Bacillus licheniformis for degrading aflatoxin and application of bacillus licheniformis in fattening pig breeding

Abstract

The invention provides bacillus licheniformis capable of degrading aflatoxin and application of the bacillus licheniformis in fattening pig breeding. The preservation number of the bacillus licheniformis is CGMCC (China General Microbiological Culture Collection Center) NO.30148. The viable count of the screened bacillus licheniformis can still reach 108 CFU / mL after high-temperature water bath, the viable count in an acid-resistant detection test is 100%, the viable count is kept unchanged compared with the viable count before and after artificial gastric juice inoculation, and the viable count in cholate-resistant detection is 83.7%; the antibacterial activity on clostridium perfringens is the strongest, the antibacterial effect is compared by adding antibiotics and JR24B-1 groups, and it is found that the antibacterial effect is good when sampling is conducted for 24 h and is better than that of the antibiotics; the drug resistance to penicillin and lincomycin is realized; meanwhile, the screened bacillus licheniformis has the effects of strongly degrading aflatoxin and promoting growth of fattening pigs.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic screening, and specifically relates to a strain of Bacillus licheniformis capable of degrading aflatoxin and application thereof in fattening pig breeding. Background Art

[0002] Aflatoxins are toxic metabolites produced by the continuous metabolism of Aspergillus flavus during the planting, harvesting, storage and processing of major feed crops such as corn, wheat and miscellaneous meal. Studies have shown that the scope of mycotoxin contamination of feed or feed products is very wide. 70% of the feed in the world is contaminated with at least one mycotoxin, the most common of which is aflatoxin. Pigs are most sensitive to aflatoxins. It is reported that feeding a low concentration of aflatoxin of 140ppb for 12 consecutive weeks can cause liver damage and gastrointestinal bleeding in pigs weighing 18-64kg, and severe cases can die within a few days. Feeding weaned piglets with 280ppb of aflatoxin for 28 consecutive days will seriously affect the weight gain of pigs. There are endless reports of aflatoxin causing huge economic losses to farms. According to the new "Feed Hygiene Standard" GB13078-2017, the permissible value of aflatoxin in the piglet's adaptability range is less than 10ppb, but the 2022 aflatoxin detection report in feed raw materials and complete feed shows that the aflatoxin exceeding the standard rate is as high as more than 10%, with the highest value reaching 406.95ppb, far exceeding the prescribed standard. Therefore, it is urgent to find a solution to aflatoxin contamination for the actual production of farms.

[0003] The degradation methods of mycotoxins mainly include chemical method, physical method and biodegradation method. Chemical methods mainly include alkali treatment, oxidation treatment, chemical degradation, etc. These chemical methods have the disadvantages of unstable effect, large loss of nutrients, and difficulty in large-scale treatment. Physical methods include raw material dilution method, grinding method, density screening method and adsorption method. Among them, the more commonly used physical method in the market is the method of adding mycotoxin physical adsorbents. The disadvantage is that the adsorbent may only have a specific adsorption effect on some toxins, and has no adsorption for other toxins with larger molecular weight. In addition, the adsorbent will also adsorb some trace nutrients while adsorbing toxins, which may cause a lack of certain trace elements and vitamins in animals. In addition, this method is not thorough in detoxification, and animal growth and development are still greatly affected. In recent years, mycotoxin biodegradation has gradually attracted the attention of the animal husbandry industry as a safe, efficient and environmentally friendly mycotoxin detoxification method, which can well make up for the defects of physical and chemical methods in removing mycotoxins.

[0004] The mycotoxin biodegradation method has a clear detoxification mechanism, that is, the toxic groups of mycotoxins are destroyed with the help of functional enzymes produced by microorganisms, and converted into non-toxic metabolites. Compared with the physical adsorption method, the biodegradation method can completely remove the toxicity of mycotoxins, will not produce toxic side effects on animals, humans and the environment, and has stable effects and good repeatability. In addition, the amount of biodegradation agents added to the feed is small, and they have no adsorption effect on small molecular organic compounds such as vitamins and trace elements, and will not affect the nutritional balance of the feed. The advantages of biodegradation are detoxification, high efficiency, strong specificity, and the protein itself does not affect the palatability of the feed and can be recycled; the degradation products are non-toxic and harmless, and will not pollute the environment when excreted from the body; biodegradation technology, due to its specificity, high efficiency and safety, represents the current development direction of mycotoxin control and has broad application prospects.

[0005] As a type of feed probiotic, Bacillus licheniformis has strong stress resistance and can produce more functional enzymes and other metabolites. The CotA laccase produced during the metabolism can not only effectively oxidize aflatoxin B1, but also decompose it into non-toxic products aflatoxin Q1 and its diastereomers. epi Q1. Studies have found that the degradation rate of aflatoxin B1 by Bacillus licheniformis is as high as 94.7%, and it can make the mutagenic effect of aflatoxin B1 disappear. In summary, Bacillus licheniformis has great potential to be developed into a feed additive for removing aflatoxin B1 from feed raw materials, and the addition of Bacillus licheniformis to feed may be an ideal choice for detoxification of mycotoxins in animals and bioremediation of the intestines. However, when the existing Bacillus licheniformis with the ability to degrade aflatoxin is used as a pig feed additive, the strain will die due to high temperature during the preparation of feed, resulting in the loss of activity of the added strain. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Bacillus licheniformis capable of degrading aflatoxin and application thereof in fattening pig breeding.

[0007] The first object of the present invention is to provide a strain of Bacillus licheniformis JR24B-1, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 26, 2024, with a deposit number of CGMCC NO.30148 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] In another aspect, the present invention provides a use of the Bacillus licheniformis JR24B-1, which is the use in degrading aflatoxin or in preparing a product for degrading aflatoxin.

[0009] The present invention also provides another use of the screened Bacillus licheniformis JR24B-1 strain, which is use as a feed additive.

[0010] In another aspect, the present invention further provides a pig feed, wherein the fermented bacterial powder of the Bacillus licheniformis JR24B-1 strain is added to the pig feed.

[0011] Furthermore, the added amount of the fermentation bacteria powder is preferably 0.2%.

[0012] Furthermore, the fermented bacteria powder also contains glucose, corn starch and rice bran meal.

[0013] The viable bacterial count of the Bacillus licheniformis JR24B-1 strain screened by the present invention can still reach 10 after being exposed to high temperature water bath at 85°C and 90°C. 8 CFU / mL, the survival rate of live bacteria in the acid-resistant test was 100%, and the number of live bacteria before and after inoculation with artificial gastric juice remained unchanged, and the survival rate of live bacteria in the bile salt resistance test was 83.7%; it had the strongest antibacterial performance against Clostridium perfringens, and the antibacterial effect was compared by adding antibiotics and JR24B-1 into groups. It was found that the antibacterial effect was good when sampling at 24h, which was better than antibiotics; it was resistant to penicillin and lincomycin; at the same time, Bacillus licheniformis JR24B-1 has a strong effect of degrading aflatoxin and promoting the growth of fattening pigs. After adding 0.2% active bacterial powder of Bacillus licheniformis JR24B-1 to the complete feed of fattening pigs, the daily weight gain of fattening pigs increased by 24.52g / day, the feed ratio decreased by 0.03%, the feed conversion rate increased by 53.11 yuan / head, the proportion of 1-3 quality meat increased by 4.18%, and the comprehensive economic benefits increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Colony morphology of Bacillus licheniformis JR24B-1 on bacterial nutrient agar medium.

[0015] Figure 2 :Bacillus licheniformis JR24B-1 bacterial morphology under microscope.

[0016] Figure 3 :The results of drug resistance experiment of Bacillus licheniformis JR24B-1.

[0017] Figure 4 : The results of the antibacterial effect experiment of Bacillus licheniformis JR24B-1, the indicator bacteria are Clostridium perfringens (CP), Staphylococcus aureus (STA), Salmonella (SE), and Escherichia coli (EC). DETAILED DESCRIPTION

[0018] The invention provides a Bacillus licheniformis JR24B-1 strain, which can be used for degrading aflatoxin.

[0019] The fermentation method of the Bacillus licheniformis JR24B-1 strain of the present invention is as follows:

[0020] 1) The method for preparing the bacterial fermentation liquid is to streak the Bacillus licheniformis JR24B-1 strain on a bacterial nutrient agar plate and culture it at a constant temperature of 37° C. for 18-24 hours.

[0021] 2) Pick a single colony of Bacillus licheniformis JR24B-1 and activate it once on a bacterial nutrient agar plate. Pick a single colony of Bacillus licheniformis JR24B-1 after activation and place it in a 50ml centrifuge tube with 2ml LB nutrient broth, and culture it at 220r / 18-24h, 37℃ with shaking to obtain Bacillus licheniformis JR24B-1 seed liquid.

[0022] 3) Add 100 ml of LB nutrient broth into a conical flask, add 2 ml of the shaken Bacillus licheniformis JR24B-1 seed solution into the flask, mix well, and culture at 220 r / 18-24 h at 37° C. to obtain a bacterial fermentation broth.

[0023] 4) The nutrient agar medium is composed of 3 g beef extract, 10 g peptone and 5 g sodium chloride. The container is shaken until the solute is dissolved, heated, and 15-20 g agar is added. After the agar is melted, 5% sodium hydroxide solution or 5% dilute hydrochloric acid is added to adjust the pH to 7.2-7.4 and the volume is made up to 1000 mL with distilled water. After high-pressure sterilization at 121°C for 30 min, 5-10 ml is dispensed into a culture dish and used after solidification.

[0024] 5) The LB medium is composed of 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl. The container is shaken until the solute is dissolved, 5% sodium hydroxide solution or 5% dilute hydrochloric acid is added, the pH is adjusted to 7.0, and the volume is made up to 1000 mL with distilled water. After steam sterilization at 121°C under high pressure for 21 min, it is ready for use.

[0025] Another aspect of the present invention provides a probiotic preparation, which is prepared using the above-mentioned active bacterial powder of Bacillus licheniformis JR24B-1 as a raw material. The specific preparation steps are as follows:

[0026] 1) The fermentation liquid was examined under a microscope for bacterial morphology, centrifuged at 10,000 r / 10 min, and the supernatant of the fermentation liquid was discarded.

[0027] 2) Add 3 ml of sterile water to the precipitated bacteria after centrifugation and mix them evenly, weigh 10 g of glucose, 30 g of corn starch, and 10 g of rice bran meal, add 3 ml of the precipitated product, knead and mix them evenly, and then place them in a 37°C oven to dry for 2 days. Use a grinder to grind to obtain active bacterial powder of Bacillus licheniformis JR24B-1.

[0028] The active bacterial powder prepared by the Bacillus licheniformis JR24B-1 provided by the present invention has strong stress resistance: good tolerance to bile salts and gastric acid; antibacterial property: it has inhibitory effects on Escherichia coli, Salmonella, Staphylococcus aureus and Clostridium perfringens; it is safe: it is resistant to tetracycline, penicillin, azithromycin and lincomycin. It has a probiotic effect: adding 0.2% to the complete pig feed can improve the production performance and economic benefits of fattening pigs.

[0029] The detection method of aflatoxin is as follows: The ELISA kit detection of aflatoxin (Shanghai Youlong Biotechnology Co., Ltd.) is as follows:

[0030] 1. Sample preparation

[0031] 1) Before use, take the kit out of the refrigerator and return it to room temperature for 10 minutes.

[0032] 2) The enzyme marker provided in this kit is a concentrated solution. Please dilute it with enzyme diluent before use. Dissolve 1 bottle of enzyme marker with 1.5 ml enzyme diluent to prepare a working solution sufficient for 24 wells.

[0033] 3) The concentrated washing solution provided in this kit can be used after being diluted 20 times with distilled water. For example: 2 ml concentrated washing solution + 38 ml distilled water is enough for 24 wells.

[0034] 2. Determination procedure

[0035] 1) Insert a sufficient number of ELISA plate microwells into the reaction plate holder according to the number of samples to be tested. Mark the blank control well, standard well, and sample well. Then add about 250 μL of washing solution to each well, leave it for 1 minute, pour out the liquid, and pat the ELISA plate dry on newspaper or wool paper.

[0036] 2) Add 50 μL of sample diluent to the blank control well, add 50 μL of aflatoxin B1 standard (0 ppb, 0.1 ppb, 0.25 ppb, 0.5 ppb, 1 ppb, 2 ppb) to each standard well, and add 50 μL of sample solution to the sample well.

[0037] 3) Add 50 μL of enzyme diluent to the blank control well, and add 50 μL of diluted enzyme marker solution to the other wells. Gently shake to mix, and then incubate at 37°C for 30 minutes.

[0038] 4) After the incubation, shake off the liquid in the microwells, then add about 250 μL of diluted washing solution to each well, let it sit for 1 minute, pour out the liquid, and repeat the washing 3 times. After pouring out the liquid, pat the ELISA plate dry on newspaper or wool paper.

[0039] 5) Add 50 μL of color developer and 50 μL of reaction substrate to each well, shake gently to mix, and incubate in a constant temperature incubator at 37°C for 15 min.

[0040] 6) After the color reaction is completed, add 50 μL of stop solution to each well. Measure at 450 nm using an ELISA reader. This must be completed within 10 minutes after adding the stop solution.

[0041] The present invention is described in detail below in conjunction with embodiments and drawings.

[0042] Example 1: Screening of strains that degrade aflatoxin

[0043] The screening and detection method of the strain provided by the present invention is as follows:

[0044] 1) Screening, separation and purification of bacterial strains

[0045] Aflatoxin selective medium: KH2PO4 0.25g, coumarin 5g, CaCl 201g, (NH4)2NO3401g, FeSO4 1g, MgSO4 0.25g, shake the container until the solute is dissolved, heat, and add 15-20g agar. After the agar is melted, add 5% sodium hydroxide solution or 5% dilute hydrochloric acid, adjust the pH to 7.2-7.4, make up to 1000mL with distilled water, and sterilize at 121℃ under high pressure for 30min.

[0046] Take 0.01 g of moldy feed sample collected from the farm and dissolve it in 1 ml of distilled water, vortex and oscillate to mix well. Take 100 μL of bacterial solution and dissolve it in 900 μL of distilled water to make the initial suspension. This dilution gradient is recorded as 10 -1 Then dilute the initial suspension 10 times with distilled water, and so on, until it is diluted to 10 -10 Gradient: For each gradient, 200 μL of the diluted bacterial suspension was evenly spread on the primary screening culture medium using a spreading rod and inverted culture at 37°C for 18-24 hours.

[0047] The colony morphology was observed and recorded, and 9 single colonies with good growth and different morphologies were picked. They were streaked multiple times on aflatoxin selective culture medium plates to separate and purify the single colonies, which were named strains JR24B-1 to 11 respectively.

[0048] 2) Determination of aflatoxin degradation rate of strains

[0049] Take moldy feed samples from the farm, crush and mix them, and mix them with normal feed to adjust the aflatoxin concentration to 10ug / mg, activate JR24B-1~11 strains, streak bacterial nutrient agar plates, and culture at 37℃ for 18-24h. Pick a single colony of strain JR24B-1~11 strains and streak three zones for activation once.

[0050] Pick out one single colony of each of the activated Bacillus licheniformis JR24B-1 to 11, place them in a 50 ml centrifuge tube with 2 ml LB nutrient broth, and culture at 220 r / 37°C for 18-24 hours.

[0051] Add 100 ml of LB liquid culture medium into a conical flask, add 2 ml of the shaken JR24B-1-11 bacterial solution into it and mix well, and culture at a constant temperature of 220r / 37℃ for 18-24h to obtain the bacterial fermentation liquid.

[0052] Centrifuge the fermentation broth of strains JR24B-1 to 11, discard the supernatant, take the live cells and adjust their concentration to 10 with sterile distilled water. 12 cfu / mL.

[0053] Take 50g of moldy feed sample with aflatoxin concentration of 10μg / mg, mix 5ml 10 12 The JR24B-1~11 bacterial solution with a concentration of cfu / mL was evenly sprayed on the moldy feed sample, mixed evenly, placed in a 37°C constant temperature incubator, and cultured for 72 hours, and the aflatoxin degradation rate of the JR24B-1~11 strains was determined. The results showed that the aflatoxin concentration of the JR24B-1 strain was less than 1ppb, that is, the aflatoxin degradation rate of the JR24B-1 strain was 100%. The degradation rate was calculated as (amount of mycotoxin added-amount of mycotoxin residue) / (amount of mycotoxin added-residual amount of the blank control group)×100%. Therefore, the JR24B-1 strain was selected as the research object and used for further research on the biodegradation of aflatoxin.

[0054] Table 1: Aflatoxin degradation rate of strains JR24B-1 to 11

[0055]

[0056] 3) Screening of strains with high temperature resistance

[0057] The above-mentioned bacteria with the function of degrading aflatoxin were cultured in LB liquid for 3-7 days, and the spore formation rate was examined under a microscope. When the bacteria produced more than 50% spores, the vegetative bodies were killed in a water bath at 80°C for 10 minutes, and the remaining spores were used to evaluate the heat resistance of the spores of the bacteria. The temperature condition was 85°C for 15 minutes. The plates were coated before and after heat treatment, and the survival rate was calculated to directly screen strains with high temperature resistance that could match those produced by feed factories. The results showed that the number of live bacteria of the JR24B-1 strain did not change after being heated at 85°C for 15 minutes, and its live bacteria count still reached 98.69%. It can withstand the influence of high temperatures on it.

[0058] Table 2: Spore rate of strains JR24B-1~11 after heat treatment for 15 minutes

[0059]

[0060] Example 2: Physicochemical properties and taxonomic identification of strain JR24B-1

[0061] 1. Morphological identification

[0062] The single colony status of the strains in the logarithmic growth phase and with stable colony size was described, mainly including the size, color, transparency, surface status and edge status of the colony. The obtained single colony size was 8-10mm, light yellow or white, round, opaque, with smooth colony edges and moist surfaces.

[0063] 2. 16S rDNA homology sequence analysis

[0064] The JR24B-1 strain was streaked and cultured, and a single colony was picked. The bacteria were used as templates for PCR, and the bacterial 16SrDNA was sequenced and compared in the NCBI database to obtain relevant data. The strain species was obtained by comparison with the database, and the strain category was determined to be Bacillus licheniformis, and was named Bacillus licheniformis JR24B-1; it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on March 26, 2024, with a deposit number of CGMCCNO.30148, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. .

[0065] Example 3: Detection of stress resistance of Bacillus licheniformis JR24B-1

[0066] 1. Heat resistance test

[0067] LB liquid culture, 3-7 days, microscopic examination of spore formation rate, after the strain produces more than 50% spores, 80℃ water bath for 10 minutes to kill the vegetative body, the remaining spores to evaluate the heat resistance of the spores of the strain, the temperature conditions are 85℃ for 2 minutes, 95℃ for 2 minutes, close to the granulation temperature, the plate is coated before and after heat treatment, and the survival rate is calculated. The results show that after the JR24B-1 strain is treated at 85℃ for 2 minutes, the number of viable bacteria can be maintained at 10 8 The survival rate of CFU / ml can reach 100%; after 2 minutes of treatment at 95℃, the number of viable bacteria can still be maintained at 10 8 CFU / ml, the survival rate is over 100%. After the JR24B-1 strain seed solution was treated in 85℃ and 95℃ water bath for 2min, the number of viable bacteria can still be cultured to 10 8 CFU / ml, it can be seen that the spores formed by Bacillus licheniformis JR24B-1 strain are tolerant to high temperature environment.

[0068] 2. Acid resistance test

[0069] LB liquid culture, microscopic examination of spore formation rate, after the strain produces more than 50% spores, 80℃ water bath for 10min to kill the vegetative body, the remaining spores are used to evaluate the acid resistance of the spores of the strain. Artificial gastric acid is sterile distilled water with a pH of 3.2. 1g of pepsin is added to every 100mL to prepare artificial gastric juice. 9mL of artificial gastric juice is added to 1mL of sample and heated at 40℃ for 2h. The plate is coated before and after treatment to calculate the survival rate.

[0070] The results showed that after 10 minutes of water bath at 80℃, the number of live bacteria remained at 4.3×108CFU / ml. After inoculation with artificial gastric juice, the number of live bacteria was 4.3×108CFU / ml, which was the same as before inoculation, and the survival rate was 100%. This shows that the strain has a strong tolerance to acid. The results suggest that the JR24B-1 strain can tolerate the effects of gastric acid.

[0071] 3. Bile salt tolerance test

[0072] LB liquid culture, microscopic examination of spore formation rate, after the strain produces more than 50% spores, 80℃ water bath for 10 minutes to kill the vegetative body, the remaining spores are used to evaluate the bile salt tolerance of the strain spores. Artificial intestinal fluid is 100mL of distilled water with 0.68g of potassium dihydrogen phosphate added, adjusted to pH 6.8, and then 0.30g of pig bile salt is added, and sterilized at 121℃ for 15min. 9mL of artificial intestinal fluid is added with 1mL of sample, and heated at 40℃ for 2h. The survival rate is calculated by coating the plate before and after treatment.

[0073] The results showed that after 10 min in 80℃ water bath, the number of viable bacteria was 4.9×10 8 CFU / ml. When bile salts were added, the number of viable bacteria was 4.1×10 8The CFU / ml showed a downward trend, and the survival rate was 83.7%, indicating that the strain of Bacillus licheniformis JR24B-1 had strong resistance to high temperature and bile salt.

[0074] Example 4: Safety testing of Bacillus licheniformis JR24B-1

[0075] 1. Antibiotic sensitivity testing

[0076] The JR24B-1 Bacillus isolate was subjected to a sensitivity test for 10 drugs according to the KB drug sensitivity paper method, and the results were judged in accordance with the 2019 edition of the standard recommended by the Clinical Laboratory Standards Institute (CLSI) of the United States.

[0077] 1) JR24B-1 resuscitation: JR24B-1 was inoculated on a bacterial nutrient plate (NA), incubated at 37°C for 8-16 hours, and then removed.

[0078] 2) JR24B-1 activation: Place the JR24B-1 strain in 3 ml LB nutrient broth and culture at 180r / 37°C for 4-8 hours before taking it out.

[0079] 3) Coating and patching: Pipette 200 μL of bacterial solution and apply it to the LB plate. After coating, directly stick the drug-sensitive paper to the surface of the plate culture medium. Use tweezers to gently press the drug-sensitive paper several times to make it stick closely to the culture medium. Place the plate in a 37°C incubator overnight, and measure the diameter of the inhibition zone (mm) with a vernier caliper the next day.

[0080] 4) Record the results: From Table 2, we can see that JR24B-1 is resistant to penicillin and lincomycin, and is sensitive to vancomycin, streptomycin, florfenicol, norfloxacin, cephalexin, azithromycin, and trimethoprim / sulfamethoxazole.

[0081] Table 3: JR24B-1 Bacillus susceptibility test results

[0082]

[0083] Note: S: sensitive; I: intermediate; R: resistant

[0084] Example 5: Detection of probiotic properties of Bacillus licheniformis JR24B-1

[0085] 1. Antibacterial ability test

[0086] The filter paper method was used to evaluate the inhibitory ability of JR24B-1 against Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens. The steps are as follows:

[0087] 1) Activation of pathogens: Four pathogens (Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens) were streaked in three zones on nutrient agar (NA) medium, incubated at 37°C, and taken out after 18-24 hours.

[0088] 2) Recovery: Inoculate JR24B-1 into NA medium, incubate at 37°C, and remove after 18-24 hours.

[0089] 3) Enrichment: Pick 4 single colonies of pathogenic bacteria respectively, place them in (5 ml, 10 ml, 20 ml) LB nutrient broth, and culture them in a shaking incubator (200 rpm) at 37°C for 4 hours before taking them out.

[0090] 4) Centrifugation: Centrifuge JR24B-1 (10000r / 10min), aspirate the supernatant (this is the bacterial solution), and resuspend the precipitated bacteria in 1ml LB liquid culture medium and mix well.

[0091] 5) Plate coating and patching: Take 200 μL of pathogenic bacteria solution and spread it on nutrient agar (NA) medium. Use sterile tweezers to pick up blank drug-sensitive paper: dip them in pure water (label 0), bacteria (label 1), and bacterial solution (label 2), respectively, and stick them evenly on the same NA plate. Invert and culture at 37℃ for 24 hours.

[0092] 6) Observation results: The results of the antibacterial test are shown in Table 3 and Figure 4 :JR24B-1 Bacillus licheniformis has significant antibacterial effect on Escherichia coli, Salmonella and Clostridium perfringens, and the bacterial solution has antibacterial effect on Salmonella and Clostridium perfringens. The diameter of the inhibition zone of JR24B-1 bacteria on Escherichia coli and Clostridium perfringens reaches the low sensitivity range, and the antibacterial performance on Clostridium perfringens is the strongest, followed by Escherichia coli and Salmonella / Staphylococcus aureus; it has a strong antagonistic effect on Gram-positive bacteria (Clostridium perfringens).

[0093] Table 4: JR24B-1 antibacterial test results

[0094]

[0095] Example 6: Comparison of antibacterial properties of Bacillus licheniformis JR24B-1

[0096] 1. JR24B-1 activation: three-zone line, 37°C, 18-24h.

[0097] 2. After 18-24 hours, take a single colony of JR24B-1 and 4 pathogens (Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens) and place it in a 50mL centrifuge tube filled with 10mL LB. After culturing at 37℃, 200r / min for 4h, 8h, and 16h, take out 2mL of the strain for detection.

[0098] 3. Test group: Take 40μL of pathogenic bacteria liquid (Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens) and add it to 1mL JR24B-1 bacterial liquid (4 groups of pathogenic bacteria liquid sampled at 4h, 8h, and 16h respectively), and add LB liquid culture medium to make the total volume 4mL (add 2.96mL LB). 1) Detection of bacterial strains (bacterial concentration): Take 100μL of bacterial liquid respectively and dissolve it in 900μL of distilled water to make the initial suspension. The dilution gradient is recorded as 10 1 Then dilute the initial suspension 10 times with distilled water, and so on, until it is diluted to 10 8 Gradient, 10 6 , 10 7 , 10 8 Gradient pipette 200 μL of diluted bacterial suspension was evenly spread on NA medium with glass beads, and cultured at 37°C for 18-24 hours before counting the number of bacteria (Table 4). 7 The bacterial solution was reserved for the next experiment (4 groups of 16h JR24B-1+8h pathogens were selected).

[0099] Table 5: JR24B-1 test group bacterial strain quantity test results

[0100]

[0101] 4. Antibiotic control group: Pipette 40 μL of pathogenic bacteria solution (Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens) and 40 μL of ampicillin (method of use: 25 mL LB + 50 μL ampicillin), and add LB liquid culture medium to make the total volume 4 mL (add 3.92 mL LB).

[0102] 5. Set up a normal control group without drug addition: pathogenic bacteria Escherichia coli / Salmonella / Staphylococcus aureus / Clostridium perfringens 40μL + 3.96mL LB.

[0103] 6. Detect the bacterial strain (bacterial concentration) of the antibiotic and no-drug control groups: Take 100 μL of bacterial solution and dissolve it in 900 μL of distilled water to make an initial suspension. The dilution gradient is 10 1 Then dilute the initial suspension 10 times with distilled water, and so on, until it is diluted to 10 8 Gradient, 10 5～8 Gradient pipette 200 μL of the diluted bacterial suspension and evenly spread it on the NA medium with glass beads. Incubate the culture medium at 37°C for 18-24 hours and then count the number of bacteria (Table 5). Select the bacterial suspension with a concentration of 107 for the next experiment.

[0104] Table 6: JR24B-1 control group bacterial strain test results

[0105]

[0106] The pathogen + JR24B-1 (4 strains), pathogen + AMP (4 strains), pathogen + LB (4 strains) were selected to reach a bacterial concentration of 10 7 The experiment was conducted on the two groups, all of which were cultured at 37°C and 180r / min. Samples were taken at 0, 4, 8, 12, 16, 20, 24 and 28h, respectively. The samples were diluted and the plate count was performed. The number of viable bacteria per milliliter of bacterial solution (CFU·mL) was determined by the plate colony count method according to GB 4789.3-2010 "National Food Safety Standard Food Microbiology Examination Coliform Group Count" -1 ).

[0107] The inhibition rate was calculated based on the live bacteria concentration in each group: Inhibition rate = (number of bacteria in the control group - number of bacteria in the test group) / number of bacteria in the control group × 100% (Table 6).

[0108] Table 7: Antibacterial rate test results of JR24B-1 test group and antibiotic control group

[0109]

[0110]

[0111] With the extension of culture time, the antibacterial rate of JR24B-1 added to the pathogen group was the best at 24h and 28h; after ampicillin was added to the pathogen, the antibacterial effect was the best at 20h and 24h; the sampling time with higher overall antibacterial rate was 24h and 28h.

[0112] The comparison of the antibacterial effects of JR24B-1 and ampicillin found that after adding Staphylococcus aureus, the antibacterial effect of JR24B-1 was higher than that of ampicillin only at 4h and 28h; after adding Salmonella, the antibacterial effect of the ampicillin group showed a lower antibacterial rate than that of the JR24B-1 group only at 28h. This shows that JR24B-1 has a certain inhibitory effect on these four pathogens, and the best inhibition rate is at 24h.

[0113] Example 7: Effect of active bacterial powder of Bacillus licheniformis JR24B-1 on production performance and economic benefits of fattening pigs

[0114] 1. Purpose of the test

[0115] To explore the effects of long-term addition of active bacterial powder of Bacillus licheniformis JR24B-1 on the production performance, health and comprehensive economic benefits of fattening pigs.

[0116] 2. Experimental breeding methods

[0117] The project intends to select 350 fattening pigs weighing 55±2kg at 85 days of age for the experiment. The site scale is 2 fattening farms with a capacity of 2,000 pigs. The animal feeding experiment is expected to last for 75 days.

[0118] 3. Experimental Grouping

[0119] During the experiment: The experiment ended when the large group feeding program of the pig farm was consumed. The weight of each pen of the control group and the experimental group was weighed and recorded. The experiment used a corn-soybean meal-based diet, and the two groups were fed and managed in the same way, with free drinking water and food.

[0120] The experiment is divided into 2 groups:

[0121] 1) Control group 1: Normally fed with corn and soybean meal based diet

[0122] 2) Experimental Group 2: The pigs were fed a normal corn-soybean meal-based diet, and 0.2% active bacterial powder of Bacillus licheniformis JR24B-1 was added daily.

[0123] 4. Production performance measurement indicators

[0124] 1) Average daily weight gain: The weight gain of each group is calculated based on the end time of the feeding number and the time of slaughter.

[0125] 2) Average feed intake: The feed consumption of each group of pigs is calculated based on the end time of the feeding number and the time of slaughter.

[0126] 3) Average mortality rate: The mortality rate of each group during the trial period

[0127] 4) Meat cost per kilogram: After the experiment, the meat cost per kilogram of each group of pigs was calculated.

[0128] 5) Drug costs: Statistical drug costs of each group during the trial

[0129] 5. Analysis and discussion of test results

[0130] 1) Analysis of 0.2% Bacillus licheniformis JR24B-1 active bacterial powder on the production performance and health of fattening pigs Table 8: Statistical table of the effect of 0.2% Bacillus licheniformis JR24B-1 active bacterial powder on the growth performance and health of fattening pigs

[0131]

[0132]

[0133] The test results show that compared with the control group, after adding 0.2% Bacillus licheniformis JR24B-1 active bacterial powder, the average daily weight gain of fattening pigs increased by 24.52g / day (increased by 3.49%); the feed-to-meat ratio decreased by 0.08 (decreased by 0.03%); in terms of improving meat quality: compared with the control group, after adding 0.2% Bacillus licheniformis JR24B-1 active bacterial powder, the proportion of 1-3 quality meat of fattening pigs increased by 4.18%. The results of this test are in line with the experimental expectations. After adding 0.2% Bacillus licheniformis JR24B-1 active bacterial powder, the daily weight gain of fattening pigs increased, the feed ratio decreased, the feed reward increased, and the comprehensive economic benefits increased.

[0134] 2) Analysis of the economic benefits of 0.2% Bacillus licheniformis JR24B-1 active bacterial powder on fattening pigs

[0135] Table 9: Analysis of the effect of 0.2% Bacillus licheniformis JR24B-1 active bacterial powder on the economic benefits of fattening pigs

[0136]

[0137]

[0138] According to the current selling price of 115-125kg standard pigs (September 2024: 17.5 yuan / kg), during the 177-day trial period, the average benefit per fattening pig fed with 0.2% Bacillus licheniformis JR24B-1 increased to 920.67 yuan / head; the feed of the 0.2% Bacillus licheniformis experimental group was 70 yuan / ton higher than that of the control group. Compared with the control group, the average economic benefit per fattening pig increased by 53.11 yuan / head after using 0.2% active Bacillus licheniformis powder, that is, the economic benefit of fattening pigs weighing 55±2kg increased by 6.12% after feeding 0.2% active Bacillus licheniformis powder. In terms of improving pork quality and enhancing the economic benefits of pork, the proportion of grade 1-3 meat of fattening pigs fed with 0.2% active Bacillus licheniformis powder was significantly increased compared with the control group, an increase of 4.34%. The ideal expectation is to increase the bargaining price by 0.3 yuan / catties (the increase in the proportion of grade 1-3 can increase the bargaining price by 1-1.6 yuan / jin). According to this calculation, the average economic benefit of fattening pigs increased by 36.47 yuan / head after using 0.2% active Bacillus licheniformis powder, that is, after fattening pigs weighing 55±2kg were fed 0.2% active Bacillus licheniformis powder, the comprehensive economic benefit of fattening pigs increased by 10.31%.

Claims

1. A strain of Bacillus licheniformis, characterized in that: The deposit number of the Bacillus licheniformis is CGMCCNO.30148.

2. Use of the Bacillus licheniformis according to claim 1 in degrading aflatoxin.

3. Use of the Bacillus licheniformis according to claim 1 in the preparation of a product for degrading aflatoxin.

4. Use of the Bacillus licheniformis according to claim 1 as a feed additive.

5. A pig feed, characterized in that: The pig feed is added with the fermented bacterial powder of Bacillus licheniformis according to claim 1.

6. The pig feed according to claim 5, characterized in that The added amount of fermentation bacteria powder in the pig feed is 0.2%.

7. The pig feed according to claim 5 or 6, characterized in that: The fermented bacteria powder also contains glucose, corn starch and rice bran meal.

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