LB agar medium was single, and a purified single colony was obtained, which was recorded as strain HBXN2020.

[0051] A loop of purified single colony was picked and inoculated into LB liquid medium, and cultured at 37℃ and 180r / min for 24h to obtain the fermentation broth of strain HBXN2020 with a bacterial activity of 1×10 9 CFU / mL.

[0052] 2. Preparation of indicator bacteria solution

[0053] Escherichia coli K88, Salmonella typhimurium ATCC14028, Salmonella enteritidis SE006, Clostridium perfringens CVCC2030, and Staphylococcus aureus ATCC25923 preserved in 30% glycerol were streaked on LB agar medium and cultured at 37°C to obtain resuscitated strains;

[0054] The revived Escherichia coli K88, Salmonella typhimurium ATCC14028, Salmonella enteritidis SE006, Clostridium perfringens CVCC2030, and Staphylococcus aureus ATCC25923 were picked up and inoculated into LB liquid culture medium with an inoculum volume of 1% of the volume of the LB liquid culture medium. After shaking culture at 37°C and 160-220 r / min for 8-10 h, the concentrations of Escherichia coli K88, Salmonella typhimurium ATCC14028, Salmonella enteritidis SE006, Clostridium perfringens CVCC2030, and Staphylococcus aureus ATCC25923 were adjusted to 10 8 CFU / mL of indicator bacteria solution.

[0055] 3. Screening of strains with antibacterial activity

[0056] The indicator bacteria liquid was spread on the LB agar medium with a sterile cotton swab, and the spread was repeated 3 times. The plates were dried at room temperature. 5 μL of the fermentation liquid of strain HBXN2020 was spotted on the LB agar plate containing the indicator bacteria liquid, dried at room temperature, and inverted in a 37°C constant temperature incubator for 14 to 16 hours. The LB liquid medium was used as negative control 1, and the sterile PBS solution was used as negative control 2. The diameter of the inhibition zone was measured. The results are shown in Table 1.

[0057] Table 1 Antibacterial activity of strain HBXN2020 against different strains

[0058]

[0059] Note: / represents no inhibition zone.

[0060] As shown in Table 1, the strain has an inhibitory effect on Escherichia coli K88, Salmonella typhimurium ATCC14028, Salmonella enteritidis SE006, Clostridium perfringens CVCC2030 and Staphylococcus aureus ATCC25923. However, there is no inhibition zone in the modified Landy medium and sterile PBS.

[0061] 4. Identification of strain HBXN2020

[0062] 1) Colony morphology of strain HBXN2020

[0063] Pick a loop of strain HBXN2020 and inoculate it on LB agar culture, invert it and culture it in a 37℃ constant temperature incubator for 12h. Figure 1 The colonies are milky white, with a smooth, transparent surface, neat edges, and are sticky and wire-like.

[0064] 2) Gram staining and microscopy

[0065] Gram staining was performed using a Gram staining kit (manufacturer: Solarbio, catalog number: G1060). The staining results are shown in Figure 4 ;Depend on Figure 4 It can be seen that the strain is Gram-positive, the bacteria are long straight rods, appearing singly or in multiples, and can form spores. Electron microscope image of Bacillus Velezii HBXN2020 (electron microscope model manufacturer: S-3400N; Hitachi, Tokyo, Japan) Fig.10 ,Depend on Fig.10 It can be seen that the strain is in the shape of a long straight rod and appears singly or in multiples.

[0066] 3) Physiological and biochemical identification of strain HBXN2020

[0067] Physiological and biochemical assays were performed according to Bergey's Manual of Bacterial Identification (Don JB, Noel RK, James TS, et al. Bergey's The detection results are shown in Table 2.

[0068] Table 2 Physiological and biochemical characteristics of the strains of the present invention

[0069]

[0070] Note: “+” represents positive, “-” represents negative.

[0071] As recorded in Table 2, the strain was positive for Gram staining, glucose utilization test, sucrose utilization test, fructose utilization test, lactose utilization test, maltose utilization test, mannose utilization test, galactose utilization test, oxidase utilization test, starch hydrolysis test, glucose fermentation test, cellobiose utilization test, glycerol utilization test, mannitol utilization test and catalase test, and negative for ethanol utilization test, methyl red, indole test, indole test and acetylmethylcarbinol test; the strain was long rod-shaped.

[0072] 4) 16S rDNA sequencing and analysis of strain HBXN2020 are as follows:

[0073] According to the instructions of the MicroElute Genomic DNA Kit, the genome of the strain of the present invention was extracted to obtain the specific sequence of the strain, as shown in SEQ ID NO.4: GTGGCTTCTGATTATGGCATTTTTACGATCGGTGATGCGATGATCACCTTAAATCCCGCAAGCACAGGGCCTTTGCGTTTTGTTACCCATTTTGAACGAAAAGCAGGAGGAGCTGAACTGAATTTTGCGATTGCATGCGCCAGGTTGAATCTGCACTCAAAGTGGGTCAGCAGACTGGGGAAGGATGAGTTCGGCAGAGTGATTTATCAGTTTGCCAGAGGAGAAGGAATTGATGTGACTGACGTCTCCTATGTTGAAGGCTTTCCTACATCTTTAAATGTCAAAGAGATCAGAAATGACGGTTCAGGCAAGACGTTTTATTACCGTTTCAATTCACCAATTCTGACTCTTGCACCGGAAGATATAACAGAGAATATGTTTGACGGCATTCACATCGTTCATCTTACCGGTGTGTTCCTCGCGCTTGATAAGAAAAACCTTGAAATTGCTGAAAGGGTGCTTTCCATCGCCAAAGCAAGAAAGATTCCCGTATCTTTTGATCCGAATATCAGGCTGAAGCTGTGGACGATTGAGGAGGCGCGTGCGGCATTTCATCAAATCTTTCCGCATACTGATATTCTTCTGGCGGGCCGGGACGAAATGAAACAGCTGACAGGTATAGGGGAAAATGAAGCATTAGCCCGGTTTGCAGAGACCTATTCAATCAGTCAGCTCGTCATAAAAGACGGTGAAGCAGGTTCTGCGCTGTATCATGATCATAAGTGGGTTCACAAAGAGGCTTTCCCGGTTGCGCCTGTTGACACGGTAGGAGCGGGTGACGGCTTTAATGCCGGCTACCTGTACAGTTATCTGCATGGTTTTGAACCGGAAAAACGCCTGGAGTTTGCCAATGCGGTCGGTGCGCTCGTCACCACAGTTTCCGGTGACAATGAAGGGCTTCCCTATCTAGAAGAGGTTCTGTCATTTGTAAACAAAGAGACTGTGATTGAACGGTAG; Then, PCR amplification was performed using the bacterial 16S rDNA universal primers: forward primer, 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and reverse primer: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO.2).

[0074] The electrophoresis results of the PCR products are shown in Figure 2, the band size is about 1500bp. The 16S rDNA sequence of strain HBXN2020 is shown in SEQ ID NO.3: TCGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGAC AAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTC AGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT; The 16S rDNA sequence of strain HBXN2020 was compared with the known sequences in NCBI by BLAST, and a phylogenetic tree was constructed, see Figure 3 Combined with the strain morphology, microscopic examination, physiological and biochemical results, the strain of the present invention was identified as Bacillus velezensis and named Bacillus velezensis HBXN2020.

[0075] Example 2

[0076] Determination of the antibacterial spectrum of Bacillus velez HBXN2020 in vitro

[0077] 1. Preservation of strains: Pick a ring of Velez Bacillus HBXN2020 and transfer it to 10 mL LB liquid culture medium, culture it at 37°C, 180 r / min and shake for 12 h, collect the bacteria by centrifugation at 4°C, 5000 r / min for 5 min, resuspend the bacteria with 30% sterile glycerol, and store it at -80°C to obtain glycerol-frozen Velez Bacillus HBXN2020 strains, wherein the formula of 30% glycerol is: glycerol and deionized water are mixed in a volume ratio of 3:7, and sterilized at 121°C for 15 min.

[0078] 2. Activation of strains: Pick a ring of single colonies of Bacillus Velezii HBXN2020 and streak it on LB agar medium, culture it in a constant temperature incubator at 37°C for 12 hours, pick a single colony and transfer it to LB liquid medium, and culture it in a constant temperature shaker at 37°C at 180 r / min for 12 hours;

[0079] 3. Preparation of indicator bacteria solution: Take glycerol-frozen Escherichia coli ATCC25922, Escherichia coli K88, Escherichia coli O157, Salmonella typhimurium ATCC14028, Salmonella enteritidis SE006, Clostridium perfringens CVCC2030, Staphylococcus aureus ATCC43300 and Staphylococcus aureus ATCC29213 as indicator bacteria, respectively. The specific preparation method refers to the preparation method of the indicator bacteria solution in Example 1;

[0080] 4. Preparation of semi-solid culture medium: TSA medium and TSB medium were mixed in a volume ratio of 5:3 and dissolved in 100 mL of distilled water, shaken to dissolve, and sterilized by autoclave at 115°C for 15 min;

[0081] 5. Preparation and collection of fermentation broth: Pick a single colony of the activated strain and transfer it to LB liquid medium, shake and culture it at 37℃ and 180r / min constant temperature shaker for 12h, then transfer it to modified Landy medium with an inoculum of 2%, shake and culture it at 28℃ and 200r / min for 36h, collect the supernatant by centrifugation at 4℃ and 8000r / min for 10min, and filter it with a 0.22μm filter to obtain sterile fermentation broth supernatant;

[0082] 6. Determination of antibacterial activity: Using LB agar plate as the basic culture medium, a sterile Oxford cup was placed in a fixed position therein, the semi-solid culture in step 4 was cooled to 50°C, 100 μL of indicator bacteria solution was added, and the mixture was immediately mixed upside down and poured into LB agar basic culture medium, cooled to room temperature until solidified, and the Oxford cup was gently pulled out using sterile hemostatic forceps, and the supernatant obtained by centrifuging the fermentation broth of Bacillus Velez HBXN2020 prepared in Example 1 at 8000 r / min for 10 min was added to the wells, 100 μL / well, and cultured upright in a constant temperature incubator at 37°C for 16 h, and the diameter of the inhibition zone was measured; the inhibition effect is shown in Table 3 and Figures 5 to 8 , Figure 5 (a) is E. coli K88, (b) is E. coli ATCC25922, and (c) is E. coli O157; Figure 6 (a) is Salmonella typhimurium ATCC14028, (b) is Salmonella enteritidis SE006; Figure 7 is Clostridium perfringens CVCC2030; Figure 8 (a) is Staphylococcus aureus ATCC43300, and (b) is Staphylococcus aureus ATCC29213.

[0083] Table 3 Inhibition results of strain HBXN2020 against common pathogenic bacteria

[0084]

[0085] From Table 3 and Figures 4 to 7 It can be seen that the Bacillus Velezii HBXN2020 strain has an antibacterial effect on a variety of pathogenic bacteria.

[0086] Example 3 Temperature tolerance experiment of spores of Bacillus velez HBXN2020 strain

[0087] 1. Preparation of DSM medium: 8 g yeast powder, 0.1 g MgSO4·7H2O, 1 g KCl, 0.02 g NaOH, 1.25 mg MnCl2, dissolved in 1000 mL distilled water, sterilized by high pressure steam at 121°C for 20 min. Before use, 1 mL of filtered sterilized CaCl2 with a concentration of 1 mol / L and 1 mL of FeSO4 with a concentration of 1 mmol / L need to be added to each liter of the above medium.

[0088] 2. Strain activation and spore preparation: The glycerol-frozen Velez Bacillus HBXN2020 strain obtained in step 1 of Example 2 was streaked on LB agar medium, cultured in a constant temperature incubator at 37°C for 12 h, and a single colony was picked and transferred to LB liquid medium, cultured at 37°C and 180 r / min to the logarithmic phase, and then transferred to DSM medium with an inoculation amount of 1% of the volume of the DSM medium, and cultured at 37°C and 180 r / min for 48 h to obtain Velez Bacillus HBXN2020 spore liquid with a spore concentration of 1×10 9 CFU / mL.

[0089] 3. Collection of strain spores: The fermentation liquid of Bacillus Velezii HBXN2020 in step 2 was centrifuged at 4°C 10000 r / min for 10 min to collect the bacteria, washed twice with sterile saline, bathed in 85°C water for 10 min, centrifuged at 4°C 10000 r / min for 10 min to collect the spores, washed twice with sterile saline, and the spore liquid of Bacillus Velezii HBXN2020 was obtained. The spore concentration was about 1×10 9 CFU / mL.

[0090] 4. Determination of temperature tolerance of strain spores: Take 1 mL of the spore solution prepared in step 3 and place it in a water bath at 75°C, 80°C, and 85°C respectively. Set a different time gradient for each temperature gradient. Perform 3 parallel experiments for each experiment and set a control group. After the experiment, dilute the samples at different temperatures and time gradients, spread them on plates, and culture them overnight at 37°C for bacterial counts. The results are shown in Table 4.

[0091] Table 4 Survival rate of Bacillus strain HBXN2020 spores at different temperatures

[0092]

[0093]

[0094] As can be seen from Table 4, the survival rate of Bacillus Velez HBXN2020 spore solution was 98.3% after being bathed in 75°C water for 10 min; the survival rate of Bacillus Velez HBXN2020 spore solution was 90.2% after being bathed in 80°C water for 10 min; the survival rate of Bacillus Velez HBXN2020 spore solution was 85.7% after being bathed in 85°C water for 10 min, indicating that Bacillus Velez HBXN2020 has good heat resistance after forming spores.

[0095] Example 4 Artificial gastric juice tolerance test of Bacillus velez HBXN2020 strain

[0096] 1. Preparation of artificial gastric juice: Take 16.4 mL of concentrated hydrochloric acid and add it to physiological saline to dilute it to pH 2.0, then add 10 g of pepsin (Solarbio, P8390), stir thoroughly to dissolve, make the volume to 1000 mL, and filter and sterilize it using a 0.22 μm filter. The mass percentage of NaCl in the physiological saline is 0.9%.

[0097] 2. Artificial gastric juice tolerance test of strains: Take 100 μL of the spore solution prepared in Example 3, inoculate it into 1 mL of the artificial gastric juice obtained in step 1, vortex to mix thoroughly, incubate in a metal bath at 37° C., take samples every 1 h for live bacteria count, and calculate the survival rate of Bacillus Velezii HBXN2020 spores. The results are shown in Table 5.

[0098] Table 5 Survival of strains in artificial gastric juice at different time conditions

[0099]

[0100] As shown in Table 5, the survival rates of Bacillus Velez HBXN2020 spores in artificial gastric juice with pH 2.0 and incubated at 37°C for 1 h and 2 h were both 84%, and the survival rate of incubation for 3 h was 70%, indicating that the spores of Bacillus Velez HBXN2020 have good tolerance to artificial gastric juice.

[0101] Example 5 Bile salt tolerance test of Bacillus velez HBXN2020 strain

[0102] 1. Preparation of bile salts: Add porcine bile salts to normal saline to concentrations of 0.1wt.%, 0.2wt.%, 0.3wt.%, 0.4wt.%, and 0.5wt.%, respectively. Vortex mix thoroughly and filter with a 0.22μm filter for sterilization. The mass percentage of NaCl in the normal saline is 0.9%.

[0103] 2. Determination of bile salt tolerance of strains: Take the spores treated with artificial gastric juice in Example 4, wash them twice with sterile saline, resuspend them, and inoculate them into the above-mentioned bile salts respectively, with the inoculation amount being 1 / 10 of the volume of the bile salts, vortex mix thoroughly, incubate in a metal bath at 37°C for 120 min, take out and count the viable bacteria, and calculate the survival rate. The results are shown in Table 6, where the mass percentage of NaCl in the sterile saline is 0.9%.

[0104] Table 6 Survival of strains in bile salts at different time conditions

[0105]

[0106]

[0107] As shown in Table 6, the survival rates of the spores of Bacillus Velez HBXN2020 in bile salt concentrations of 0.1wt.%, 0.2wt.%, 0.3wt.%, 0.4wt.%, and 0.5wt.% after incubation in a water bath at 37°C for 120 min were 94%, 91%, 88%, 78%, and 68%, respectively, indicating that the spores of Bacillus Velez HBXN2020 have good tolerance to bile salts at concentrations below 0.3wt.%, and have moderate tolerance to bile salts at concentrations above 0.3wt.%.

[0108] Example 6 Artificial intestinal fluid tolerance test of Bacillus velez HBXN2020 strain

[0109] 1. Preparation of artificial intestinal fluid: Accurately weigh 6.8 g of KH2PO4 and dissolve it in 800 mL of distilled water, then add 10 g of trypsin, adjust the pH to 6.8 with NaOH, make up to 1000 mL, stir thoroughly until completely dissolved, and filter with a 0.22 μm filter for sterilization.

[0110] 2. Determination of tolerance of Bacillus Velez subtilis HBXN2020 to artificial intestinal fluid: Take the spores treated with artificial gastric fluid in Example 4, wash them twice with sterile physiological saline (same as Example 5), resuspend them, and inoculate them into the above-mentioned artificial intestinal fluid. The inoculation amount is 1 / 10 of the volume of the artificial intestinal fluid. Vortex mix thoroughly, incubate in a water bath at 37°C, take samples every 30 minutes for live bacteria count, and calculate the survival rate of Bacillus Velez subtilis HBXN2020 spores. The results are shown in Table 7.

[0111] Table 7 Survival of strains in artificial intestinal fluid at different time conditions

[0112]

[0113] As recorded in Table 7, the survival rates of Bacillus Velezii HBXN2020 spores incubated at 37°C in artificial intestinal fluid with a pH of 6.8 for 0.5 h, 1 h, 2 h, 4 h, and 6 h were 96.36%, 104.31%, 112.82%, 130.25%, and 131.09%, respectively, indicating that the spores of strain HBXN2020 can survive and proliferate in artificial intestinal fluid, indicating that the spores of strain HBXN2020 have good survival ability in artificial intestinal fluid.

[0114] Example 7 Qualitative test of cellulase production by Bacillus Velezii HBXN2020

[0115] 1. Preparation of culture medium: Add 1 wt.% sodium carboxymethyl cellulose to LB agar medium, sterilize with high pressure steam at 121°C for 20 min, cool to 55°C, and pour into a sterile culture dish until solidified.

[0116] 2. Activation of Bacillus Velezii HBXN2020 strain: streak the frozen Bacillus Velezii HBXN2020 strain on LB agar medium and culture it in a 37°C constant temperature incubator for 12 hours until a single colony appears.

[0117] 3. Qualitative determination of cellulase production by Bacillus Velezii HBXN2020 strain: Pick a single colony from the above LB agar medium with a sterile pipette tip and spot it on the LB agar medium containing sodium carboxymethyl cellulose. Repeat three times and culture in a 37°C constant temperature incubator for 24 hours. The plate cultured for 24 hours was stained with Congo red for 5 minutes, then washed twice with 2 mol / L NaCl. The results of cellulase production by Bacillus Velezii HBXN2020 strain are shown in Fig. 9 The cellulase produced by the HBXN2020 strain decomposes the carboxymethyl cellulose in the LB agar medium, causing Congo red to not stain. Therefore, a transparent circle appears around the HBXN2020 strain colony.

[0118] Example 8 Determination of antibiotic sensitivity of Bacillus Velezii HBXN2020 strain

[0119] 1. Activation and concentration adjustment of Bacillus Velezii HBXN2020 strain: streak the frozen Bacillus Velezii HBXN2020 strain on LB agar medium, culture it in a constant temperature incubator at 37℃ for 12h, pick a single colony and transfer it to LB liquid medium, culture it at 37℃180r / min overnight, then transfer the overnight cultured bacterial solution to fresh LB liquid medium, culture it at 37℃180r / min with shaking for 2-4h until it reaches the logarithmic growth phase, collect the bacteria by centrifugation, resuspend it in sterile PBS, and adjust the bacterial solution concentration to 10 using a McFarland turbidimeter. 5 CFU / mL.

[0120] 2. Determination of antibiotic sensitivity of Bacillus Velezii HBXN2020 strain: Use commercial drug sensitivity tablets to evaluate the sensitivity of the strain to minocycline, doxycycline, tetracycline, piperacillin, meropenem, gentamicin, chloramphenicol, sulfisoxazole, erythromycin, rifampicin and nitrofurantoin. Spread the HBXN2020 bacterial solution prepared above on LB agar medium and dry it. Stick different types of drug sensitivity tablets on the culture medium coated with HBXN2020 bacterial solution, invert it, and culture it at 37℃ for 12±2h.

[0121] 3. Criteria for determining antibiotic sensitivity of Bacillus Velezii HBXN2020 strain: Referring to the National Committee for Clinical Laboratory Standards (NCCLS) drug sensitivity test standard, the diameter of the inhibition zone above 20 mm is extremely sensitive, 15 mm to 20 mm is highly sensitive, 10 mm to 14 mm is moderately sensitive, and below 10 mm is lowly sensitive.

[0122] 4. The antibiotic sensitivity results of Bacillus Velezii HBXN2020 strain are shown in Table 8.

[0123] Table 8 Antibiotic sensitivity of strains

[0124]

[0125] Note: “+” means sensitive, “-” means insensitive

[0126] As shown in Table 8, the Bacillus Velezii HBXN2020 strain is not resistant to any antibiotics.

[0127] Example 9 Preparation method of Bacillus Velez HBXN2020 fermentation broth

[0128] 1. Preparation of seed solution: Pick a single colony of Bacillus Velezii HBXN2020 cultured in NA medium for 14 h, inoculate it into LB liquid medium, and culture it at 37°C and 180 r / min for 16 h to obtain the seed solution of Bacillus Velezii HBXN2020. The bacterial activity is 1×10 9 CFU / mL.

[0129] 2. Preparation of fermentation broth: Take the seed liquid of Bacillus Velezii HBXN2020 and transfer it to the modified Landy medium at an inoculation rate of 2% of the volume of the modified Landy medium. Cultivate at 28°C and 200 r / min for 48 h to obtain the fermentation broth with a bacterial activity of 2×10 10 CFU / mL.

[0130] Example 10 Bacillus Velezii HBXN2020 alleviates Salmonella-induced colitis in mice

[0131] 1. Experimental design: After the mice were fed with free drinking water and buffered food for 7 days, they were randomly divided into 3 groups (8 mice in each group): control group, PBS+ATCC14028 group and HBXN2020+ATCC14028 group. On the 1st to 7th day of the experiment, all mice in the HBXN2020+ATCC14028 group were orally administered 100 μL of Bacillus Velezii HBXN2020 bacterial solution every day. The bacterial solution was obtained from the fermentation liquid prepared in step 2 of Example 9. The bacterial activity of the bacterial solution was about 5×10 7 CFU / mL, the mice in the control group and PBS+ATCC14028 group were orally administered with 100 μL sterile PBS every day. On the 8th day of the experiment, the mice in the PBS+ATCC14028 group and the HBXN2020+ATCC14028 group were intragastrically administered with 100 μL of ATCC14028 bacterial solution, which was obtained by diluting the indicator bacteria Salmonella typhimurium ATCC14028 in step 2 of Example 1. The bacterial activity of the bacterial solution was about 5×10 7 CFU / mL, and mice in the control group were gavaged with 100 μL sterile PBS.

[0132] 2. Collection of fecal samples: After ATCC14028 infection, fecal samples of mice in the PBS+ATCC14028 group and HBXN2020+ATCC14028 group were collected every day, weighed, and resuspended in sterile PBS.

[0133] 3. Collection of intestinal tissue samples: On the 5th day of ATCC14028 infection, the mice were killed, and the ileum, cecum and colon tissues of the mice in the PBS+ATCC14028 group and HBXN2020+ATCC14028 group were collected.

[0134] 4. Determination of the number of Salmonella typhimurium in feces: The fecal samples of mice in the PBS+ATCC14028 group and the HBXN2020+ATCC14028 group were weighed and resuspended in sterile PBS. After homogenization with a 1 mm sterile steel ball, each mixture was diluted to 1×, 10×, 100×, 10 3 ×, 10 4 × and 10 5 ×. Then, 100 μL of each dilution was evenly spread on an LB agar plate containing 50 μg / mL kanamycin. The plate was aerobically cultured at 37°C for 12 h. The number of Salmonella typhimurium in the feces was counted and analyzed.

[0135] 5. Determination of the number of Salmonella typhimurium in the intestine of mice: The ileum, cecum, and colon of mice in the PBS+ATCC14028 group and the HBXN2020+ATCC14028 group were weighed and resuspended in sterile PBS. After homogenization with a 1 mm sterile steel ball, each mixture was diluted to 1×, 10×, 100×, and 10× in sequence. 3 ×, 10 4 × and 10 5 ×. Then, 100 μL of each dilution was evenly spread on LB agar plates containing 50 μg / mL kanamycin. Incubate aerobically at 37°C for 12 h. Count and analyze the number of Salmonella typhimurium in the ileum, cecum, and colon.

[0136] 6. Mouse body weight determination: During the experiment, the body weights of mice in the control group, PBS+ATCC14028 group, and HBXN2020+ATCC14028 group were monitored daily and recorded accurately.

[0137] The results of the number of Salmonella typhimurium in mouse feces are shown in Table 9.

[0138] Table 9 Number of Salmonella Typhimurium in mouse feces

[0139]

[0140] As shown in Table 9, after infection with ATCC14028 Typhimurium, the amount of ATCC14028 Typhimurium excreted in the feces of mice in the HBXN2020+ATCC14028 group was significantly lower than that in the PBS+ATCC14028 group. This indicates that Bacillus Velezii HBXN2020 can inhibit or kill ATCC14028 Typhimurium in the intestine, thereby effectively reducing the amount of ATCC14028 Typhimurium excreted in the feces of mice.

[0141] The results of the changes in mouse body weight are shown in Table 10.

[0142] Table 10 Effects of strain HBXN2020 on mouse body weight

[0143]

[0144] Note: Different shoulder marks in the same column indicate significant differences (P<0.05).

[0145] As shown in Table 10, after infection with Typhimurium ATCC14028, the weight loss rate of mice in the HBXN2020+ATCC14028 group was significantly slower than that of mice in the PBS+ATCC14028 group, indicating that Bacillus Velezii HBXN2020 can improve the disease resistance of mice and thus slow down the weight loss of mice.

[0146] The results of the number of Salmonella colonization in the mouse intestine are shown in Table 11.

[0147] Table 11 Number of Salmonella colonization in mouse ileum

[0148]

[0149] As shown in Table 11, compared with the PBS+ATCC14028 group, the number of Salmonella typhimurium in the ileum, cecum, and colon of mice in the HBXN2020+ATCC14028 group was significantly reduced, indicating that Bacillus Velezii HBXN2020 can maintain the balance of intestinal flora in mice, thereby reducing the colonization of ATCC14028 typhimurium in the intestines of mice.

[0150] In summary, the Bacillus Velez HBXN2020 of the present invention is isolated from the intestine of healthy piglets. The strain has a broad-spectrum antibacterial effect, can survive under high temperature and high pressure, strong acid and strong alkali, can produce cellulase, and has no antibiotics and drug resistance; the strain can also help maintain the balance of animal gastrointestinal flora, prevent animal diarrhea, and thus improve the disease resistance of animals. From the above, it can be seen that Bacillus Velez HBXN2020 is a pig-derived probiotic spore-forming bacteria with great application potential, which is of great significance to the development of animal health, animal husbandry and feed industry.

[0151] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacillus velezensis HBXN2020, characterized in that: The deposit number of the Bacillus Velez is CCTCC M 20221720.

2. The Bacillus Velezii according to claim 1, characterized in that The specific sequence of Bacillus Velez is shown in SEQ ID No.

4.

3. A microecological preparation or animal feed additive, characterized in that: The active ingredients of the microecological preparation or animal feed additive include the Bacillus Velez subtilis HBXN2020 described in claim 1, or the fermentation liquid of Bacillus Velez subtilis HBXN2020.

4. The microecological preparation or animal feed additive according to claim 3, characterized in that: The bacterial activity of Bacillus Velezii HBXN2020 in the microecological preparation or animal feed additive is 1×10 10 CFU / g~3×10 10 CFU / g.

5. A method for preparing a fermentation broth of Bacillus Velez HBXN2020, characterized in that: The following steps are involved: The Bacillus Velez subsp. HBXN2020 was inoculated into a modified Landy medium for fermentation to obtain a fermentation liquid of the Bacillus Velez subsp. HBXN2020.

6. Use of the Bacillus Velezii HBXN2020 described in claim 1 or 2 or the probiotic preparation or animal feed additive described in claim 3 or 4 in the preparation of a product for preventing Salmonella-induced colitis.

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