Bacillus subtilis BS-21001 and application thereof

Abstract

The invention provides bacillus subtilis BS-21001 and application thereof, belongs to the technical field of biological prevention and treatment, and can solve the technical problems that a traditional riemerella anatipestifer disease treatment method is not ideal in immune effect and drug residues exist. The bacillus subtilis BS-21001 is named as bacillus subtilis in taxonomy, the preservation number of the bacillus subtilis BS-21001 is CGMCC (China General Microbiological Culture Collection Center) No.26444, and the bacillus subtilis BS-21001 is preserved in the China General Microbiological Culture Collection Center on March 23, 2023. The fermentation supernatant of the bacillus subtilis BS-21001 can be used as a single active component or compounded with other active components to prepare a microecological preparation, and the microecological preparation can effectively inhibit riemerella anatipestifer in duck bodies and in a breeding environment and has no chemical pollution to the environment.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and in particular relates to Bacillus subtilis BS-21001 and application thereof. Background Art

[0002] Duck infectious serositis (also known as "Riemerella anatipestifer disease") is an acute or chronic contact infectious disease of ducks caused by Riemerella anatipestifer, which mainly affects ducklings aged 2-7 weeks and is characterized by neurological symptoms and fibrinous pericarditis, perihepatitis and air sac inflammation. Riemerella anatipestifer is a serious threat to the breeding industry and can directly cause animal illness and death. In addition, ducks infected with Riemerella anatipestifer have reduced feed conversion rates and slowed growth and development, resulting in very serious economic losses.

[0003] The disease is distributed worldwide and seriously affects the healthy development of waterfowl farming. Drugs play an important role in the prevention and treatment of this disease, but with the increasing number of drug-resistant strains of Riemerella anatipestifer, the emergence of multi-drug resistant strains, and the continuous changes in drug resistance mechanisms, the application of drugs has been greatly limited.

[0004] The abuse of antibiotics has become a global problem. Due to unreasonable medication regimens, drug resistance of Riemerella anatipestifer is common, with a high and widespread resistance rate, and certain problems such as drug residues. Moreover, Riemerella anatipestifer invades the air sacs and the back of the brain, making it difficult for drugs to reach and thus difficult to remove. At present, an important means of preventing the disease is to vaccinate with Riemerella anatipestifer vaccine. Inactivated vaccines for infectious serositis have been commercialized. When the serotype of the vaccine strain is the same as that of the epidemic strain, the inactivated vaccine can provide effective protection. However, Riemerella anatipestifer has more than 21 serotypes. Once the serotype of the epidemic strain changes, the immune effect is reduced. Therefore, it is urgent to find an effective method that can effectively inhibit Riemerella anatipestifer and prevent and treat Riemerella anatipestifer disease. Summary of the invention

[0005] Aiming at the technical problems of unsatisfactory immune effect and drug residue in traditional treatment methods for Riemerella anatipestifer disease, the invention provides a Bacillus subtilis BS-21001 and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] Bacillus subtilis BS-21001, whose taxonomic name is Bacillus subtilis, has a deposit number of CGMCC No.26444. It was deposited in the General Microbiology Center of China National Culture Collection Administration on March 23, 2023, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] In one embodiment, the 16S rDNA sequence of Bacillus subtilis BS-21001 is shown as SEQ ID No.1.

[0009] The microecological preparation comprises the Bacillus subtilis BS-21001 described in the above embodiment.

[0010] In one embodiment, the probiotic preparation is a single preparation using the fermentation supernatant prepared by Bacillus subtilis BS-21001 as described in the above embodiment or a compound preparation using the fermentation supernatant prepared by Bacillus subtilis BS-21001 as described in the above embodiment as the main ingredient and compounded with other active ingredients.

[0011] In one embodiment, when the probiotic preparation is prepared with the fermentation supernatant prepared by Bacillus subtilis BS-21001 as described in the above embodiment as a single preparation, the concentration of the fermentation supernatant of Bacillus subtilis BS-21001 in the probiotic preparation is 250-350 IU / mL.

[0012] In one embodiment, the other active ingredient is selected from Riemerella anatipestifer phage, and the probiotic preparation is prepared by compounding the Riemerella anatipestifer phage with the fermentation supernatant of Bacillus subtilis BS-21001.

[0013] Preferably, the Riemerella anatipestifer phage described in the present invention refers to Riemerella anatipestifer phage RKP-RA-22009, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 20, 2022, with a deposit number of CGMCC No.45095, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its host bacteria is RA-22009. The full genome length of the phage RKP-RA-22009 is 45828bp, and is composed of the sequences shown in the sequence list SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4, SEQ ID NO.5 and SEQ ID NO.6 connected in sequence. Because the length of a single sequence of the genome is too large, the nucleotide sequence table machine-readable carrier cannot be submitted through the electronic application client, so it is split and processed.

[0014] In one embodiment, the concentration of the fermentation supernatant of Bacillus subtilis BS-21001 is 250-350 IU / mL, and the concentration of the Riemerella anatipestifer phage is 5.0×10 6 -5.0×10 8 pfu / mL.

[0015] In one embodiment, the fermentation supernatant is obtained by inoculating the Bacillus subtilis BS-21001 into a liquid culture medium for fermentation.

[0016] Wherein, the liquid culture medium is TSB liquid culture medium, the culture temperature of the Bacillus subtilis BS-21001 is 35-37°C, the rotation speed is 120-250rpm, and the culture time is 20-44h.

[0017] Application of Bacillus subtilis BS-21001 or microecological preparations in inhibiting Riemerella anatipestifer.

[0018] Feed / cleaner / disinfectant, comprising Bacillus subtilis BS-21001 described in the above embodiment or the probiotic preparation described in the above embodiment, is used to inhibit Riemerella anatipestifer in ducks or in breeding environments.

[0019] Compared with the prior art, the advantages and positive effects of the present invention are:

[0020] 1. The present invention provides a Bacillus subtilis BS-21001, whose fermentation supernatant has an antibacterial effect on Riemerella anatipestifer in ducks and breeding environments. The antibacterial activity of the fermentation supernatant of Bacillus subtilis BS-21001 on Riemerella anatipestifer measured by the double-layer plate Oxford cup method reaches 400 IU / mL;

[0021] 2. The present invention provides a Bacillus subtilis BS-21001 having a wide lysis spectrum against Riemerella anatipestifer. The probiotic produces an inhibition zone for 72 of the 76 strains of Riemerella anatipestifer, and the lysis rate is as high as 94.74%;

[0022] 3. The present invention provides a Bacillus subtilis BS-21001 and its application. The fermentation supernatant of Bacillus subtilis BS-21001 is used to prepare a microecological preparation, which can not only effectively inhibit Riemerella anatipestifer in ducks and breeding environments, but also has no chemical pollution to the environment.

[0023] 4. The present invention provides a Bacillus subtilis BS-21001 and its application. The microecological preparation prepared by compounding the fermentation supernatant of Bacillus subtilis BS-21001 with Riemerella anatipestifer phage has a very obvious inhibitory effect on Riemerella anatipestifer in vitro, and can inhibit the growth of Riemerella anatipestifer for a long time. It has a good therapeutic effect on diseases caused by Riemerella anatipestifer and has extremely high safety characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The inhibition zone of the fermentation supernatant of Bacillus subtilis BS-21001 provided by the present invention with Riemerella anatipestifer as the indicator bacteria;

[0025] Figure 2 This is a colony morphology diagram of Bacillus subtilis BS-21001 provided by the present invention after being cultured in LB solid medium;

[0026] Figure 3 This is the antibacterial activity change curve of the fermentation supernatant of Bacillus subtilis BS-21001 provided by the present invention;

[0027] Figure 4 This is the antibacterial activity change curve of the fermentation supernatant of Bacillus subtilis BS-21001 provided by the present invention;

[0028] Figure 5 This is an electron microscope image of the bacteriophage RKP-SR-22003 provided by the present invention;

[0029] Figure 6 This is a curve showing the difference in effects of the fermentation supernatants of different probiotics combined with the bacteriophage provided by the present invention;

[0030] Figure 7 This is a curve showing the difference in effects of different concentrations of phage-compounded probiotic fermentation supernatants provided by the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In order to more clearly and in detail introduce the Bacillus subtilis BS-21001 and its application provided by the embodiments of the present invention, a description will be given below in conjunction with specific embodiments.

[0033] Example 1

[0034] This example is about the acquisition and identification of Bacillus subtilis BS-21001, specifically:

[0035] (1) The acquisition of Bacillus subtilis BS-21001 is carried out by dilution gradient separation method, which includes the following steps:

[0036] Weigh 5 g of duck feces into a 100 mL centrifuge tube, add 20 mL of sterile saline, shake and mix, let stand, take 100 μL of the bacterial suspension, add sterile saline, and dilute to a concentration of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7Spread on LB (Luria-Bertani medium) solid plates and PDA solid plates (potato dextrose agar medium) respectively, invert and place in an incubator at 37°C for cultivation, wait for the isolate to grow, and separate and purify the single colonies grown on the plates according to the colony morphology to obtain the strain to be verified, and place in TSB containing 20% ​​glycerol and store at -20°C.

[0037] The strains that inhibit Riemerella anatipestifer were screened by the Oxford cup agar diffusion method, and a strain with strong inhibitory ability against Riemerella anatipestifer was obtained (referred to as "antibacterial strain"). Figure 1 Shown: The indicator bacteria is Riemerella anatipestifer, and the sample is the fermentation supernatant of BS-21001.

[0038] Figure 2 The colony morphology of the antibacterial strain provided in this embodiment after being cultured on the LB solid plate is white, with irregular edges, flat, dry surface to form wrinkles, and a wrinkled tip in the center of the colony. The specific culture conditions are inoculation on the LB solid medium, streak culture, inverted at 37°C, and cultured for 14-16h.

[0039] (2) Identification of Bacillus subtilis BS-21001 using molecular biological identification methods, including the following steps:

[0040] Pick out the above-mentioned single bacterial colony with antibacterial effect and put it into 10 μl of sterile water, use a pipette to blow and mix evenly, use it as a template for PCR reaction, and perform PCR process according to the amplification system shown in Table 1:

[0041] Table 1 PCR system for identification of Bacillus subtilis BS-21001

[0042]

[0043] Among them, in the above table: 2×TaqPCRStarMixwithLoadingDye is a premix containing optimized concentrations of Genstar high-concentration TaqDNAPolymerase, dNTPs, Mg 2+ , reaction buffer and stabilizer in a 2-fold concentration of ready-to-use PCR reagent. The primers used in this example are 27F and 1492R, and the specific sequences of the primers are:

[0044] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0045] 1492R:5'-TACGGCTACCTTGTTACGACTT-3'

[0046] PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s, repeated for 30 cycles; and reaction at 72°C for 10 min.

[0047] Figure 3 This is an agarose gel electrophoresis diagram of the PCR amplification product of the antibacterial strain provided in this example. The DNA product obtained by PCR amplification was separated by 1% agarose gel electrophoresis, and there was an obvious band near 1500 bp.

[0048] The PCR amplification product was bidirectionally sequenced to obtain the gene sequence. The gene sequence obtained by sequencing was compared with the nucleotide sequence in the NCBI database (https: / / www.ncbi.nlm.nih.gov), and the results showed that the antibacterial strain had a 100% similarity with the strain Bacillus subtilis strain JCL16. Combining the colony morphological structure characteristics of the antibacterial strain and the 16S sequencing comparison results, it was determined to be Bacillus subtilis and named Bacillus subtilis BS-21001. The Bacillus subtilis is a probiotic that can inhibit Riemerella anatipestifer. The 16s rDNA sequence is shown in SEQ ID NO.1.

[0049] Example 2

[0050] This example is a determination of the antibacterial effect of the fermentation supernatant of Bacillus subtilis BS-21001, specifically:

[0051] In this example, Bacillus subtilis BS-21001 strain was cultured in TSB culture medium (1% v / v) at 37°C, and samples were taken out every 2 hours, centrifuged at 10000-12000 rpm, and filtered through a 0.22 μm filter to determine the antibacterial activity of the fermentation supernatant. The method used was the double-layer plate Oxford cup method. The antibacterial activity of the probiotic fermentation supernatant is expressed in arbitrary units per milliliter (IU / mL), which is defined as the highest dilution multiple that has a certain inhibitory effect on the indicator.

[0052] Antibacterial effect test results: Figure 4 As shown, at 20 h, the antibacterial activity of the fermentation supernatant was detected, which was about 100 IU / mL, reaching a maximum value of about 400 IU / mL at 44 h, and the antibacterial activity reached a stable state.

[0053] Example 3

[0054] This example is a test for the lysis spectrum determination of the fermentation supernatant of Bacillus subtilis BS-21001, specifically:

[0055] The Oxford cup double-layer plate method was used to determine the lysis spectrum of the fermentation supernatant of Bacillus subtilis BS-21001 of the present invention on 76 strains of Riemerella anatipestifer isolated in the laboratory.

[0056] Preparation of Riemerella anatipestifer bacterial liquid: inoculate Riemerella anatipestifer into TSB medium, add 2% fetal bovine serum, and culture for 24 hours to obtain Riemerella anatipestifer bacterial liquid.

[0057] Introduce 1.5% solid soybean casein agar (TSA) culture medium to the bottom of the plate, wait for the culture medium to solidify, use sterilized tweezers to place the Oxford cup on the surface of the solid culture medium, take 1mL of the overnight cultured bacterial solution to be tested and add it to 0.7% tryptic soybroth (TSB) (2% fetal bovine serum) semi-solid culture medium, mix well, pour it into the plate, let it stand and solidify, then take out the Oxford cup with tweezers to obtain the Oxford cup hole. Take 50μL of Bacillus subtilis fermentation supernatant that has been sterilized by filtering with a 0.22μL filter and drop it into the Oxford cup hole, culture at 37℃ for about 16h, and observe whether there is an inhibition zone.

[0058] Among the 76 strains of Riemerella anatipestifer used in this experiment, 72 strains produced inhibition zones, indicating that the lysis rate of the Bacillus subtilis fermentation supernatant of the present invention on the 76 strains of Riemerella anatipestifer was as high as 94.74%. It can be seen that the Bacillus subtilis fermentation supernatant has a wide lysis spectrum for Riemerella anatipestifer.

[0059] Example 4

[0060] This example is to prepare a microecological preparation by compounding Bacillus subtilis BS-21001 with Riemerella anatipestifer phage, and to determine the inhibitory / bactericidal effect of the preparation on Riemerella anatipestifer, specifically:

[0061] (1) Isolation, purification and preparation of bacteriophages:

[0062] Riemerella anatipestifer phage RKP-RA-22009 was isolated from sewage samples from duck farms. Duck sewage samples were added with 10 mL SM buffer was soaked overnight, and then the overnight leaching solution was centrifuged at 10000rpm for 5min, the supernatant was filtered with a 0.22μm filter, and the filtrate was stored at 4°C for standby use; 1mL of Riemerella anatipestifer suspension and 1mL of filtrate were added to 10mL TSB (2% fetal bovine serum), cultured at 37°C for 24h, and then centrifuged at 10000rpm for 10min, the supernatant was filtered with a 0.22μm filter, and the filtrate was used for standby use; the double plate method was used to separate the bacteriophage, 1mL of Riemerella anatipestifer suspension was mixed with 0.7% TSB soft agar and spread on a TSB solid plate, after the soft agar solidified, 0.1mL of the filtrate was spotted on the plate, and it was left to stand until the mixed solution was absorbed, and after being cultured at 37°C for 24h, a transparent plaque was picked up with an inoculation loop and placed in 2mL SM buffer, and stored at 4°C for overnight to allow the phage to be completely released, thereby obtaining the phage.

[0063] The double plate method was used to separate the phage. 0.1 mL of the mixed bacterial suspension filtrate and 0.2 mL of the host bacterial suspension were mixed evenly, and then soft agar cooled to about 50°C was added to mix evenly. Then, double plates were spread and placed in a 37°C incubator for 24 hours. Then, a single transparent spot with smooth edges was picked up and placed in 1 mL of SM buffer and stored at 4°C overnight. 0.1 mL of the overnight stored extract was mixed with 0.1 mL of the bacterial suspension, and soft agar cooled to about 50°C was added to mix evenly, and double plates were spread. The plaques were cultured in a 37°C incubator for 24 hours. Purification was performed according to this step until the plaques were uniform in size and smooth in edges, and the purified phages were obtained. 0.2 mL of Riemerella anatipestifer RA-22009 bacterial suspension and 1 mL of purified bacteriophage RKP-RA-22009 suspension were added to 50 mL of tryptic soy broth (TSB) (2% fetal bovine serum), cultured overnight at 37°C with shaking, and then centrifuged at 12000 rpm for 10 min. The supernatant was filtered with a 0.22 μm filter, and the phage filtrate was titered to 5.0 × 10 8 pfu / mL.

[0064] (2) Phage sequencing and analysis:

[0065] After enrichment and cultivation of a single phage, centrifuge at 8000g for 15 minutes at 4°C, add 10% PEG8000 and 0.5M NaCl and let stand overnight, then add an equal amount of chloroform and mix well, let stand and separate, centrifuge at 5000g for 10 minutes, remove the chloroform layer and PEG layer, add restriction endonuclease for digestion, and suspend the phage under gradient density cesium chloride conditions, then dialyze 3 times with TM buffer, 30 minutes each time, and send a part to a biological sequencing company for whole genome sequencing. The sequencing method is as follows:

[0066] Using Illumina TruSeq TM The Nano DNA Sample Prep Kit method was used to construct the library. The specific steps are as follows:

[0067] 1. Build the library with 1 μg DNA as the starting amount;

[0068] 2. Covaris M220 ultrasonically breaks DNA into 300-500 bp;

[0069] 3. Fill in the 3' end and add A, connect the index adapter (TruSeq TM Nano DNA Sample Prep Kit);

[0070] 4. Library enrichment and PCR amplification for 8 cycles;

[0071] 5. Recover the target band with 2% agarose gel (Certified Low Range Ultra Agarose);

[0072] 6. TBS380 (Picogreen) quantification, mixing according to the data ratio;

[0073] 7. Bridge PCR amplification is performed on the cBot solid phase carrier to generate clusters;

[0074] 8. Illumina HiSeq sequencing platform, performing 2×150bp sequencing.

[0075] The total length of the phage genome is 45828 bp, and is composed of the sequences shown in the sequence list SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 connected in order. Since the length of a single sequence of the genome is too large, the nucleotide sequence table machine-readable carrier cannot be submitted through the electronic application client, so it is split. According to the sequence comparison and annotation results of databases such as NCBI (Nr library) and CARD (antibiotic resistance gene database), the phage RKP-RA-22009 screened in this example does not have virulence genes and lysogen genes.

[0076] Phage morphology observation: Take 20 μL of liquid containing crude phage particles and drop it on a copper mesh. Let it precipitate naturally for 15 minutes, and use filter paper to absorb excess liquid from the side. Add a drop of 2% phosphotungstic acid (PTA) on the copper mesh to stain the phage for 10 minutes, then use filter paper to absorb the staining liquid from the side. After the sample is dry, use an electron microscope to observe the phage morphology, such as Figure 5 The electron microscope image of bacteriophage RKP-RA-22009 is shown. Figure 5 It can be seen that the bacteriophage RKP-RA-22009 has a polyhedral three-dimensional symmetrical head, which is wrapped with nucleic acid, with a diameter of about 20.92nm, a tail of about 42.80nm long, a tail sheath, and a neck connecting the head and tail. According to the ninth report on virus classification of the International Organization for Taxonomy of Viruses, the bacteriophage is classified as the family Myoviridae of the order Caudovirales.

[0077] The host bacteria RA-22009 suspension cultured overnight was inoculated into 1000 mL TSB liquid medium (supplemented with 2% fetal bovine serum) at a 1% inoculum and cultured at 37°C for 8 h (OD was about 0.15, about 5.0×10 8 pfu / mL, or use TSB liquid medium to adjust the bacterial solution to an OD of 0.15.

[0078] In order to screen out the optimal ratio of the above-mentioned bacteriophage and Bacillus subtilis BS-21001 for preparing the microecological preparation, this experiment was divided into two groups, namely Experimental Group 1 and Experimental Group 2. 50 mL of bacterial suspension was dispensed into 18 sterile conical bottles, all marked with numbers 1-9. The specific operations of each group were as follows:

[0079] Experimental group 1: Effects of phage combined with different amounts of probiotic fermentation supernatant

[0080] No. 1 is the negative control, without any treated bacterial solution;

[0081] No. 2 added 1 mL of bacteriophage RKP-RA-22009 (5.0×10 8 pfu / mL, the following phage concentrations are all 5.0×108 pfu / mL);

[0082] No. 3, 1 mL of bacteriophage RKP-RA-22009 and 100 IU / mL of probiotic fermentation supernatant were added;

[0083] No. 4, 1 mL of bacteriophage RKP-RA-22009 and 150 IU / mL of probiotic fermentation supernatant were added;

[0084] No. 5, 1 mL of bacteriophage RKP-RA-22009 and 200 IU / mL of probiotic fermentation supernatant were added;

[0085] No. 6, 1 mL of bacteriophage RKP-RA-22009 and 250 IU / mL of probiotic fermentation supernatant were added;

[0086] No. 7, 1 mL of bacteriophage RKP-RA-22009 and 300 IU / mL of probiotic fermentation supernatant were added;

[0087] At step 8, 1 mL of bacteriophage RKP-RA-22009 and 350 IU / mL of probiotic fermentation supernatant were added;

[0088] At No. 9, 1 mL of bacteriophage RKP-RA-22009 and 400 IU / mL of probiotic fermentation supernatant were added.

[0089] Experimental Group 2: Effects of different concentrations of phage combined with probiotic fermentation supernatant

[0090] No. 1 is the negative control, without any treated bacterial solution;

[0091] No. 10, 200 IU / mL of probiotic fermentation supernatant was added;

[0092] 11. Add 200 IU / mL of probiotic fermentation supernatant and 5.0×10 2 pfu of bacteriophage RKP-RA-22009;

[0093] No. 12, 200 IU / mL of probiotic fermentation supernatant and 5.0×10 3 pfu of bacteriophage RKP-RA-22009;

[0094] 13. Add 200 IU / mL of probiotic fermentation supernatant and 5.0×10 4 pfu of bacteriophage RKP-RA-22009;

[0095] 14. Add 200 IU / mL of probiotic fermentation supernatant and 5.0×10 5 pfu of bacteriophage RKP-RA-22009;

[0096] On the 15th, 200 IU / mL of probiotic fermentation supernatant and 5.0×10 6 pfu / of bacteriophage RKP-RA-22009;

[0097] 16, add 200 IU / mL of probiotic fermentation supernatant and 5.0×10 7 pfu / of bacteriophage RKP-RA-22009;

[0098] On the 17th, 200 IU / mL of probiotic fermentation supernatant and 5.0×10 8 pfu / of bacteriophage RKP-RA-22009.

[0099] OD of the culture system after adding the microecological preparation test 600 Value, compare bacterial solution OD 600 The changes in the value of , thus obtaining the lysis effect of the microecological preparations in different dosage combinations on the strain. Among them, Figure 6 This is the difference curve of the effect of phage compounding with different probiotic fermentation supernatants. Figure 7 This is the difference curve of the effects of different concentrations of phage combined with probiotic fermentation supernatant.

[0100] Depend on Figure 6 It can be seen that when bacteriophage and Bacillus subtilis fermentation supernatant acted alone on Riemerella anatipestifer, they both showed inhibitory effects; compared with the use of bacteriophage and Bacillus subtilis supernatant alone, the microecological preparation composed of bacteriophage and Bacillus fermentation supernatant can significantly enhance the inhibitory effect on Riemerella anatipestifer; with the increase of the antibacterial activity unit of Bacillus subtilis fermentation supernatant, the antibacterial effect of the microecological preparation compounded with bacteriophage also increased accordingly; when the fermentation supernatant with an antibacterial activity of 350IU was compounded with bacteriophage, the antibacterial effect was obvious, which extended the delay period of Riemerella anatipestifer to 480min and delayed the appearance of the logarithmic phase by 200min; within the range of 100-350IU, it can be compounded with bacteriophage to achieve antibacterial effect, and the combination of the two can enhance the antibacterial effect. In vitro experiments have shown that the antibacterial effects of bacteriophage and probiotic Bacillus subtilis fermentation supernatant on Riemerella anatipestifer are mutually reinforcing; and the combined microecological preparation of the two can inhibit Riemerella anatipestifer for a long time, with a more obvious synergistic inhibitory effect.

[0101] Depend on Figure 7 It can be seen that different concentrations of phages were compounded on the basis of Bacillus fermentation supernatant. When the phage concentration was 5.0×10 6 When the concentration of pfu / mL was 1.54, the antibacterial effect was the best.

[0102] In summary, the use of 250-350 IU / mL of fermentation supernatant in synergy with 5.0×10 6-5.0×10 8 pfu / mL phage has a significant antibacterial effect. Among them, the probiotic fermentation supernatant 250IU / mL can be selected to cooperate with Riemerella anatipestifer phage concentration 5.0×10 6 pfu / mL, achieving better inhibition of Riemerella anatipestifer with low-cost reagents.

[0103] Example 5

[0104] This example is a treatment experiment of artificially infected Muscovy ducks with a probiotic preparation, specifically:

[0105] In this experiment, 140 10-day-old Muscovy ducks were selected and isolated for 2 days before animal testing. 100 of the ducks were injected subcutaneously with 0.5 mL of Riemerella anatipestifer RA-22009 solution (the bacterial content was 1×10 8 cfu / mL), and then the 40 ducks that were not challenged with bacteria were divided into groups 1 and 2, and the 100 ducks that were challenged with bacteria were randomly divided into 5 groups. The 140 ducks that were treated were raised for 2 days before the experiment. The grouping is as follows:

[0106] Group 1 received no treatment and was fed normally;

[0107] 2 groups of 1×10 6 pfu Bacillus subtilis 1mL;

[0108] Group 3 was only subjected to bacterial attack treatment without injection of bacteriophage or addition of probiotic fermentation supernatant;

[0109] Group 4 was immediately injected subcutaneously with 0.5 mL of bacteriophage RKP-RA-22009 (the content of bacteriophage was 2×10 8 pfu);

[0110] Group 5 was immediately injected subcutaneously with 0.5 mL of bacteriophage RKP-RA-22009 (the content of bacteriophage was 2×10 8 pfu), 1×10 6 cfu Bacillus subtilis 1mL;

[0111] Group 6 immediately took 1×10 6 cfu Bacillus subtilis 1 mL, 4 h later, 0.5 mL of bacteriophage RKP-RA-22009 (the content of bacteriophage was 2×10 8 pfu);

[0112] 7 groups of 1×10 6 pfu Bacillus subtilis 1mL.

[0113] The ducks were isolated and raised under the same conditions and observed for 7 days. The number of deaths and mortality rates of the ducks were counted. The weight changes of the ducks in groups 1 and 2 were compared. The surviving ducks in the five groups were dissected to detect the presence of RKP-RA-22009 phage in the blood and brain. The treatment effects are shown in Table 2:

[0114] Table 2 The therapeutic effect of each test group after artificial infection with Riemerella anatipestifer

[0115]

[0116] Combined with the test results in Table 2, the results of Experimental Group 1 and Experimental Group 2 indicate that the probiotics and the fermented liquid are safe for the body, and the weight gain index of the Muscovy ducks in Experimental Group 2 is higher than that in the Muscovy ducks in Control Group 1; in the surviving Muscovy ducks in Experimental Groups 4 and 5, the phage RKP-RA-22009 can be detected and isolated in the blood and brain.

[0117] After attacking Riemerella anatipestifer, the number of deaths in each experimental group with different treatment measures was different, as shown in Table 1. The results of experimental groups 4 and 6 show that the use of bacteriophage or Bacillus subtilis alone can play a certain role in the treatment and prevention of Riemerella anatipestifer; experimental groups 5 and 6 confirmed that the order of administration of probiotics and bacteriophages had no difference in the treatment effect; the results of experimental group 5 show that the mortality rate of Muscovy ducks using the ecological preparation of bacteriophage and Bacillus micro-compound is much lower than that of Muscovy ducks using bacteriophage or Bacillus alone, indicating that the combined effect of bacteriophage and Bacillus has a better therapeutic effect on Muscovy ducks artificially infected with Riemerella anatipestifer.

[0118] In summary, the Bacillus subtilis provided by the present invention is a probiotic that can regulate the balance of intestinal flora, promote nutrient absorption and intestinal development, and mainly exerts an effect on Riemerella anatipestifer in the intestine; and the bacteriophage can enter the blood and can cross the blood-brain barrier to exert an effect at the site where Riemerella anatipestifer colonizes. The coupling of the two can inhibit Riemerella anatipestifer, and utilize the therapeutic and preventive effects of Bacillus subtilis on Riemerella anatipestifer and the characteristics of bacteriophage colonizing the blood-brain barrier to inhibit the proliferation of Riemerella anatipestifer, which has a synergistic preventive and therapeutic effect on diseases caused by Riemerella anatipestifer.

Claims

1. Bacillus subtilis BS-21001, characterized in that Its taxonomic name is Bacillus subtilis, and its preservation number is CGMCC No.26444. It was deposited in the General Microbiology Center of China Culture Collection Administration on March 23, 2023.

2. The Bacillus subtilis BS-21001 according to claim 1, characterized in that Its 16S rDNA sequence is shown as SEQ ID No.

1.

3. Microecological preparation, characterized in that: The method comprises the Bacillus subtilis BS-21001 as claimed in claim 1 or 2.

4. The microecological preparation according to claim 3, characterized in that: The microecological preparation is a single preparation using the fermentation supernatant prepared by Bacillus subtilis BS-21001 as claimed in claim 1 or 2, or a compound preparation using the fermentation supernatant prepared by Bacillus subtilis BS-21001 as claimed in claim 1 or 2 as the main ingredient and compounded with other active ingredients.

5. The microecological preparation according to claim 4, characterized in that: When the probiotic preparation is prepared with the fermentation supernatant prepared by Bacillus subtilis BS-21001 as claimed in claim 1 or 2 as a single preparation, the concentration of the fermentation supernatant of Bacillus subtilis BS-21001 in the probiotic preparation is 250-350 IU / mL.

6. The microecological preparation according to claim 4, characterized in that: The other active ingredients are selected from Riemerella anatipestifer bacteriophage, and the probiotic preparation is prepared by compounding the Riemerella anatipestifer bacteriophage with the fermentation supernatant of Bacillus subtilis BS-21001.

7. The microecological preparation according to claim 6, characterized in that: The concentration of the fermentation supernatant of Bacillus subtilis BS-21001 is 250-350 IU / mL, and the concentration of the Riemerella anatipestifer phage is 5.0×10 6 -5.0×10 8 pfu / mL.

8. The microecological preparation according to claim 4, characterized in that: The fermentation supernatant is obtained by inoculating the Bacillus subtilis BS-21001 into a liquid culture medium for fermentation; Wherein, the liquid culture medium is TSB liquid culture medium, the culture temperature of the Bacillus subtilis BS-21001 is 35-37°C, the rotation speed is 120-250rpm, and the culture time is 20-44h.

9. Use of the Bacillus subtilis BS-21001 described in claim 1 or 2 or the probiotic preparation described in claim 3 in inhibiting Riemerella anatipestifer.

10. Feed / cleaner / disinfectant, characterized in that The invention comprises the Bacillus subtilis BS-21001 described in claim 1 or 2 or the microecological preparation described in claim 3, and is used for inhibiting Riemerella anatipestifer in a duck body or a breeding environment.

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