Feed containing bacillus paralicheniformis carrying quorum sensing related genes and preparation method thereof

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a feed containing bacillus paralicheniformis carrying quorum sensing related genes and a preparation method of the feed. The bacillus parlicheniformis is bacillus parlicheniformis Shannan.BP-803, and the bacillus parlicheniformis is bacillus parlicheniformis Shannan.BP-803. The invention also discloses a preparation method of the bacillus parlicheniformis. The viable count of the bacillus paralicheniformis contained in each kg of the feed is 2.1 * 10 < 9 >-7.3 * 10 < 9 > CFU. The bacillus paralicheniformis Shann.BP-803 strain used in the feed disclosed by the invention has the capability of efficiently inhibiting pathogenic bacteria and pathogenic fungi and the capability of degrading mycotoxin, so that after the feed containing the bacillus paralicheniformis Shann.BP-803 strain is eaten, the diarrhea rate of animals is reduced, and the feeding effect is better.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a feed containing Bacillus paralicheniformis and a preparation method thereof. Background Art

[0002] In the process of livestock and poultry breeding, livestock and poultry feed is easily infected by various pathogenic bacteria and pathogenic fungi. Among them, eating livestock and poultry feed infected with pathogenic bacteria can easily cause livestock and poultry to have symptoms such as coughing, tracheitis and bronchitis, pulmonary edema, bleeding, dyspnea, suffocation, etc.; eating livestock and poultry feed infected with pathogenic fungi can easily cause livestock and poultry to have symptoms such as decreased feed intake, growth stagnation, and reduced immunity.

[0003] The Chinese patent application with publication number CN117778266A discloses a strain of Bacillus paralicheniformis LN33 and its application. Specifically, it discloses that Bacillus paralicheniformis LN33 is used as a fermentation agent in feed to inhibit the growth of pathogenic bacteria Escherichia coli, Salmonella and Staphylococcus aureus in feed. However, the Bacillus paralicheniformis LN33 used in the patent has a weak inhibitory ability against Escherichia coli, Salmonella and Staphylococcus aureus. At the same time, the patent does not disclose the inhibitory effect of feed containing Bacillus paralicheniformis LN33 on pathogenic fungi and the effect of degrading fungal toxins. Summary of the invention

[0004] The problem existing in the prior art is that there is a lack of feeds that have the function of inhibiting pathogenic bacteria, inhibiting pathogenic fungi, and degrading fungal toxins.

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a feed containing Bacillus paralicheniformis and a preparation method thereof.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a feed containing Bacillus paralicheniformis carrying a quorum sensing-related gene, wherein the Bacillus paralicheniformis is Bacillus paralicheniformis Shannan.BP-803.

[0008] Preferably, the number of viable bacteria of Bacillus paralicheniformis contained in each kg of the feed is 2.1×10 9 -7.3×10 9 CFU.

[0009] Preferably, the quorum sensing-related gene contains the gene sequence shown in SEQ ID NO:4 and / or SEQ ID NO:5.

[0010] Preferably, the amino acid sequence encoded by the gene sequence shown in SEQ ID NO:4 is the amino acid sequence shown in SEQ ID NO:6.

[0011] Preferably, the amino acid sequence encoded by the gene sequence shown in SEQ ID NO:5 is the amino acid sequence shown in SEQ ID NO:7.

[0012] Preferably, the Bacillus paralicheniformis has the functions of forming biofilm, and / or producing extracellular protease, and / or inhibiting pathogenic bacteria, and / or inhibiting the performance of pathogenic fungi, and / or degrading toxins produced by pathogenic fungi.

[0013] Preferably, the pathogenic bacteria include one or more of Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis and Aeromonas hydrophila; more preferably, the pathogenic bacteria include one or more of Clostridium perfringens, Streptococcus suis and Aeromonas hydrophila.

[0014] Preferably, the pathogenic fungi include one or more of Aspergillus flavus, Fusarium graminearum, Fusarium trilineatum, Fusarium verticillium and Penicillium verrucosum; more preferably, the pathogenic fungi include one or more of Fusarium trilineatum, Fusarium verticillium and Penicillium verrucosum.

[0015] Preferably, the toxin comprises: one or more of AFB1 toxin, ZEA toxin, DON toxin, T-2 toxin, OTA toxin and FB1 toxin.

[0016] Preferably, the enzymatic activity of the extracellular protease of the Bacillus paralicheniformis is 10-30U, wherein U is one enzymatic activity unit of the extracellular protease, wherein, when 1 mL of the enzyme solution of the Bacillus paralicheniformis is hydrolyzed at 40°C, the amount of casein produced by 1 pg of tyrosine per minute is 1 enzymatic activity unit U of the extracellular protease.

[0017] In a second aspect, the present invention provides a method for preparing the feed containing Bacillus paralicheniformis carrying quorum sensing related genes, comprising the following steps: mixing the bacterial suspension of Bacillus paralicheniformis with a basic feed to obtain the feed containing Bacillus paralicheniformis carrying quorum sensing related genes.

[0018] Preferably, the preparation method comprises the following steps:

[0019] (1) expanding the culture of the Bacillus paralicheniformis;

[0020] (2) adding the expanded cultured Bacillus paralicheniformis to a liquid culture medium, preferably, culturing at 35-42° C. to obtain a bacterial suspension of Bacillus paralicheniformis;

[0021] (3) Mixing the bacterial suspension of Bacillus paralicheniformis with a basic feed to obtain a feed containing Bacillus paralicheniformis.

[0022] In a third aspect, the present invention provides a feed containing Bacillus paralicheniformis, which is prepared by the preparation method.

[0023] Preferably, the feed containing Bacillus paralicheniformis is pig feed and / or chicken feed.

[0024] Beneficial effects of the present invention:

[0025] (1) The Bacillus paralicheniformis Shannan.BP-803 strain used in the feed of the present invention has the ability to effectively inhibit pathogenic bacteria and pathogenic fungi and degrade mycotoxins, so that after eating the feed containing the Bacillus paralicheniformis Shannan.BP-803 strain, the diarrhea rate of animals is reduced, and the feeding effect is better.

[0026] (2) The whole genome sequencing of the Bacillus paralicheniformis Shannan.BP-803 used in the feed of the present invention revealed that it contains two unique genes related to quorum sensing. This characteristic enables Bacillus paralicheniformis Shannan.BP-803 to have strong biofilm formation and extracellular protease synthesis capabilities, thereby giving it better antibacterial function. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a line graph showing the change of toxin degradation rate of Bacillus paralicheniformis Shannan.BP-803 at 0h, 2h, 4h, 8h, 12h, 1d, 3d, 7d and 14d;

[0028] Figure 2 Shown is a line graph showing changes in toxin degradation rate by Bacillus paralicheniformis CICC 10102 at 0h, 2h, 4h, 8h, 12h, 1d, 3d, 7d and 14d.

[0029] Strain deposit information

[0030] The Bacillus paralicheniformis Shannan.BP-803 (Bacillus paralicheniformis Shannan.BP-803) provided by the present invention was deposited in the China Center for Type Culture Collection on April 3, 2024, with a deposit number of CCTCCNO: M 2024624, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. DETAILED DESCRIPTION

[0031] In view of the lack of efficient antibacterial solutions for pathogenic bacteria and fungi in animal feed in the prior art, and the need to degrade or detoxify fungal toxins in animal feed, the present invention provides a feed containing Bacillus paralicheniformis and a preparation method.

[0032] The inventors have screened out a new strain of Bacillus paralicheniformis through intensive research, which can effectively inhibit pathogenic bacteria and fungi in animal feed, and can degrade or detoxify fungal toxins in feed. The Bacillus paralicheniformis is Bacillus paralicheniformis Shannan.BP-803.

[0033] The inventors then used the strain to make animal feed, which was able to inhibit pathogenic bacteria and reduce the diarrhea rate of animals in the growth effect experiment of piglets and broilers. Specifically, the diarrhea rate of piglets was reduced from 18% to 4%, and the diarrhea rate of broilers was reduced from 8% to 1%, which improved the feeding effect and improved the economic benefits of breeding.

[0034] The inventors also found through sequencing studies that the whole genome of Bacillus paralicheniformis Shannan.BP-803 used in the present invention contains two unique genes related to quorum sensing compared with 20 strains of Bacillus paralicheniformis (Control_20) whose complete genomes have been published in the NCBI database. This characteristic enables Bacillus paralicheniformis Shannan.BP-803 to have stronger biofilm formation and extracellular protease synthesis capabilities, thereby giving it better antibacterial function.

[0035] The inventors also found that the Bacillus paralicheniformis Shannan.BP-803 used in the present invention has a high ability to inhibit pathogenic bacteria, and specifically has a good inhibitory effect on pathogenic bacteria including Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, and Aeromonas hydrophila. Among them, it has the best inhibitory effect on Escherichia coli, which is the most harmful to animal husbandry. The diameter of its transparent circle is 24.24 mm, which is 3 times higher than that of Bacillus paralicheniformis CICC 10102.

[0036] The paralicheniformis Shannan.BP-803 used in the present invention has high efficiency in inhibiting pathogenic fungi and detoxifying ability, and has good inhibitory effect on pathogenic fungi including Aspergillus flavus, Fusarium graminearum, Fusarium trilineatum, Fusarium verticillioides, and Penicillium verrucosus, among which the inhibitory effect on Aspergillus flavus, which is the most common in animal husbandry, is the best, with an inhibition rate of 96.2%, which is 3.19 times higher than that of Bacillus paralicheniformis CICC 10102. In addition, Bacillus paralicheniformis Shannan.BP-803 also has a high efficiency in detoxifying the fungal toxin AFB1 produced by Aspergillus flavus, with a detoxification rate of 71%, which is 3.6 times higher than that of Bacillus paralicheniformis CICC 10102.

[0037] Glossary

[0038] Quorum sensing is a form of intercellular communication that regulates a wide range of activities in microorganisms, including the formation of biofilms and the synthesis of extracellular proteases. The formation of biofilms can effectively control the structural stability of probiotics, improve the ability of bacteria to resist external environmental stress, and promote the colonization and longer residence of beneficial bacteria in the intestinal mucosa, strengthen the exchange of nutrients between the microbiome and the host, thereby preventing the colonization of pathogens and enhancing the host's defense capabilities. Extracellular proteases can be synthesized to inhibit the formation of biofilms outside the body of pathogens and destroy the cell wall structure, thus playing an important role in antibacterial effects. Extracellular proteases mainly include: alkaline proteases, neutral proteases, metalloproteases, serine proteases, and cell wall-bound proteases.

[0039] AFB1 toxin: also known as aflatoxin B1 (AflatoxinB1), is a secondary metabolite produced by toxin-producing strains such as Aspergillus flavus and Aspergillus parasiticus.

[0040] ZEA toxin: also known as Zearalenone, is a fungal toxin produced by specific molds, especially secondary metabolites produced by molds such as Fusarium graminearum and Gibberella zeae.

[0041] DON toxin: also known as Deoxynivalenol, is a trichothecenes compound produced by specific fungi, mainly from Fusarium graminearum, Fusarium graminearum, etc.

[0042] T-2 toxin: It belongs to the trichothecenes family of compounds. It is mainly produced by a variety of fungi, especially specific strains of Fusarium such as Fusarium trilineatum.

[0043] OTA toxin: also known as ochratoxin A (Ochratoxin A), is a fungal toxin mainly produced by Penicillium verrucosum and other fungi.

[0044] FB1 toxin: also known as fumonisin B1, is a mycotoxin produced by certain fungi, including Fusarium verticillium.

[0045] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0046] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercially available products. The information sources of the experimental materials and experimental instruments used in the present invention are shown in Table 1:

[0047] Table 1 Reagent / Instrument Information

[0048]

[0049]

[0050]

[0051] The preparation methods of the reagents or culture media used in the embodiments are as follows:

[0052] 10% methanol solution: add 100 ml of methanol to 900 ml of water and mix thoroughly.

[0053] 33% glacial acetic acid solution: Take 33mL of glacial acetic acid and add 67mL of water and mix thoroughly.

[0054] PDA solid culture medium: Put 200 g of peeled potatoes into 1 L of boiling water and boil for 20 min. Then filter the filtrate with gauze to collect the filtrate. Then mix the filtrate, 20 g of glucose, 15 g of agar and distilled water and make up to 1 L with distilled water. Sterilize at 115°C for 30 min.

[0055] Example 1 Isolation, purification and identification of strains

[0056] Screening and identification of strains

[0057] (1) Strain enrichment

[0058] Take 25 g of the digestive tract contents of Tibetan pigs from herdsmen's homes in Yadui Township, Naidong District, Shannan City, Tibet, and mix them with 225 mL of sterile saline to obtain a uniform sample solution.

[0059] (2) Cultivation of single colonies

[0060] The sample solution was diluted in a gradient manner, and 10 -3 , 10 -4 , 10 -5 , 10 -6The dilution of the concentration was spread on LB medium plates for culture. After culturing at 37°C for 48 hours, single colonies were picked according to the different bacterial morphologies.

[0061] (3) Strain screening and isolation

[0062] The single colony picked in step (2) was purified and separated for three generations. When the colony morphology was consistent, the strain was cultured in LB liquid culture medium, mixed with 50% glycerol in equal proportions, and stored at -80°C.

[0063] Morphological features: Figure 1 As shown, the strain isolated in step (3) grows in LB agar medium in the form of light yellow colonies with smooth surfaces, protruding bacterial bodies, and sometimes transparent colonies formed around the colonies;

[0064] Gram staining preliminary identification: Gram staining was performed on the strain isolated in step (3). The identification results showed that the bacteria were stained purple and were Gram-positive rods. The morphological characteristics of the strain under microscopic examination were as follows: Figure 2 shown.

[0065] (4) Identification of strains

[0066] High-fidelity enzymes were used to perform 16S rDNA sequencing and bacterial species identification on the strains to be identified. The primer information and PCR amplification reaction system and conditions used are shown in Tables 2 and 3.

[0067] Table 2 Primer information

[0068]

[0069] Table 3 PCR amplification reaction system and conditions

[0070]

[0071] After the PCR product was tested qualified, it was sent to a sequencing company for sequencing, and the 16srRNA sequence was obtained as shown in SEQ ID NO: 3.

[0072]

[0073]

[0074] The sequencing results were compared with the NCBI GenBank database for homology analysis, and the strain was identified as Bacillus paralicheniformis, named Bacillus paralicheniformis Shannan.BP-803, which was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO:M 2024624.

[0075] Example 2 Whole genome sequencing analysis of Bacillus paralicheniformis Shannan.BP-803

[0076] Bacillus paralicheniformis Shannan.BP-803 was entrusted to Shenzhen BGI Co., Ltd. for whole genome sequencing and comparative genomics analysis. The whole genome of Bacillus paralicheniformis Shannan.BP-803 was compared with 20 strains of Bacillus paralicheniformis (Control_20) whose complete genomes were published in the NCBI database. The results showed that the strain contained two genes related to quorum sensing, and the gene IDs were: 80LB-3GL000179 and 80LB-3GL002199. The gene sequence of 80LB-3GL000179 is shown in SEQ ID NO:4, and the gene sequence of 80LB-3GL002199 is shown in SEQ ID NO:5.

[0077]

[0078]

[0079] The gene IDs of the unique genes related to quorum sensing are 80LB-3GL000179 and 80LB-3GL002199, and their ID numbers in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database are K06354 and K07800, respectively. These two unique genes related to quorum sensing enable Bacillus paralicheniformis Shannan.BP-803 to have higher biofilm formation ability and extracellular protease synthesis ability, improve the antibacterial performance of the strain, and enable it to better exert its probiotic properties.

[0080] The amino acid sequence encoded by the 80LB-3GL000179 gene is shown in SEQ ID NO:6, and the amino acid sequence encoded by the 80LB-3GL002199 gene is shown in SEQ ID NO:7.

[0081]

[0082]

[0083] Example 3 Determination of biofilm forming ability of Bacillus paralicheniformis Shannan.BP-803

[0084] The strains preserved in the glycerol tube were activated. The first activation was inoculated into 10 mL of LB liquid medium at a 2% inoculation amount and cultured at 37°C for 24 hours. The second activation was inoculated into 10 mL of LB liquid medium at a 2% inoculation amount and cultured at 37°C for 12 hours. Then, the activated Bacillus paralicheniformis Shannan.BP-803 and Bacillus paralicheniformis CICC 10102 were inoculated into 96-well plates containing 200uL of LB liquid medium and cultured in a 37°C incubator. The culture solution was then aspirated, and 200μL of sterile PBS buffer (1x) was added to each well to wash the plate wells 3 times, and then 100μL of 10% methanol solution was added to the wells for fixation for 15min, and then the methanol was aspirated and dried naturally. Add 100 μL of 1% crystal violet solution to each well, stain at room temperature for 15 minutes, aspirate the crystal violet solution, and wash with sterile distilled water three times until colorless. Invert the culture plate on filter paper, absorb the surface moisture, and then place it in a 37°C oven to dry the moisture. After the plate is completely dry, add 100 μL of 33% glacial acetic acid solution to each well, place it in a 37°C incubator for 30 minutes, and measure the absorbance value (i.e. OD) at 580 nm using LB liquid culture medium as a control. 580 ), and the results are shown in Table 3.

[0085] Table 2 Results of determination of biofilm forming ability of strains

[0086] Bacillus paralicheniformis Shannan.BP-803 Bacillus paralicheniformis CICC10102 <![CDATA[OD 580nm ]]> 1.2305±0.156 0.4542±0.174

[0087] As shown in Table 2, the results showed that the biofilm-forming ability of strain Shannan.BP-803 was 1.2305±0.156, which was much higher than that of Bacillus paralicheniformis CICC 10102, indicating that Bacillus paralicheniformis Shannan.BP-803 had a strong biofilm-forming ability.

[0088] Example 4 Determination of the enzyme activity of extracellular protease produced by Bacillus paralicheniformis Shannan.BP-803

[0089] (1) The strain preserved in the glycerol tube was activated. The first activation was inoculated into LB liquid culture medium at an inoculum size of 2% and cultured at 37° C. for 24 h. The second activation was inoculated into LB liquid culture medium at an inoculum size of 2% and cultured at 37° C. for 12 h. Then, the activated Bacillus paralicheniformis Shannan.BP-803 and Bacillus paralicheniformis CICC 10102 were inoculated into 200 mL of LB liquid culture medium at an inoculum size of 2% by volume, respectively. The culture solution was placed in a constant temperature shaking incubator at 37° C. and 160 rpm for 7 days to obtain a bacterial solution of Bacillus paralicheniformis Shannan.BP-803 (OD 600nm =2.5), and Bacillus paralicheniformis CICC 10102 culture (OD600nm =2.5), then a portion of the bacterial solution was taken out and centrifuged at 9000 rpm and 4°C for 15 min, and the supernatant, i.e., the enzyme solution, was retained to obtain the enzyme solution of Bacillus paralicheniformis Shannan.BP-803 and the enzyme solution of Bacillus paralicheniformis CICC 10102, respectively.

[0090] (2) Determine the enzymatic activity of the extracellular protease produced by Bacillus paralicheniformis Shannan.BP-803 and Bacillus paralicheniformis CICC 10102. The specific determination method is shown in steps (2.1) and (2.2):

[0091] (2.1) Preparation of solution:

[0092] L-tyrosine standard solution with a concentration of 1 mg / mL: weigh 0.1000 g of L-tyrosine that has been dried to constant weight at 105°C, accurate to 0.0002 g, dissolve it in 1 mol / L HCl 60 mL, and make up to 100 mL;

[0093] 100 μg / mL L-tyrosine standard solution: Pipette 10.00 mL of the above standard solution and dilute to 100 mL with 0.1 mol / L HCl;

[0094] Solution for using Folin: Mix Folin phenol reagent and water in a ratio of 1:2, shake well, and store away from light.

[0095] Phosphate buffer (pH = 7.5): Dissolve 6.02 g Na2HPO4·12H2O and 0.5 g NaH2PO4·2H2O in water and make up to 1000 mL.

[0096] 10.0g / L casein solution: Accurately weigh (1.000±0.001)g of casein, add 2-3 drops of 0.5mol / L sodium hydroxide solution to wet it, then add 80mL of phosphate buffer, heat in a boiling water bath for 30min, and continue stirring until the casein is completely dissolved. After cooling to room temperature, check the pH value and adjust it to 6-8, and finally dilute to 100mL with phosphate buffer. The solution is stored at 4℃ and is valid for 3 days.

[0097] Trichloroacetic acid (TCA) solution with a concentration of 0.4 mol / L: weigh 6.5365 g of trichloroacetic acid solid and add 100 mL of water to dissolve it.

[0098] (2.2) Draw the standard curve:

[0099] Add 0mL, 1mL, 2mL, 3mL, 4mL or 5mL of 100μg / mL L-tyrosine standard solution to 6 test tubes respectively, make up the volume to 10mL with distilled water, and name the 6 test tubes corresponding to the volume of L-tyrosine standard solution added, and name them as Tube 0, Tube 1, Tube 2, Tube 3, Tube 4 and Tube 5 in sequence, add 1mL of 1mg / mL L-tyrosine standard solution to 6 test tubes respectively, then add 5mL of 0.4mol / L Na2CO3 solution to 6 test tubes respectively, and add 1mL of Folin-phenol reagent to 6 test tubes respectively, shake to mix, place in a 40±0.2℃ water bath and heat for 20min, and then use a spectrophotometer to measure the absorbance at 680nm, i.e. OD680nm.

[0100] Tube 0 was used as blank control, and the OD values ​​of tubes 1-5 were 680nm Value minus OD of 0 680nm Value is Net OD 680nm The net absorbance value is used as the ordinate and the tyrosine concentration is used as the abscissa to draw a standard curve. The regression equation is obtained using the standard curve. According to the regression equation, the absorption constant K, i.e. the amount of L-tyrosine (μg) when the absorbance is 1, is calculated. If the K value is within the range of 95 to 100, it means that the curve is qualified, otherwise the test needs to be repeated.

[0101] (2.3) Determination of the enzymatic activity of extracellular protease produced by Bacillus paralicheniformis Shannan.BP-803:

[0102] Preheat the enzyme solution of Bacillus paralicheniformis Shannan.BP-803 and the casein solution in a 40℃ water bath for 5 minutes. Then mix 1mL of the enzyme solution of Bacillus paralicheniformis Shannan.BP-803 and 1mL of 10g / L casein solution, and then heat in a water bath at 40℃ for 10 minutes for enzymatic reaction. Immediately after 10 minutes, add 3mL of 0.4mol / L trichloroacetic acid solution. Let stand for 10 minutes, and then centrifuge at 10000r / min for 15 minutes. Pipette 1mL of supernatant into a new test tube, and add 5mL of 0.4mol / L sodium carbonate solution and 1mL of Folin solution. Mix quickly and color in a 40℃ water bath for 20 minutes. Then use a spectrophotometer to measure the absorbance at 680nm, i.e. OD 680nm Then, the enzymatic activity of the extracellular protease produced by Bacillus paralicheniformis Shannan.BP-803 was calculated according to the following extracellular protease activity calculation formula.

[0103] The method for determining the enzymatic activity of the extracellular protease produced by the control group is different from the method for determining the enzymatic activity of the extracellular protease produced by Bacillus paralicheniformis Shannan.BP-803 in that 1 mL of the enzyme solution of Bacillus paralicheniformis Shannan.BP-803 and 3 mL of a 0.4 mol / L trichloroacetic acid solution are mixed, and then heated in a water bath at 40°C for 10 minutes. Immediately after 10 minutes, 1 mL of a 10 g / L casein solution is added.

[0104] (2.4) The method for determining the enzymatic activity of the extracellular protease produced by Bacillus paralicheniformis CICC 10102 is the same as step (2.3).

[0105] The calculation formula of extracellular protease activity is: X = 5 × A × K × n / 10

[0106] Where: X represents the extracellular protease activity (U); A represents the net OD 680nm value; K represents the absorption constant; n represents the dilution multiple of the enzyme solution; 10 represents the reaction time of the enzymatic reaction, which is 10 min; 5 represents the amount of 5 mL of the reaction solution, wherein the reaction solution is the total volume of: 1 mL of the enzyme solution of Bacillus paralicheniformis Shannan.BP-803, 1 mL of the casein solution with a concentration of 10 g / L and 3 mL of the trichloroacetic acid solution with a concentration of 0.4 mol / L.

[0107] One unit of extracellular protease activity is defined as: 1 mL of enzyme solution at 40°C, the amount of casein hydrolyzed per minute to produce 1 pg of tyrosine is 1 unit of extracellular protease activity U.

[0108] Table 3 Determination results of the enzyme activity of extracellular proteases produced by strains

[0109]

[0110]

[0111] As shown in Table 3, the results showed that the enzymatic activity of the extracellular protease of Bacillus paralicheniformis Shannan.BP-803 was 12.3U after 7 days of culture, which was higher than that of Bacillus paralicheniformis CICC 10102, indicating that the enzymatic activity of the extracellular protease produced by Bacillus paralicheniformis Shannan.BP-803 was higher.

[0112] Example 5 Determination of the ability of Bacillus paralicheniformis Shannan.BP-803 to inhibit pathogenic bacteria

[0113] (1) Preparation of bacterial liquid of animal pathogen indicator bacteria: Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, and Aeromonas hydrophila after activation were inoculated at an inoculum size of 2% in 10 mL of LB liquid culture medium, and cultured at 37° C. and 180 rpm for 24 h to obtain bacterial liquid of Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, and Aeromonas hydrophila.

[0114] (2) Determination of the ability of Bacillus paralicheniformis Shannan.BP-803 to inhibit pathogenic bacteria: The activated Bacillus paralicheniformis Shannan.BP-803 was inoculated into 200 mL of LB liquid culture medium at a 2% inoculum amount and cultured at 37°C and 180 rpm for 24 h to obtain a bacterial culture of Bacillus paralicheniformis Shannan.BP-803 (OD 600nm =2.5). The bacterial solutions of Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, and Aeromonas hydrophila obtained in step (1) were diluted 1000 times with physiological saline, and 100 μL was applied on different NB agar media. The bacterial solution of Bacillus paralicheniformis Shannan.BP-803 was centrifuged at 5000 rpm for 20 min, and the supernatant was collected for antibacterial test. The specific method of the antibacterial test is: a hole with a diameter of 0.7 cm was punched on the NB agar medium coated with the bacterial solutions of Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, and Aeromonas hydrophila, 3 holes were punched on each NB agar medium, which were used as 3 biological replicates, 100 μL of supernatant was added to each well until it did not overflow, and the culture was incubated at 37°C for 24 h to observe and determine the size of each inhibition zone. The measurement results are shown in Table 4.

[0115] (3) The method for determining the ability of Bacillus paralicheniformis CICC 10102 to inhibit pathogenic bacteria is the same as step (2).

[0116] Table 4. Results of the test on the ability of strains to inhibit pathogenic bacteria

[0117]

[0118]

[0119] As shown in Table 4, the results show that Bacillus paralicheniformis Shannan.BP-803 has good inhibitory effects on common animal pathogens such as Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis, Aeromonas hydrophila, etc., and the inhibitory effects are better than those of Bacillus paralicheniformis CICC 10102.

[0120] Example 6 Determination of the inhibitory and detoxification abilities of Bacillus paralicheniformis Shannan.BP-803 against pathogenic fungi

[0121] Isolation of Aspergillus flavus, Fusarium graminearum, Fusarium trilineatum, Fusarium verticillioides and Penicillium verrucosum from moldy pig and chicken feed. The specific isolation method is as follows:

[0122] (1) Treatment method of mycelium in the treatment group of Bacillus paralicheniformis Shannan.BP-803: The mycelium block of the pathogenic fungus was inoculated in the center of the plate of PDA solid culture medium, and the bacterial solution of Bacillus paralicheniformis Shannan.BP-803 was inoculated at 3 points 3 cm away from the center of the PDA plate, and repeated 3 times. After culturing at 28°C in the dark for 5 days, the mycelium diameter of the pathogenic fungus was counted. At the same time, the PDA solid culture medium inoculated with only the mycelium block of the pathogenic fungus was used as the mycelium of the control group, and the antibacterial rate was calculated. The results are shown in Table 5. Among them, the pathogenic fungi were Aspergillus flavus, Fusarium graminearum, Fusarium trilineatum, Fusarium verticillioides and Penicillium verrucosus.

[0123] The treatment method of mycelia of the Bacillus paralicheniformis CICC 10102 treatment group was the same as the treatment method of mycelia of the Bacillus paralicheniformis Shannan.BP-803 treatment group.

[0124] The calculation formula of the antibacterial rate is as follows:

[0125] Bacterial inhibition rate (%) = (diameter of mycelium in the control group - diameter of mycelium in the treatment group) / diameter of mycelium in the control group × 100%. The mycelium in the treatment group is mycelium in the treatment group of Bacillus paralicheniformis Shannan.BP-803 or mycelium in the treatment group of Bacillus paralicheniformis CICC 10102.

[0126] Table 5 Inhibitory effect of strains on pathogenic fungi

[0127]

[0128]

[0129] As shown in Table 5, the results showed that Bacillus paralicheniformis Shannan.BP-803 had good inhibitory effects on Aspergillus flavus, Fusarium graminearum, Fusarium trilineatum, Fusarium verticillium and Penicillium verrucosum, and the effect was better than that of Bacillus paralicheniformis CICC 10102.

[0130] (2) Determination of toxin degradation rate of Bacillus paralicheniformis Shannan.BP-803: The activated Bacillus paralicheniformis Shannan.BP-803 was inoculated into 100 mL of LB liquid culture medium at a 1% inoculation amount and cultured at 37°C and 180 rpm for 24 h to obtain a bacterial solution of Bacillus paralicheniformis Shannan.BP-803. Then, 1 mg of each of AFB1 toxin produced by Aspergillus flavus, ZEA toxin and DON toxin produced by Fusarium graminearum, T-2 toxin produced by Fusarium trilineatum, OTA toxin produced by Penicillium verrucosum, and FB1 toxin produced by Fusarium verticillium were taken and mixed with 100 mL of cultured bacterial liquid of Bacillus paralicheniformis Shannan.BP-803 to obtain 6 mixed solutions, and the 6 mixed solutions were cultured at 37°C. The contents of corresponding toxins in the 6 mixed solutions were detected at 0h, 2h, 4h, 8h, 12h, 1d, 3d, 7d and 14d of culture, and the detoxification curve was plotted with the culture time as the horizontal axis and the degradation rate as the vertical axis. The results are shown in the figure. Figure 1 As shown, degradation rate (%) = (toxin content in the mixed solution at the 0th hour of culture - residual toxin content in the mixed solution after the corresponding culture time) / toxin content in the mixed solution at the 0th hour of culture × 100%.

[0131] (3) The method for determining the degradation rate of toxins by Bacillus paralicheniformis CICC 10102 is the same as that for determining the degradation rate of toxins by Bacillus paralicheniformis Shannan.BP-803. The detoxification curve is plotted as follows: Figure 2 shown.

[0132] like Figure 1 and Figure 2 As shown, Figure 1 It is a line graph showing the change of toxin degradation rate of Bacillus paralicheniformis Shannan.BP-803 at 0h, 2h, 4h, 8h, 12h, 1d, 3d, 7d and 14d;

[0133] Figure 2 It is a line graph showing the changes in the degradation rate of toxins by Bacillus paralicheniformis CICC 10102 at 0h, 2h, 4h, 8h, 12h, 1d, 3d, 7d and 14d, where: Figure 1 and Figure 2 In the table, AFB1 represents AFB1 toxin produced by Aspergillus flavus, ZEA represents ZEA toxin produced by Fusarium graminearum, DON represents DON toxin produced by Fusarium graminearum, T-2 represents T-2 toxin produced by Fusarium trilineatum, OTA represents OTA toxin produced by Penicillium verrucosus, and FB1 represents FB1 toxin produced by Fusarium verticillium.

[0134] The results showed that Bacillus paralicheniformis Shannan.BP-803 had a good degradation effect on AFB1 toxin produced by Aspergillus flavus, ZEA toxin and DON toxin produced by Fusarium graminearum, T-2 toxin produced by Fusarium trilineatum, ochratoxin produced by Penicillium verrucosum, and fumonisin B1 produced by Fusarium verticillium, and the degradation rate increased with time. After 14 days of fermentation, the degradation rate of these six toxins could reach more than 60%, which was better than that of Bacillus paralicheniformis CICC10102.

[0135] Example 7 Experiment on the effect of Bacillus paralicheniformis Shannan.BP-803 as feed additive on the growth of piglets

[0136] Healthy piglets with similar weight were selected for the experiment and divided into an experimental group and a control group, with 50 pigs in each group. The feeds of the experimental group and the control group were as follows:

[0137] Control group: fed with basic pig feed;

[0138] Experimental group: pigs were fed with feed containing Bacillus paralicheniformis Shannan.BP-803, where the number of viable bacteria of Bacillus paralicheniformis Shannan.BP-803 per 1 kg of the feed was 4.2×10 9 CFU, the pig feed is prepared by the following steps:

[0139] (1) The strain preserved in the glycerol tube was activated. For the first activation, the strain was inoculated into 10 mL of LB liquid culture medium at a 2% inoculation amount and cultured at 37° C. for 24 h. For the second activation, the strain was inoculated into 10 mL of LB liquid culture medium at a 2% inoculation amount and cultured at 37° C. for 12 h to obtain the activated Bacillus paralicheniformis Shannan.BP-803.

[0140] (2) The activated Bacillus paralicheniformis Shannan.BP-803 obtained in step (1) was inoculated into 200 mL LB liquid culture medium at an inoculum size of 2%, and cultured at 37° C. for 24 h to obtain a bacterial suspension of Bacillus paralicheniformis Shannan.BP-803. The number of viable bacteria in the bacterial suspension of Bacillus paralicheniformis Shannan.BP-803 was 4.2×10 9 CFU / mL;

[0141] (3) 100 mL of the bacterial suspension of Bacillus paralicheniformis Shannan.BP-803 was mixed with 100 kg of basic pig feed to obtain pig feed containing Bacillus paralicheniformis Shannan.BP-803.

[0142] Each group of piglets was fed three times a day for 120 days. During the trial period, they were free to eat and provided with sufficient drinking water. Other daily management measures were carried out in accordance with the routine management of the farm. After 120 days, the number of Escherichia coli in the feces of each group of piglets was detected. The results are shown in Table 6. At the same time, the incidence of pigs in the experimental group and the control group during the trial period was statistically analyzed, and the diarrhea rate and mortality rate of piglets in each group were calculated. The results are shown in Table 6.

[0143] Table 6. Results of the number of Escherichia coli in piglet feces and the diarrhea rate and mortality rate of piglets

[0144] <![CDATA[Escherichia coli (10 7 / g)]]> Diarrhea rate (%) mortality rate(%) Control group 4.57 18 8 Experimental Group 1.04 4 0

[0145] As shown in Table 6, the results showed that the diarrhea rate and mortality rate of the experimental group piglets were significantly lower than those of the control group, and the number of Escherichia coli in the feces of the experimental group piglets was less, while the number of Escherichia coli in the feces of the control group piglets was more, indicating that Bacillus paralicheniformis Shannan.BP-803 as a feed additive can significantly reduce the diarrhea rate of piglets, enhance the resistance of piglets, and reduce their mortality.

[0146] Example 8 Experiment on the effect of Bacillus paralicheniformis Shannan.BP-803 as feed additive on the growth of broilers

[0147] (1) 300 healthy broilers of similar weight at 30 days of age were selected and randomly divided into 3 groups, namely the control group, experimental group 1, and experimental group 2, with 100 chickens in each group. Except for the different feeding feeds, all other feeding management methods were the same, and the chickens were fed in a tethered house, with temperature and time controlled throughout the whole process, and free access to food and water. The formal experiment began after each group was fed with basic chicken feed for 3 days. The dosage and time of gavage for each broiler were kept consistent every day, and the gavage was performed once a day. The experiment lasted for 30 days, and the average daily weight gain of each group of broilers within 30 days, the daily feed intake of each group of broilers within 30 days, and the number of broilers in each group that had diarrhea within 30 days were counted, that is, the diarrhea rate (%), and the results are shown in Table 7. Among them, the feeding feeds of the control group, experimental group 1 and experimental group 2 are as follows:

[0148] Control group: fed with basic chicken feed daily;

[0149] Experimental group 1: Chickens were fed with feed containing Bacillus paralicheniformis Shannan.BP-803, wherein the number of viable bacteria of Bacillus paralicheniformis Shannan.BP-803 per 1 kg of the chicken feed was 4.2×10 9 CFU, the chicken feed is prepared by the following steps:

[0150] (1) The strain preserved in the glycerol tube was activated. For the first activation, the strain was inoculated into 10 mL of LB liquid culture medium at a 2% inoculation amount and cultured at 37° C. for 24 h. For the second activation, the strain was inoculated into 10 mL of LB liquid culture medium at a 2% inoculation amount and cultured at 37° C. for 12 h to obtain the activated Bacillus paralicheniformis Shannan.BP-803.

[0151] (2) The activated Bacillus paralicheniformis Shannan.BP-803 obtained in step (1) was inoculated into 200 mL of LB liquid culture medium at an inoculum size of 2%, and cultured at 37° C. for 24 h to obtain a bacterial suspension of Bacillus paralicheniformis Shannan.BP-803. The number of viable bacteria in the bacterial suspension of Bacillus paralicheniformis Shannan.BP-803 was: 4.2×10 9 CFU / mL;

[0152] (3) 100 mL of the bacterial suspension of Bacillus paralicheniformis Shannan.BP-803 was mixed with 100 kg of basic chicken feed to obtain chicken feed containing Bacillus paralicheniformis Shannan.BP-803.

[0153] Experimental group 2: fed with basic chicken feed + 0.05g / mL oxytetracycline daily.

[0154] Table 7 Growth of broiler chickens

[0155] Average daily weight gain (g) Daily feed intake (g) Diarrhea rate (%) Control group 28.03 43.56 8％ Experimental Group 1 33.27 45.25 1％ Experimental Group 2 31.53 44.78 3％

[0156] As shown in Table 7, the results showed that compared with experimental group 2, experimental group 1 could increase the average daily weight gain and daily feed intake of broilers, reduce the diarrhea rate, improve the quality of broilers, have a good feeding effect, and improve the economic benefits of breeding to a certain extent.

[0157] (2) During the 30-day test period, fresh fecal samples of broilers in the control group, test group 1 and test group 2 were randomly collected. Three broilers were collected from each group, and three fecal samples were collected from each broiler. Finally, a total of 9 fecal samples were collected from each group. 5 g of each fecal sample was weighed and thoroughly mixed in 45 mL of normal saline. 100 μL was taken for gradient dilution and spread on eosin-methylene blue agar medium (for culturing Escherichia coli), BBL agar medium (for culturing Bifidobacterium), TSC agar medium (for culturing Clostridium perfringens), and LBS agar medium (for culturing Lactobacillus). Escherichia coli and Bifidobacterium were cultured at 37°C for 24 h, and Clostridium perfringens and Lactobacillus were cultured at 42°C for 24 h. The colony morphology in eosin-methylene blue agar medium, BBL agar medium, TSC agar medium and LBS agar medium was observed and examined under a microscope, and the target colonies on the plate were counted. The results are shown in Table 8.

[0158] Table 8. Determination of bacterial flora in broiler feces

[0159]

[0160] As shown in Table 8, the results show that the addition of Bacillus paralicheniformis Shannan.BP-803 to the basic chicken feed of the first group of experiments significantly increased the content of lactobacillus and bifidobacterium in the broiler feces compared with the control group, while the content of Escherichia coli and Clostridium perfringens decreased, which shows that the addition of Bacillus paralicheniformis Shannan.BP-803 can promote the abundance of beneficial bacteria and reduce the abundance of harmful bacteria. In the basic chicken feed of the second group of experiments, antibiotics were administered externally. It can be seen that the content of lactobacillus, bifidobacterium, Escherichia coli and Clostridium perfringens in the broiler feces was reduced compared with the content of the control group, which shows that the addition of antibiotics to the basic chicken feed will not only inhibit the content of pathogens, but also inhibit the growth of probiotics. Compared with the antibiotic group, the inhibition of pathogens in the group added with Bacillus paralicheniformis Shannan.BP-803 was significantly higher than that in the antibiotic group, which may be the result of resistance caused by continuous intake of antibiotic solution.

[0161] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not limitations of the present invention. Any non-prominent substantial features and non-significant improvements made by those skilled in the art on this basis should fall within the scope of protection of the present invention.

Claims

1. A feed containing Bacillus paralicheniformis carrying a quorum sensing-related gene, characterized in that: The Bacillus paralicheniformis is Bacillus paralicheniformis Shannan. BP-803.

2. The feed according to claim 1, characterized in that The number of viable bacteria of Bacillus paralicheniformis contained in each kg of the feed is 2.1×10 9 -7.3×10 9 CFU.

3. The feed according to claim 1 or 2, characterized in that The quorum sensing-related gene contains the gene sequence shown in SEQ ID NO: 4 and / or SEQ ID NO:

5.

4. The feed according to claim 3, characterized in that The amino acid sequence encoded by the gene sequence shown in SEQ ID NO:4 is the amino acid sequence shown in SEQ ID NO:

6.

5. The feed according to claim 3, characterized in that The amino acid sequence encoded by the gene sequence shown in SEQ ID NO:5 is the amino acid sequence shown in SEQ ID NO:

7.

6. The feed according to any one of claims 1 to 5, characterized in that The Bacillus paralicheniformis has the functions of forming biofilm, and / or producing extracellular protease, and / or inhibiting pathogenic bacteria, and / or inhibiting the performance of pathogenic fungi, and / or degrading toxins produced by pathogenic fungi.

7. The feed according to claim 6, characterized in that The pathogenic bacteria include one or more of Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Streptococcus suis and Aeromonas hydrophila; preferably, the pathogenic bacteria include one or more of Clostridium perfringens, Streptococcus suis and Aeromonas hydrophila.

8. The feed according to claim 6, characterized in that The pathogenic fungi include one or more of Aspergillus flavus, Fusarium graminearum, Fusarium trilineatum, Fusarium verticillium and Penicillium verrucosum; preferably, the pathogenic fungi include one or more of Fusarium trilineatum, Fusarium verticillium and Penicillium verrucosum.

9. The feed according to claim 6, characterized in that The toxins include one or more of AFB1 toxin, ZEA toxin, DON toxin, T-2 toxin, OTA toxin and FB1 toxin.

10. The feed according to claim 6, characterized in that The enzymatic activity of the extracellular protease of the Bacillus paralicheniformis is 10-30U, wherein U is one enzymatic activity unit of the extracellular protease, wherein, when 1mL of the enzyme solution of the Bacillus paralicheniformis is hydrolyzed at 40°C, the amount of 1pg of tyrosine produced by casein per minute is 1 enzymatic activity unit U of the extracellular protease.

11. A method for preparing a feed containing Bacillus paralicheniformis carrying a quorum sensing-related gene according to any one of claims 1 to 10, characterized in that: The method comprises the following steps: mixing the bacterial suspension of the Bacillus paralicheniformis with a basic feed to obtain a feed containing the Bacillus paralicheniformis carrying a quorum sensing related gene.

12. The preparation method according to claim 11, characterized in that: The steps include: (1) expanding the culture of the Bacillus paralicheniformis; (2) adding the expanded cultured Bacillus paralicheniformis to a liquid culture medium, preferably, culturing at 35-42° C. to obtain a bacterial suspension of Bacillus paralicheniformis; (3) Mixing the bacterial suspension of Bacillus paralicheniformis with a basic feed to obtain a feed containing Bacillus paralicheniformis.

13. A feed containing Bacillus paralicheniformis, characterized in that: It is prepared by the preparation method according to any one of claims 11 or 12.

14. The feed containing Bacillus paralicheniformis according to any one of claims 1 to 10 or claim 13, characterized in that: The feed is pig feed and / or chicken feed.

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