Bacillus amyloliquefaciens and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses bacillus amyloliquefaciens and application thereof. The bacillus amyloliquefaciens K1 has been preserved in the Guangdong Microbial Culture Collection Center on August 5, 2024, and the preservation number of the bacillus amyloliquefaciens K1 is GDMCC (China General Microbiological Culture Collection Center) NO: 64954, and the bacillus amyloliquefaciens K1 has been preserved in the Guangdong Microbial Culture Collection Center on August 5, 2024; fermentation supernate of the strain contains antibacterial substances, has a good effect of inhibiting clostridium perfringens and can be used for preparing products for inhibiting clostridium perfringens; besides, the generated surfactin can inhibit the replication of TGEV and PEDV viruses, so that the surfactin can be used for preparing medicines for treating or preventing porcine epidemic diarrhea and porcine infectious gastroenteritis, or other surfactin production products, and can effectively solve the problems in the current products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a Bacillus amyloliquefaciens strain and application thereof. Background Art

[0002] Diarrhea is one of the important causes of piglet mortality, causing huge economic losses to the world's pig industry. In recent years, Clostridium perfringens (C. perfringens) has been considered to be the main pathogen causing diarrhea in piglets, and has become a major problem hindering the healthy development of the pig industry. Porcine epidemic diarrhea (PED) and porcine transmissible gastroenteritis (TGE) are gastrointestinal infectious diseases caused by porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV), respectively. Both viruses can infect small intestinal epithelial cells, leading to cell death and shedding, thereby causing vomiting and diarrhea in piglets. Therefore, a probiotic whose metabolites can produce antibacterial and antiviral substances has very important application value.

[0003] Surfactin is widely used in animal husbandry and aquaculture, agricultural production, food, medicine, petroleum, etc. due to its good emulsification and antibacterial properties. Its emulsification performance is 10 times stronger than that of lysophospholipids. Natural biological emulsifiers can improve the digestion and utilization rate of fat in the diet by livestock and poultry. Surfactin can significantly reduce the infection ability of porcine transmissible gastroenteritis virus (TGEV) and prevent the symptoms of porcine epidemic diarrhea virus (PEDV). It has the potential to be used as an antiviral vaccine; Surfactin can also regulate animal intestinal flora and promote animal growth.

[0004] However, there is currently a lack of drugs and feed additives that can effectively utilize surfactant to prevent and treat porcine transmissible gastroenteritis and porcine epidemic diarrhea. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a strain of Bacillus amyloliquefaciens. The fermentation supernatant of the strain contains antibacterial substances, and the strain has a good effect of inhibiting Clostridium perfringens, and can be used to prepare products that inhibit Clostridium perfringens; in addition, since the produced surfactin can inhibit the replication of TGEV and PEDV viruses, it can be used to prepare drugs for treating or preventing porcine epidemic diarrhea and porcine transmissible gastroenteritis, or used in other production of surfactin products.

[0006] In order to achieve the above invention objectives, the present invention is implemented by the following technical solutions:

[0007] One of the purposes of the invention of this application is to protect a strain of Bacillus amyloliquefaciens K1, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 5, 2024, with the collection number GDMCC NO: 64954; the address of the depository unit is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] Furthermore, the 16S rDNA sequence of the Bacillus amyloliquefaciens K1 strain described above is shown in SEQ ID NO.1.

[0009] Furthermore, the primers used to amplify the 16S rDNA of Bacillus amyloliquefaciens K1 include 27F and 1492R, the nucleotide sequence of 27F is shown in SEQ ID NO.2, and the nucleotide sequence of 1492R is shown in SEQ ID NO.3.

[0010] The second invention objective of the present invention is to protect the use of the above-mentioned Bacillus amyloliquefaciens K1 in the preparation of products for inhibiting Clostridium perfringens, such as inhibitors, etc. Preferably, the production of surfactin by Bacillus amyloliquefaciens K1 reaches 0.138 g / L.

[0011] The third invention objective of the present invention is to protect the use of the above-mentioned Bacillus amyloliquefaciens K1 in the preparation of drugs for treating or preventing porcine epidemic diarrhea and porcine transmissible gastroenteritis.

[0012] Preferably, the drug for treating or preventing porcine epidemic diarrhea and porcine transmissible gastroenteritis comprises Bacillus amyloliquefaciens K1 and pharmaceutically acceptable additives.

[0013] Furthermore, the pharmaceutically acceptable additives include physiological saline, glucose solution, interleukin-12, and cationic liposomes.

[0014] The fourth invention objective of the present invention is to protect the use of the above-mentioned Bacillus amyloliquefaciens K1 in the production of surfactin products.

[0015] Furthermore, the products include emulsifiers, stomach drinks, lozenges, health products, feed and milk powder.

[0016] Furthermore, the emulsifier also includes additives, which include any one or more mixtures of lecithin, monoglyceride and sucrose ester.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Bacillus amyloliquefaciens grows fast, has a high spore conversion rate, large yield and is easy to preserve during industrial production, and has good application value.

[0019] (ii) The fermentation supernatant of the strain contains antibacterial substances. The strain has a good effect of inhibiting Clostridium perfringens and can be used to prepare products that inhibit Clostridium perfringens.

[0020] (iii) Since the produced surfactant can inhibit the replication of TGEV and PEDV viruses, it can be used to prepare drugs for treating or preventing porcine epidemic diarrhea and porcine infectious gastroenteritis.

[0021] (3) Bacillus amyloliquefaciens can also be used in other surfactant products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison chart of the antibacterial effects of the initially screened strains in the embodiments of the present invention;

[0023] Figure 2 The colony morphology and bacterial morphology of strain K1 in the embodiment of the present invention are shown in FIG.

[0024] Figure 3 This is a phylogenetic analysis diagram of strain K1 in the embodiment of the present invention;

[0025] Figure 4 This is a standard curve diagram of the surfactin standard product in the embodiment of the present invention;

[0026] Figure 5 The amount of Surfactin produced by strain K1 at different culture times in the present invention (A is 22h, B is 24h, C is 30h, and D is 48h fermentation broth sample);

[0027] Figure 6 This is the emulsification effect of K1 bacterial powder in the embodiment of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with embodiments, which are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in the field without creative work are all within the scope of protection of the present invention.

[0029] In the present application, % not specifically indicated indicates the mass percentage, i.e. wt%; reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0030] The experimental materials involved in the following examples are as follows:

[0031] RCM plate: peptone 10g, beef powder 10g, yeast powder 3g, soluble starch 1g, sodium chloride 5g, sodium acetate 5g, glucose 5g, L-cysteine ​​hydrochloride 0.5g, agar 15g, ddH2O to 1L, adjust pH to 6.8±0.1, autoclave at 121℃ for 15min to prepare RCM plate.

[0032] RCM liquid culture medium: peptone 10g, beef powder 10g, yeast powder 3g, soluble starch 1g, sodium chloride 5g, sodium acetate 5g, glucose 5g, L-cysteine ​​hydrochloride 0.5g, make up to 1L with ddH2O, adjust the pH to 6.8±0.1, autoclave at 121℃ for 15min to prepare RCM liquid culture medium.

[0033] Preparation of anaerobic PBS: weigh 0.27g of potassium dihydrogen phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride, and 0.2g of potassium chloride, dissolve in 1L of distilled water, heat to boil, cool to room temperature, add 0.55g of cysteine ​​hydrochloride, stir to dissolve, adjust the pH to 6.5, install a quantitative dispenser and pass N2, heat to boiling, keep at a slight boiling state for 30min, cool and package into 500mL / bottle, sterilize at 121℃ high temperature and wet heat for 30min, store in a cool and dry place for later use.

[0034] Spore-forming medium: peptone 10 g, yeast powder 10 g, ammonium sulfate 0.6 g, magnesium sulfate (heptahydrate) 0.12 g, calcium chloride (dihydrate) 0.08 g, potassium carbonate 3.48 g, potassium dihydrogen phosphate 2.6 g, make up to 1 L with ddH2O, adjust the pH to 7.9±0.1, autoclave at 121°C for 15 min to prepare the spore-forming medium.

[0035] Embodiment 1:

[0036] Screening and Preservation of Inhibitory Bacteria against Clostridium perfringens

[0037] The dilution coating and plate streak method was used to screen the intestinal feces samples discharged by a healthy chicken in Chengdu, Sichuan, China. The bacteria obtained from the initial screening were subjected to antibacterial tests, and a strain that could inhibit the growth of Clostridium perfringens was selected.

[0038] Activation of indicator bacteria: After Clostridium perfringens was activated by anaerobic culture at 37°C, the OD600nm value was measured using a UV-visible spectrophotometer. The culture was stopped when the OD value was around 0.50.

[0039] Activation of the initial screening strains: After multiple separation and purification of the strains obtained from the intestinal feces of healthy chickens, the strains were cultured in a shake flask at 37°C overnight, and the supernatant was taken as the test solution after centrifugation at 10,000 r / min for 5 minutes.

[0040] Oxford cup method to detect antibacterial effect: add 1mL indicator bacteria to 100mL sterilized nutrient agar, mix well to make bacterial nutrient agar. Place 9 Oxford cups in a sterilized culture dish and pour 15mL of bacterial nutrient agar. After the culture medium solidifies, use tweezers to take out the Oxford cups, mark them, and add 100μL of sample solution to each well (0.85% phosphoric acid is used for the control group). After the sample is added, let it stand for 1 hour and then culture it in a 37℃ incubator. If an antibacterial circle appears, it indicates an antibacterial effect, otherwise there is no antibacterial effect.

[0041] The results are as follows Figure 1 As shown in Table 1:

[0042] Table 1 Diameters of inhibition zones of various strains screened against Clostridium perfringens

[0043]

[0044] according to Figure 1 As shown in the results in Table 1, among the six bacterial strains, strain K1 had the best in vitro antibacterial effect against Clostridium perfringens, with an inhibition zone diameter of 22.09 mm.

[0045] Molecular identification of Clostridium perfringens inhibitory bacteria

[0046] The primers for amplifying the 16S rDNA sequence are 27F and 1492R, the nucleotide sequence of 27F is shown in SEQ ID NO.2, and the nucleotide sequence of 1492R is shown in SEQ ID NO.3; wherein SEQ ID NO.2 is: 5'-AGTTTGATCMTGGCTCAG-3';

[0047] SEQ ID NO.3 is: 5'-GGTTACCTTGTTACGACTT-3'.

[0048] Wuhan Jinkairui Bioengineering Co., Ltd. was commissioned to sequence and its 16S rDNA sequence was determined to be as shown in SEQ ID NO.1:

[0049]

[0050] The sequencing results were compared with the sequence information published on the NCBI website by Blast, and the phylogenetic tree was constructed using the neighbor-joining method using MEGA7.0 software. The results are shown in the figure. Figure 2 and Figure 3 shown.

[0051] according to Figure 2 The results showed that strain K1 was a Gram-positive bacterium with a rod-shaped morphology.

[0052] according to Figure 3 The results showed that strain K1 was identified as Bacillus amyloliquefaciens by 16S rDNA molecular identification and phylogenetic analysis.

[0053] Deposit of the strain: The bacterium was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 5, 2024, with the deposit number GDMCC NO: 64954; the address of the deposit unit is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0054] Embodiment 2:

[0055] Based on Example 1, the characteristics of Bacillus amyloliquefaciens K1 were studied:

[0056] A 3L Fermentation Test of Bacillus amyloliquefaciens K1

[0057] The preserved Bacillus amyloliquefaciens K1 strain was activated, and then the activated Bacillus amyloliquefaciens strain was used for a small test in a 3L fermenter, using LB medium (tryptone 10g / L, yeast extract, 5g / L sodium chloride, NaCl 10g / L), a culture temperature of 37°C, and a culture time of 7-48h. Table 2 shows the fermentation parameters of Bacillus amyloliquefaciens K1 strain in a 3L fermenter:

[0058] Table 2 Fermentation conditions of 3L fermentation system

[0059]

[0060]

[0061] The OD of the fermentation broth of Bacillus amyloliquefaciens K1 cultured at different times was measured. 600nm The results are shown in Table 3. After 48 hours of fermentation, the OD value of the fermentation liquid of Bacillus amyloliquefaciens K1 strain was 16.96.

[0062] Table 3 OD of fermentation broth of Bacillus amyloliquefaciens K1 strain at different culture times 600nm value

[0063] Cultivation time 7h 22h 24h 30h 48h <![CDATA[OD 600nm ]]> 1.565 16.6 16.8 17.36 16.96

[0064] Embodiment 3:

[0065] Based on Example 2, the surfactin content in the fermentation broth of Bacillus amyloliquefaciens K1 strain was tested at different culture times.

[0066] Preparation of standard solution: Accurately weigh 1.8 mg of surfactin standard and dissolve it in 1 mL of methanol to prepare a standard stock solution (1800 mg / L). According to experimental needs, dilute the surfactin standard into 5 different concentrations of standard solutions: 100 mg / L, 80 mg / L, 40 mg / L, 20 mg / L, and 10 mg / L.

[0067] Chromatographic conditions: chromatographic column: COSMOSIL 5C18-AR-Ⅱ (4.6×250 mm, Nacalai Tesque, Japan); injection volume: 20 μL; column temperature: 35°C; detection wavelength: 210 nm; flow rate: 0.84 mL / min; solvent: A: acetonitrile (containing 0.1% TFA) and B: water (containing 0.1% TFA).

[0068] Treatment of the fermentation broth sample of Bacillus amyloliquefaciens K1 to be tested: take 50mL of the fermentation broth of Bacillus amyloliquefaciens K1 strain, centrifuge at 10000r / min for 10min, remove the bacteria, and take the supernatant; adjust the pH value of the supernatant to 2, and let it stand for 12h; centrifuge at 10000r / min for 5min, and retain the precipitate; repeatedly extract the precipitate with 100mL of methanol, and adjust the pH to 7; centrifuge at 10000r / min for 5min and retain the supernatant; take 2mL of the sample, vacuum concentrate and dry, then dissolve it with 1mL of methanol, and perform HPLC content detection.

[0069] The constructed standard curve is shown in Figure 4 As shown in Figure 2, the surfactin content at different times was Figure 5 The results are shown in Table 4.

[0070] Table 4 Surfactin production of strain K1 at different culture times

[0071] Cultivation time 22h 24h 30h 48h Surfactin content (g / L) 0.128 0.072 0.094 0.138

[0072] According to Table 4 and Figure 5 The results show that the production of surfactin in the fermentation broth of strain K1 was 0.138 g / L after 48 hours of fermentation. Surfactin with a concentration higher than 0.015 g / L can almost completely inhibit the replication of TGEV and PEDV viruses. Surfactin can exert a strong antiviral effect in the concentration range of 0.015 g / L to 0.02 g / L without causing damage to cells. Therefore, K1 fermentation broth can effectively prevent porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus.

[0073] Example 4: Emulsification of bacterial powder

[0074] Bacillus amyloliquefaciens K1 was cultured in a 500 L fermenter (culture conditions were the same as in the previous example). After 48 h, a fermentation broth of Bacillus amyloliquefaciens K1 was obtained, which was then spray-dried to obtain a viable cell count of 1 × 10 14 CFU / g of bacterial powder. The obtained bacterial powder was subjected to an emulsification experiment. 100g water + 150g oil + 3.5g Bacillus amyloliquefaciens K1 bacterial powder was homogenized with an emulsifier for 5 minutes and then left to stand for 30 minutes. No stratification occurred. Uniform oil droplets were observed under a 45x microscope, proving that the emulsification effect was good (the results are shown in the figure). Figure 6 shown).

[0075] The above basic examples of the present invention and their further selected examples can be freely combined to form multiple embodiments, all of which are embodiments that can be adopted and claimed by the present invention. In the scheme of the present invention, each selected example can be arbitrarily combined with any other basic example and selected example. Those skilled in the art will know that there are many combinations.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens K1 was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 5, 2024, with the deposit number GDMCC NO:64954.

2. The Bacillus amyloliquefaciens K1 according to claim 1, characterized in that The 16S rDNA sequence of the strain is shown as SEQ ID NO.

1.

3. Use of Bacillus amyloliquefaciens K1 according to claim 1 or 2 in the preparation of a product for inhibiting Clostridium perfringens.

4. Use of Bacillus amyloliquefaciens K1 as claimed in claim 1 or 2 in the preparation of a medicament for treating or preventing porcine epidemic diarrhea and porcine transmissible gastroenteritis.

5. The use according to claim 4, characterized in that: The drug for treating or preventing porcine epidemic diarrhea and porcine transmissible gastroenteritis comprises Bacillus amyloliquefaciens K1 as claimed in claim 1 or 2 and pharmaceutically acceptable additives.

6. The use according to claim 5, characterized in that: The pharmaceutically acceptable additives include physiological saline, glucose solution, interleukin-12, and cationic liposomes.

7. Use of Bacillus amyloliquefaciens K1 as claimed in claim 1 or 2 in the production of surfactin products.

8. The use according to claim 7, characterized in that: The products include emulsifiers, stomach drinks, lozenges, health products, feed and milk powder.

9. The use according to claim 8, characterized in that The emulsifier also includes additives.

10. The use according to claim 9, characterized in that The additive comprises a mixture of any one or more of lecithin, monoglyceride and sucrose ester.

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