TWI729523B - Bacillus amyloliquefaciens and use of the bacillus amyloliquefaciens for manufacturing compositions to inhibit or kill pathogenic bacteria and its metabolites for manufacturing compositions to inhibit or kill pathogenic bacteria - Google Patents

Abstract

A bacillus amyloliquefaciens is provided, deposited at Food Industry Research and Development Institute (FIRDI) under accession number BCRC 910926 and named as N43, including a 16S rRNA, and an nucleotide sequence of the gene coding for 16s rRNA including SEQ ID NO: 1. A use of the bacillus amyloliquefaciens as described above is also provided, for inhibiting or killing at least one pathogenic bacteria of a group including livestock pathogenic bacteria, plant pathogenic bacteria, and aquatic pathogenic bacterium. A use of metabolites of the bacillus amyloliquefaciens as described above is further provided, for inhibiting or killing at least one pathogenic bacteria of a group including livestock pathogenic bacteria and aquatic pathogenic bacterium.

Description

Liquefied Bacillus amylolus and Liquefied Bacillus amylolus are used in the preparation of inhibitors or The use of the composition for killing pathogenic bacteria and the use of its metabolites for the preparation of the composition for inhibiting or killing pathogens

The present invention relates to a use of liquefied Bacillus amylolus and Bacillus liquefied for preparing a composition for inhibiting or killing pathogenic bacteria and the use of their metabolites for preparing a composition for inhibiting or killing pathogenic bacteria.

Plants, animals and aquatic products are susceptible to bacterial or fungal infections during their growth. However, in the past, the method of inhibiting plant, animal and aquatic infections was the use of chemically synthesized agents such as fungicides and bactericides. However, these chemically synthesized agents not only cannot be decomposed in water, but also make bacteria and fungi resistant. It is medicinal and has toxins for some organisms. This will cause pollution and harm to organisms, human bodies and the environment, and there are shortcomings that need to be improved urgently.

Therefore, it is necessary to provide a novel and progressive use of liquefied Bacillus amylolus, liquefied Bacillus amylolus for preparing compositions for inhibiting or killing pathogenic bacteria and their metabolites for preparing compositions for inhibiting or killing pathogenic bacteria. Use to solve the above-mentioned problems.

The main purpose of the present invention is to provide a use of liquefied Bacillus amylolus and Bacillus liquefied for preparing compositions for inhibiting or killing pathogenic bacteria and their metabolites for preparing compositions for inhibiting or killing pathogenic bacteria. Or kill aquatic pathogens, livestock pathogens or plant pathogens.

In order to achieve the above objective, the present invention provides a Bacillus amyloliquefaciens N43, deposited at the Food Industry Development Institute of the Consortium, with the deposit number: BCRC 910926, which includes a 16S rRNA, which includes a nucleotide sequence It is the same nucleotide sequence as SEQ ID NO:1.

In order to achieve the above-mentioned object, the present invention also provides a use of liquefied starch spore bar for preparing a composition for inhibiting or killing pathogenic bacteria. The pathogenic bacteria used for inhibiting or killing include aquatic pathogenic bacteria and livestock pathogenic bacteria. The aquatic pathogenic bacteria include the following At least one pathogen is formed into a group, and the group includes: Vibrio alginolyticus, Vibrio harveyi, Streptococcus iniae, and Lactococcus garvieae; Animal pathogens include Brachyspira hyodysenteriae.

In order to achieve the above-mentioned object, the present invention further provides a use of a metabolite of liquefied Bacillus amylovora for the preparation of a composition for inhibiting or killing pathogenic bacteria, which is used to inhibit or kill at least one pathogenic bacteria in the following constituent groups. Group includes: Aeromonas guinea pig (Aeromonas cavieae), Aeromonas veronii (Aeromonas veronii), Streptococcus iniae (Streptococcus iniae).

Figure 1 is an analysis diagram of the sensitivity of Bacillus amyloliquefaciens to antibiotics of the present invention.

Fig. 2 is an analysis diagram of the inhibitory effect of Bacillus amyloliquefaciens against Vibrio alginolyticus according to the present invention.

Fig. 3 is an analysis diagram of the inhibitory efficacy of Bacillus amyloliquefaciens against Vibrio parahaemolyticus according to the present invention.

Fig. 4 is an analysis diagram of the inhibitory efficacy of Bacillus amyloliquefaciens against Vibrio harveyi according to the present invention.

Fig. 5 is an analysis diagram of the inhibitory effect of Bacillus amyloliquefaciens on hydrophilic Aeromona hydrophila according to the present invention.

Fig. 6 is an analysis diagram of the inhibitory effect of Bacillus amyloliquefaciens on Streptococcus iniae of the present invention.

Fig. 7 is an analysis diagram of the inhibitory effect of Bacillus amyloliquefaciens on Lactococcus garvieae according to the present invention.

Fig. 8 is an analysis diagram of the inhibitory effect of the metabolites of Bacillus amyloliquefaciens against Aeromonas cavieae in the present invention.

Figure 9 is a metabolite of the present invention of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Analysis graph of inhibitory efficacy against hydrophilic Aeromona hydrophila.

Fig. 10 is an analysis diagram of the inhibitory effect of the metabolites of Bacillus amyloliquefaciens against Aeromonas veronii according to the present invention.

Fig. 11 is an analysis diagram of the inhibitory efficacy of the metabolites of Bacillus amyloliquefaciens of the present invention against Streptococcus iniae.

Fig. 12 is an analysis diagram of the test results of Bacillus amyloliquefaciens and lactic acid bacteria antibacterial protein of the present invention.

Figures 13 and 14 are graphs showing the analysis results of the test results of the inhibition of gram-negative and positive bacteria by metabolites of Bacillus amyloliquefaciens according to the present invention.

15 and 16 are diagrams showing the results of the antibacterial test of Salmonella in laying hens actually administered by Bacillus amyloliquefaciens according to the present invention.

The following examples are only used to illustrate the possible implementation aspects of the present invention, but they are not intended to limit the scope of protection of the present invention.

The present invention is a liquefied Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is deposited in the Food Industry Development Institute of the consortium, with the deposit number: BCRC 910926, and its taxonomic name is (Bacillus amyloliquefaciens, N43), which includes a 16S rRNA, the 16S rRNA includes the same nucleotide sequence as SEQ ID NO:1. The partial nucleotide sequence (SEQ ID NO: 1) of the 16S rRNA is a special and important technical feature of N43.

Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is selected from natto food and further cultivated. Bacillus amyloliquefaciens is a single colony Type and cultivation method: Use LB medium/liquid (Luria-Bertani agar/broth), cultivate at 37°C for 24 hours, then colonies can be observed.

N43 is resistant to at least one antibiotic in the following group consisting of antibiotics of Amoxicillin, Colistin and Tiamulin.

<N43 Drug Sensitivity Test>

1. Preparation of indicator bacteria solution: Take N43 liquefied Bacillus amylovora (N43, 1×10 ¹¹ CFU/g) bacterial powder, and prepare a suspension as the indicator bacteria solution (the number of bacteria is 1×10 ¹⁰ CFU/mL (per ml) The total number of bacterial colonies contained in the sample)).

2. Preparation of test culture dishes: the nutrient agar is sterilized at 122℃/20min (minutes) and then cooled to 45-50℃, and mixed with 100.00mL (ml) medium and 100.00μL (microliter) indicator bacterial solution. After homogenization (to make the cell concentration in the medium reach 1×10 ⁸ CFU/mL), pour about 20.00mL into a sterile plastic petri dish with a diameter of 9.00cm (cm) (built-in 4 stainless steel rings with an outer diameter of 8.00mm (mm)) sterilized ) And after solidification, take out the stainless steel and use it to make the sterile plastic petri dish have multiple holes.

3. Detect the sensitivity of N43 to antibiotics: take Amoxicillin, Colistin and Tiamulin with ultrapure water individually configured to 1000ppm (parts per million concentration), 500.00ppm, 250.00ppm and 100.00ppm concentration, and then filtered with a sterile 0.20μm (micron) filter membrane respectively, and then set aside. Take Amoxicillin and mycobacteria in each of the above concentrations (1000.00ppm, 500.00ppm, 250.00ppm and 100.00ppm). Inject 150.00μL of Colistin and Tiamulin antibiotics into one of the holes in the prepared test plate (take out the stainless steel sterile plastic petri dish), incubate in an incubator at 37°C for 24 hours, and observe if there is any inhibition The creation of the circle.

Please refer to Figure 1, which is the test result, showing the test dose of Amoxicillin (Amoxicillin), Colistin (Colistin) and Tiamulin at a test dose of 100.00ppm to 1000.00ppm (Tiamulin) has no obvious inhibitory effect on N43. Amoxicillin, Colistin and Tiamulin are commonly used antibiotics in feed, so N43 can be used in conjunction with the above antibiotics. To enhance the role in the intestinal tract. To further illustrate, the number corresponding to the inhibition circle on each sterile plastic petri dish in Figure 1 is expressed as the test dose concentration (upper left is 1000.00ppm, upper right is 500.00ppm, lower left is 100.00ppm, and lower right is 250.00ppm).

N43 is used to prepare a composition that inhibits or kills pathogenic bacteria, the pathogens include aquatic pathogens and livestock pathogens, the use of the liquefied Bacillus amylolus is also used to inhibit or kill plant pathogens, and the livestock pathogens include Brachyspira hyodysenteriae (Brachyspira hyodysenteriae) , As detailed below.

The aquatic pathogens include at least one/plural pathogens of the following group consisting of: Vibrio alginolyticus, Vibrio harveyi, Streptococcus iniae and lactic acid chain Lactococcus garvieae, in this way, can extensively inhibit or kill pathogenic bacteria that are common in marine, freshwater, brackish water aquaculture, etc. The aquatic pathogen further includes at least one pathogen in the following group consisting of: Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Aeromona hydrophila (Aeromona hydrophila).

The livestock pathogens include at least one/plural pathogens of the following constituent groups, and the constituent groups additionally include: Salmonella, Staphylococcus, Escherichia coli, and Clostridium perfringens.

<The efficacy test of N43 bacterial powder in inhibiting aquatic pathogens in vitro>

1. Configure N43 bacterial powder (tested N43 bacterial liquid): prepare different concentrations of N43 bacterial powder, 1.00mg/mL (concentration), 5.00mg/mL and 10.00mg/mL, and dissolve them in 1X PBS buffer solution. (0.01M phosphate buffer), obtain 1.00mg/mL (mg/mL), 5.00mg/mL and 10.00mg/mL of N43 bacterial solution and activate (overnight) at 28°C for use.

2. Dilute different pathogens to be tested: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Aeromona hydrophila, and bottle nose The pathogenic bacteria such as Streptococcus iniae and Lactococcus garvieae were diluted to about 10 ⁵ CFU/mL, and then spread on a medium suitable for the growth of these pathogens.

3. Make a well that can hold about 20 uL (microliter) of liquid on the medium coated with the pathogenic bacteria. In addition, 1X PBS was used as a negative control (Negative control).

4. Take 20.00μL of N43 bacterial solution of different concentrations (1mg/mL, 5mg/mL and 10mg/mL) and add it to the groove of the medium coated with the pathogenic bacteria (step 2), and culture it at 28°C for 24 From hours to 48 hours, observe the formation of the antibacterial ring. Perform 3 replicates for each tested N43 bacterial solution.

Please refer to Figures 2 to 4 and Table 1 that the size of the antibacterial ring produced by the tested N43 bacterial solution on different aquatic pathogens is different, to show the different degree of inhibitory efficacy of the tested N43 bacterial solution on aquatic pathogens, as described in detail below. Figures 2 to 4 show the effect of the tested N43 bacterial solution on Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi as the test concentration increases, and the antibacterial ring transparent area Larger, the concentration from left to right in each figure is 1.00mg/mL, 5.00mg/mL and 10.00mg/mL, so the higher the concentration of the tested N43 bacterial solution, the better the antibacterial effect.

Please refer to Figure 5 and Table 1. The tested N43 bacterial solution is more effective against the hydrophilic Aeromona hydrophila (Aeromona hydrophila) than against Vibrio spp. from the preliminary test results. It shows that the size of the inhibitory ring does not vary significantly with the change of the bacterial powder concentration. The size of the inhibitory ring of the tested N43 bacterial solution at a concentration of 1.00mg/mL and the tested N43 bacterial solution at a concentration of 10.00mg/mL are both about 2.00cm. , Which means that the tested N43 bacterial solution can have an inhibitory effect on the hydrophilic Aeromona hydrophila (Aeromona hydrophila) at a low concentration.

Please refer to Figures 6 to 7 and Table 1, which show that the tested N43 bacterial solution has the effect of the Gram-positive bacteria Streptococcus iniae (Figure 6) and Lactococcus garvieae (Figure 6). 7) The growth inhibition test showed that the growth of bottlenose Streptococcus iniae and Lactococcus garvieae had obvious bacteriostasis zona pellucida respectively, and the growth was larger with the increase of bacterial powder concentration. Test N43 bacterial solution has obvious growth inhibitory effect on bottlenose Streptococcus iniae and Lactococcus garvieae.

The metabolites (without microorganisms) of the liquefied Bacillus amyloliquefaciens are used to prepare a composition that inhibits or kills at least one pathogenic bacteria in the following composition groups, the composition group includes at least one pathogenic bacteria in the following composition groups, and the composition group includes ：Streptococcus iniae, Aeromonas cavieae, Aeromonas veronii and Swine dysentery Brachyspira hyodysenteriae (Brachyspira hyodysenteriae), so that it can broadly inhibit or kill pathogens commonly found in fresh water, brackish water, and livestock.

<N43 metabolites (without microorganisms) in vitro inhibition of aquatic pathogens efficacy test>

1. Configure sterilization filtrate: take 20.00mg (mg) of N43 bacterial powder, and dissolve it in 1mL (ml) reverse osmosis (RO) water, after acting at 28℃ for 30 minutes, use 0.22μm (micron) filter membrane Filter and take the filtrate for subsequent antibacterial testing.

2. A stainless steel ring is placed in the petri dish in advance to form a groove. Dilute the test pathogens such as hydrophilic Aeromona hydrophila, Streptococcus iniae, Aeromonas cavieae and Aeromonas veronii After reaching about 10 ³ -10 ⁶ CFU/mL (the dilution factor depends on the bacterial species), mix them with 20 mL of sterilized, non-solidified appropriate medium, and pour it into a petri dish (place multiple stainless steel rings) After the culture medium is solidified, take out the plurality of stainless steel rings to form a plurality of grooves.

3. Add 150μL of test solution (3mg/mL) into the groove of each medium containing different pathogenic bacteria. The test solution includes sterilization filtrate (numbered 1 to 3 in Figure 9 to Figure 10, each test solution is 3 Repeat), positive control (Positive control, broad-acting antibiotic: 0.03mg/mL chloramphenicol; number F in Figure 9 to Figure 10), negative control (Negative control, including media filtrate (Figure 9 to Figure 10) No. M on 10) and water for dissolving N43 bacterial powder). Incubate at 28°C for 24 hours to 48 hours to observe the formation of the antibacterial ring.

Please refer to Figure 8 to Figure 10 and Table 2, the metabolites of N43 against Aeromonas spp. including hydrophilic Aeromona hydrophila (Figure 9), Guinea pig Aeromonas The growth of Aeromonas cavieae (Figure 8) and Aeromonas veronii (Figure 10) has a very obvious inhibitory effect, especially for Aeromonas cavieae in guinea pigs. ) And hydrophilic Aeromona hydrophila (Aeromona hydrophila) formed a bacteriostatic ring no less than the positive control (Positive control, broad-effect antibiotic). Please refer to Figure 11 and Table 2, for the sterilization filtrate to perform a growth inhibition test on the Gram-positive bacterium Streptococcus iniae. The test shows that the sterilization filtrate is against Streptococcus iniae. The growth has an inhibitory effect, and the size of the formed bacteriostatic ring is no less than that of antibiotics. Therefore, the metabolites of N43 can inhibit aquatic pathogens and contain antibacterial substances that inhibit aquatic pathogens.

<N43 Inhibition of Spirochete Swine Surgery Bacteriostasis Test>

The medium mentioned in the following experiment is Blood Agar (Blood Agar, including Blood Agar Base, defiberized sheep blood, and diluted product); the strain is Brachyspira hyodysenteriae (Brachyspira hyodysenteriae). A total of 57 strains were isolated in the field.

N43 and lactic acid bacteria inhibitor protein were serially diluted by two-fold dilution to 8 different concentrations (diluted concentration range 62.50mg/L to 8,000mg/L). The sensitivity test is as follows:

1. Dilute the Brachyspira hyodysenteriae (Brachyspira hyodysenteriae) strains isolated from livestock farms with agar according to the standard operating procedures of the American Clinical & Laboratory Standards Institute (CLSI, 2014) for anaerobic bacteria Method to carry out the minimum concentration inhibition test.

2. Incubate for 96 hours in an anaerobic environment at 37°C for interpretation.

N43 test results: Brachyspira hyodysenteriae (Brachyspira hyodysenteriae) minimum inhibitory concentration (MIC) value distribution range is 1,000.00 ppm to 62.50 ppm, and its MIC ₅₀ and MIC ₉₀ are both 500.00 ppm, as shown in Figure 12. Therefore, when N43 is used for Brachyspira hyodysenteriae (Brachyspira hyodysenteriae) infection, it should have a good therapeutic effect.

<In vitro antibacterial test of livestock pathogens of N43 and its metabolites>

The well plate mentioned in the following experiment is a 96 well cell culture plate-U type (96 well cell culture plate-U type); culture medium: Mueller-Hinton II broth (MH II broth, BBL ^TM ); pathogenic bacteria strain: pig origin 100 strains of Salmonella and 100 strains of E. coli, 50 strains of Staphylococcus, and 29 strains of Clostridium from chicken.

1. Prepare a culture medium (MH II broth) with a concentration of 22.00g/L and sterilize it at 121°C for 15 minutes.

2. After the sterilized culture medium (MH II broth) is cooled to room temperature, add 100.00μL MH II broth to the second row to the twelfth row of the well plate.

3. Add 180.00μL culture medium (MH II broth) to the first row of the well plate, then add 20.00μL ten times diluted N43 filtrate (without microorganisms) or probiotic extract, mix evenly and draw 100.00μL to the second Well, perform a two-fold dilution of the concentration of each row in the previous row in sequence.

^{4. Add 5.00 μL of the bacterial solution (about 1.5 x 10 8} CFU/mL) of the above-mentioned pathogenic bacteria strains configured with wheat standard turbidity (0.5 McFarland 0.5) into each hole of the orifice plate.

5. Put the orifice plate in a constant temperature incubator at 37.00°C, incubate for 16 to 20 hours, and perform interpretation.

Please refer to Figure 13 to Figure 14. The above test results are based on the minimum inhibitory concentration test results. The metabolites of N43 are against both Gram-positive bacteria (Staphylococcus and Clostridium) and Gram-negative bacteria (Salmonella and Escherichia coli). It has inhibitory properties, and has high sensitivity to Staphylococcus and Clostridia, especially the best sensitivity to Clostridia.

<Salmonella test of N43 bacterial powder actually administered to laying hens>

1. Sampling samples: The feces of 20 laying hens were collected from the experimental layer farm on September 4, 102, as the basic control group before the N43 bacterial powder was administered, and the N43 bacteria were administered to the batch of chickens on September 8, 102 Powder, and the N43 bacteria has passed the Disk-diffusion and minimum inhibitory concentration (MIC) laboratory tests of the antibacterial agent, and confirmed that it can inhibit the growth of Salmonella. On September 25, 102 , October 2, 102 and October 16, 102 were collected from the same batch of chickens, the feces of 30 laying hens.

2. Salmonella isolation: Put about 10.00g (gram) of the feces collected above into Tetrathionate broth (TT), incubate at 37℃ for 18 hours; then take 100.00μL of Tetrathionate broth (TT) Tetrathionate broth (TT) broth was added to magnesium chloride malachite green broth (Rappaport-Vassiliadis R10 Broth; R10) and placed in an incubator at 42°C for 24 hours; then a sterile fishing ring was used to dip the magnesium chloride malachite green broth (Rappaport-Vassiliadis R10 Broth) was inoculated into BPLS medium (Brilliant-green phenol-red lactose sucrose agar). After culturing in a 37°C incubator for 24 hours, white colonies with a red background can be seen in the streaked area of the medium. Scrape BPLS medium The single colony above was inoculated into TSA medium (Tryptic Soy Agar) for purification, and cultured in an incubator at 37°C for 24 hours.

3. Salmonella identification: (a). Extraction of nucleic acid: Use a sterile fishing ring to scrape a single colony on TSA medium and use it according to the bacterial and fungal genome nucleic acid purification kit (Genomic DNA Purification Kit; BioKit ^® ) The steps described extract nucleic acid. (b) Polymerase chain reaction (PCR): the isolated and extracted bacterial nucleic acid is amplified with the primer team designed for Salmonella spp., and finally the product is stored at 4°C for preparation After electrophoresis analysis, the expected product size is 243bp. (c) Electrophoresis analysis: The polymerase chain reaction product (PCR product) of Salmonella spp. was electrophoresed with 2% agarose gel (Agarose gel) and then placed in ethidium bromide ( After staining with Ethidium bromide; EtBr), it was observed in a UV light box, and the result showed that Salmonella was positive. The results are shown in Figures 15-16. Two weeks after the N43 bacterial powder was administered, the Salmonella isolation rate dropped significantly from 45% to 3%, and it was stopped one week after the drug was stopped (four weeks after the N43 bacterial powder was administered) ( The separation result on October 16th) showed that the drug still had a sustainable effect. It is shown that N43 bacterial powder can inhibit the growth of Salmonella, which has reduced the number of Salmonella in the intestines of chickens, and enhances the healthy growth of chickens and the safety of laying eggs. Before the N43 bacterial powder was administered, only the feces appeared moist and soft and unshaped. The chicken feces after N43 bacteria powder are relatively dry, shaped and have distinct particles. During the separation process, it can be seen that the cultured liquid of Magnesium Chloride Malachite Green Broth (Rappaport-Vassiliadis R10 Broth) changed from a turbid light blue to a clear dark blue, indicating that the intestinal flora of the chicken was not only Salmonella but also other harmful bacteria reduced. .

The aforementioned Salmonella includes at least one pathogenic bacteria in the following group consisting of: Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Derby, Salmonella sera Type (Salmonella Schwarzengrund), Salmonella Livingstone (Salmonella Livingstone), Salmonella Newport (Salmonella Newport), Salmonella Hadar, Salmonella Albany, Salmonella Potsdam. The above-mentioned staphylococcus includes at least one pathogenic bacterium in the following group consisting of: Staphylococcus honeycomb/Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus epidermidis, Pasteurella Staphylococcus pasteuri, Staphylococcus agnetis, Staphylococcus saprophyticus, Staphylococcus cohnii.

In detail, the plant pathogen includes at least one pathogen in the following group consisting of: Rhizoctonia solani, Sclerotium rolfsii Saccardo, and Verticillium oxysporum (Fusarium oxysporum). f.sp. Cepae), Fusarium oxysporum f.sp.lycopersici, Fusarium oxysporum f.sp.pisi, and Erwinia carotovora subsp.carotovora.

N43 is a bacterial and virus bactericide used in animal feed additives, plant growth promoters, and aquaculture.

【Biological Material Deposit】

TW Food Industry Development Research Institute of the Republic of China 2019/8/20 BCRC 910926

Claims (12)

A kind of Bacillus amyloliquefaciens N43, deposited in the Food Industry Development Institute of the consortium, deposit number: BCRC 910926, which includes a 16S rRNA, the 16S rRNA includes the same nucleotide sequence as SEQ ID NO:1 Nucleotide sequence. The Bacillus amyloliquefaciens N43 according to claim 1, which is resistant to at least one antibiotic in the following group consisting of: Amoxicillin, Colistin, and The antibiotic of Tiamulin. A use of the liquefied Bacillus amylolus according to any one of claims 1 to 2 for preparing a composition for inhibiting or killing pathogenic bacteria, which is used for inhibiting or killing pathogenic bacteria, and the pathogenic bacteria include aquatic pathogens and livestock pathogens The aquatic pathogens include at least one pathogen in the following group consisting of: Vibrio alginolyticus, Vibrio harveyi, Streptococcus iniae, Streptococcus lactis (Lactococcus garvieae); The livestock pathogens include Brachyspira hyodysenteriae. The use of the liquefied Bacillus amylolique in claim 3 for preparing a composition that inhibits or kills pathogenic bacteria, wherein the aquatic pathogenic bacteria further includes at least one pathogenic bacteria in the following composition group, the composition group including: Vibrio enteritidis (Vibrio parahaemolyticus), hydrophilic Aeromona hydrophila (Aeromona hydrophila). The use of the liquefied Bacillus amylolus in claim 3 for preparing a composition for inhibiting or killing pathogenic bacteria, wherein the livestock pathogenic bacteria further includes at least one pathogenic bacteria in the following composition group, the composition group including: Salmonella, Staphylococcus, Escherichia coli, Clostridium perfringens. The use of the liquefied Bacillus amylolus according to claim 5 for preparing a composition for inhibiting or killing pathogenic bacteria, wherein the Salmonella includes at least one pathogenic bacteria in the following group consisting of: Salmonella typhimurium (Salmonella Typhimurium), Salmonella cholerae (Salmonella Choleraesuis, Salmonella Derby, Salmonella Schwarzengrund, Salmonella Livingstone, Salmonella Newport, Salmonella Hadar, Salmonella Hadar, Salmonella Schwarzengrund (Salmonella Albany), Potsdam Salmonella (Salmonella Potsdam). The use of the liquefied Bacillus amylolique in claim 5 for the preparation of a composition for inhibiting or killing pathogenic bacteria, wherein the staphylococcus includes at least one pathogenic bacterium in the following group consisting of: Staphylococcus almonds/Swine grape Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pasteuri, Staphylococcus agnetis, Staphylococcus saprophyticus, Staphylococcus Staphylococcus cohnii. The use of the liquefied Bacillus amylolus described in claim 3 for preparing a composition for inhibiting or killing pathogenic bacteria, and for inhibiting or killing plant pathogenic bacteria. The plant pathogenic bacteria includes at least one pathogenic bacteria in the following group: The group includes: Rhizoctonia solani, Sclerotium rolfsii Saccardo, Fusarium oxysporum f.sp.Cepae, Fusarium oxysporum f.sp.lycopersici ), Fusarium oxysporum f.sp.pisi, and Erwinia carotovora subsp.carotovora. The use of the liquefied Bacillus amylolus according to claim 8 for the preparation of a composition for inhibiting or killing pathogenic bacteria, which is used as an animal feed additive and a plant growth promoting agent; wherein the aquatic pathogenic bacteria include the following constituent groups plural of the pathogenic bacteria , This group includes: Vibrio parahaemolyticus, Hydrophilic Aeromona hydrophila, Aeromonas cavieae and Aeromonas veronii The livestock pathogenic bacteria includes the following group of pathogens, the group includes: Salmonella, Staphylococcus, Escherichia coli, Clostridium perfringens; The Salmonella includes the following groups A group of at least one pathogenic bacteria, the group includes: Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Derby, Salmonella Schwarzengrund , Salmonella Livingstone (Salmonella Livingstone), Salmonella Newport (Salmonella Newport), Salmonella Hadar (Salmonella Hadar), Salmonella Albany (Salmonella Albany), Salmonella Potsdam (Salmonella Potsdam); the Staphylococcus includes the following group Plural pathogenic bacteria, the group includes: Staphylococcus hyicus/Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pasteuri, Argenti Staphylococcus agnetis, Staphylococcus saprophyticus, Staphylococcus cohnii. A use of the metabolite of liquefied Bacillus amylolus according to any one of claims 1 to 2 for preparing a composition for inhibiting or killing pathogenic bacteria, which is used for inhibiting or killing at least one of the following constituent groups Pathogenic bacteria, this group includes: Aeromonas cavieae, Aeromonas veronii, Streptococcus iniae. The metabolite of the liquefied Bacillus amylolus described in claim 10 is used for the preparation of a composition for inhibiting or killing pathogenic bacteria, and also for inhibiting or killing Salmonella, and the Salmonella includes at least one pathogenic bacteria from the following composition group, the composition Groups include: Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Derby, Salmonella Schwarzengrund, Salmonella Livingstone ), Salmonella Newport, Salmonella Hadar, Salmonella Albany, Salmonella Potsdam. The metabolite of the liquefied Bacillus amylolus described in claim 10 is used for preparing a composition for inhibiting or killing pathogenic bacteria, and for inhibiting or killing staphylococci. The staphylococcus includes at least one pathogenic bacteria in the following composition group: The group includes: Staphylococcus honeycomb/Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pasteuri, Staphylococcus agnetis ), Staphylococcus saprophyticus, Staphylococcus cohnii.

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