CN115181690B - Bacillus amyloliquefaciens with antagonism to cow mastitis pathogenic bacteria and application thereof - Google Patents
Bacillus amyloliquefaciens with antagonism to cow mastitis pathogenic bacteria and application thereof Download PDFInfo
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- CN115181690B CN115181690B CN202210699668.4A CN202210699668A CN115181690B CN 115181690 B CN115181690 B CN 115181690B CN 202210699668 A CN202210699668 A CN 202210699668A CN 115181690 B CN115181690 B CN 115181690B
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 77
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K1/00—Housing animals; Equipment therefor
- A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
- A01K1/0152—Litter
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K1/00—Housing animals; Equipment therefor
- A01K1/015—Floor coverings, e.g. bedding-down sheets ; Stable floors
- A01K1/0152—Litter
- A01K1/0155—Litter comprising organic material
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention relates to the field of fecal sewage treatment, in particular to bacillus amyloliquefaciens with antagonism on cow mastitis pathogenic bacteria and application thereof. The bacillus amyloliquefaciens provided by the invention is preserved in the China general microbiological culture Collection center, and the serial number is: CGMCC No.24846. The bacillus amyloliquefaciens has antagonism on dairy cow streptococcus agalactiae, staphylococcus aureus and escherichia coli, has better cellulose, protein and starch decomposition capacity, and can be used as an inhibiting bacteria and a microbial inoculum in the production and use process of cow dung regeneration padding.
Description
Technical Field
The invention relates to the field of fecal sewage treatment, in particular to bacillus amyloliquefaciens with antagonism on cow mastitis pathogenic bacteria and application thereof.
Background
The cow dung regenerated padding is a cow dung bed padding produced by taking cow dung as a raw material and adopting the technical means of aerobic fermentation and the like, and has become an important technical means for the resource utilization of the cow dung. Compared with the traditional padding, the cow dung regenerated padding has the congenital advantages of convenient and cheap raw material acquisition, but compared with inorganic padding such as sand and the like, cow dung carries a plurality of pathogenic bacteria and is used as organic matters to cause the propagation of microorganisms more easily, so that sterilization is an indispensable step in the production process of the cow dung regenerated padding.
However, in the actual production process, cow mastitis pathogenic bacteria cannot be thoroughly killed, and in the use process of cow dung regeneration padding, in order to save cost, the padding cannot be replaced every day in a dairy farm, and pathogenic microorganisms entering the padding through cow bodies, air flow and the like in the process can gradually increase along with accumulation and reproduction, including cow mastitis pathogenic bacteria such as Streptococcus (Streptococcus), staphylococcus aureus (Staphylococcus aureus), escherichia coli and the like. Cow mastitis can lead to reduced milk yield, reduced quality, cow death and elimination, and is considered to be one of the most costly diseases in the dairy industry. Therefore, a microbial agent for inhibiting cow mastitis pathogenic bacteria, which is suitable for the production and use process of cow dung regeneration padding, is needed in the market at present.
Disclosure of Invention
The invention aims to provide bacillus amyloliquefaciens with antagonism to cow mastitis pathogenic bacteria and application thereof.
In a first aspect, the invention provides bacillus amyloliquefaciens with a preservation number of CGMCC No.24846, which can be rapidly proliferated in a cow dung environment.
The invention screens out the strains with the largest bacteriostasis circle for the streptococcus agalactiae, staphylococcus aureus and escherichia coli of cow sources from cow dung biogas residues, carries out 16s rRNA sequencing, establishes a phylogenetic tree, and determines the strains with the largest bacteriostasis circle as bacillus amyloliquefaciens DN-1 according to the 16s rRNA sequencing result, the phylogenetic tree result and morphological analysis.
The bacillus amyloliquefaciens DN-1 provided by the invention is preserved in China general microbiological culture collection center (CGMCC for short, address: north Chen Xie Lu No. 1, 3 of the area of Chaoyang in Beijing, post code 100101) in 2022, 5 and 9 days, and is classified and named: bacillus amyloliquefaciens Bacillus amyloliquefaciens with the preservation number of CGMCC No.24846.
In a second aspect, the invention provides a microbial inoculum comprising the bacillus amyloliquefaciens.
In a third aspect, the present invention provides a fermentation method, wherein the bacillus amyloliquefaciens is used as a fermentation bacterium, and cellulose, protein or starch is used as a carbon source for fermentation.
In a fourth aspect, the invention provides a bacillus amyloliquefaciens fermentation broth or fermentation supernatant, wherein the bacillus amyloliquefaciens fermentation broth is obtained by fermentation through the fermentation method; the bacillus amyloliquefaciens fermentation supernatant is obtained by centrifuging and filtering bacillus amyloliquefaciens fermentation liquid.
The use of the above bacillus amyloliquefaciens or the above microbial inoculum or the above fermentation method or the above bacillus amyloliquefaciens fermentation broth or fermentation supernatant for inhibiting the pathogenic bacteria of dairy cows' mastitis is also claimed according to the understanding of the person skilled in the art.
In the application provided by the invention, the cow mastitis pathogenic bacteria comprise cow streptococcus agalactiae, staphylococcus aureus and escherichia coli.
In the application provided by the invention, the bacillus amyloliquefaciens or the microbial inoculum or the bacillus amyloliquefaciens fermentation liquid or fermentation supernatant can inhibit cow mastitis pathogenic bacteria when the environmental temperature is 30-35 ℃.
In the application provided by the invention, the diameter of the inhibition zone of the bacillus amyloliquefaciens fermentation supernatant on the dairy cow source streptococcus agalactiae is more than 14.14 mm.
The invention also claims the application of the bacillus amyloliquefaciens or the microbial inoculum or the fermentation method or the bacillus amyloliquefaciens fermentation liquid or fermentation supernatant in preparing cow dung regeneration padding.
In a fifth aspect, the present invention provides a cow dung recycling pad, which contains the bacillus amyloliquefaciens or the microbial inoculum, or the bacillus amyloliquefaciens fermentation liquid or fermentation supernatant, or a compound microbial inoculum of bacillus and lactobacillus, bacillus subtilis and other strains, and can inhibit the increase of mastitis pathogenic bacteria in the cow dung pad.
The invention has the beneficial effects that:
the bacillus amyloliquefaciens DN-1 is separated from cow dung biogas residues, and fermentation liquor of the bacillus amyloliquefaciens DN-1 can inhibit streptococcus agalactiae, staphylococcus aureus and escherichia coli simultaneously in one treatment; the supernatant of the fermentation liquid of the bacillus amyloliquefaciens DN-1 after centrifugation can also realize the inhibition effect on streptococcus agalactiae, and the diameter of the inhibition zone of the fermentation supernatant of the bacillus amyloliquefaciens DN-1 on the streptococcus agalactiae of the dairy cow source is more than 14.14 mm.
Drawings
FIG. 1 is a phylogenetic tree of Bacillus amyloliquefaciens DN-1 provided by the invention.
FIG. 2 shows the colony morphology of Bacillus amyloliquefaciens DN-1 provided by the invention.
Fig. 3 shows a microscopic examination form of bacillus amyloliquefaciens DN-1 provided by the invention.
Fig. 4 is a photograph showing the decomposing ability of bacillus amyloliquefaciens DN-1 provided by the invention to cellulose (left), protein (middle) and starch (right).
Fig. 5 is a diagram showing the bacteriostatic effect of bacillus amyloliquefaciens DN-1 in cow dung.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention may be made without departing from the spirit and nature of the invention and are intended to be within the scope of the present invention.
Unless otherwise indicated, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are commercially available; all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art unless specifically indicated.
EXAMPLE 1 screening procedure for Bacillus amyloliquefaciens DN-1
This example provides a screening procedure for bacillus amyloliquefaciens DN-1 and the medium used.
Wherein the carboxymethyl is usedThe formula of the sodium cellulose (CMC-Na) culture medium is as follows: CMC-Na 15.0g, KH 2 PO 4 1.0g，MgSO 4 〃7H 2 O0.5 g, naCl 0.5g, agar 14.0g, 1000mL of deionized water, and natural pH value, heating to melt, sterilizing at 121deg.C for 20min, cooling to 50deg.C, and pouring into a plate.
0.9% NaCl solution: 9.0g of NaCl, 1000mL of deionized water and sterilizing at 121 ℃ for 20min.
The formula of the LB broth culture medium is as follows: 10.0g of tryptone, 5.0g,NaCl 10.0g,1000mL g of yeast extract and deionized water, and sterilizing at 121 ℃ for 20min after dissolution.
The formula of the LB agar medium is as follows: 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl, 15.0g of agar and 1000mL of deionized water, sterilizing for 15min at 121 ℃ after dissolution, and cooling to 50 ℃ to a flat plate.
The screening step comprises the following steps:
(1) 50g of cow dung biogas residues are placed in a conical flask, sealed by a sealing film and then placed in a constant temperature box for enrichment for 5d at 37 ℃.
(2) Placing 5g enriched cow dung biogas residue into a conical flask, adding 45g 0.9% NaCl solution, shaking thoroughly, and diluting 1mL supernatant with 0.9% NaCl solution to 10 -2 ～10 -5 Concentration gradient 100. Mu.L of dilutions of different concentration gradients were applied to CMC-Na medium. After inversion culture for 24 hours in a biochemical incubator, single colonies with different forms are selected for streak separation for a plurality of times until the single strains are purified.
(3) Inoculating the strain to be tested on an LB plate for 24 hours for activation, then picking the strain to be tested by an inoculating loop, inoculating the strain to be tested on an LB liquid culture medium, placing the strain to be tested in a constant-temperature shake incubator for culturing at 37 ℃ and 150r/min until the strain is in a logarithmic phase, and performing an antibacterial test.
(4) Inoculating a strain to be tested on an LB flat plate, culturing for 24 hours for activation, then picking the strain to be tested by an inoculating loop, inoculating the strain to be tested on an LB liquid culture medium, placing the LB liquid culture medium in a constant-temperature shaking incubator at 37 ℃ for culturing for 24 hours at 150r/min, centrifuging the fermentation liquor at 4 ℃ for 12 minutes at 4000r/min, filtering the supernatant after centrifugation by a 22m water-based filter membrane for 2 times, and performing an antibacterial test.
(5) Diluting 100 μL to 10 7 Pathogenicity of cfu/mLCoating bacterial liquid on an LB flat plate, punching 4 holes on the flat plate by using a puncher with the diameter of 6mm after the bacterial liquid is dried, wherein 30 mu L of LB liquid culture medium is injected into 1 hole to serve as a control group, and 30 mu L of the bacterial liquid to be tested in the step (3) or the supernatant obtained after filtering in the step (4) is injected into the other 3 holes, placing the flat plate in an ultra-clean workbench until the bacterial liquid is completely diffused, placing the flat plate at 37 ℃ for culturing for 24 hours, and measuring the bacteriostasis zone of the strain with the best bacteriostasis effect in the strain to be tested, wherein the bacteriostasis zone is shown in table 1.
TABLE 1 inhibition zone of the best bacteriostatic strain
| Streptococcus agalactiae/mm | Staphylococcus aureus/mm | Coli/mm | |
| Diameter of bacteria liquid inhibition zone | 13.16±0.09 | 11.69±0.04 | 8.22±0.04 |
| Diameter of supernatant inhibition zone | 14.14±0.99 | - | - |
And selecting the strain with the largest inhibition zone for 16s rRNA sequencing, and establishing a phylogenetic tree, wherein the phylogenetic tree is shown in figure 1. The strain with the largest inhibition zone is determined to be bacillus amyloliquefaciens and named as bacillus amyloliquefaciens DN-1 by the 16s rRNA sequencing result, the phylogenetic tree result and morphological analysis. Colony morphology and microscopic examination morphology of bacillus amyloliquefaciens DN-1 are shown in FIG. 2 and FIG. 3.
Example 2 Bacillus amyloliquefaciens DN-1 has multiple substance decomposing Capacity
This example provides a test for the multiple substance decomposing ability of Bacillus amyloliquefaciens DN-1, using the following media and procedure.
The formula of the protein culture medium is as follows: beef extract 3g, casein 10g, naCl 5g, K 2 HPO 4 2g, bromothymol blue 0.05g, agar powder 15g, deionized water 1000mL, pH value of 7.3-7.5, sterilizing at 121 ℃ for 20min, and pouring the mixture into a flat plate after cooling to 50 ℃.
Congo red staining solution: congo red 1g, deionized water 1000mL.
Decolorization liquid: 58.5g NaCl, 1000mL deionized water.
The formula of the starch culture medium is as follows: 10.0g of peptone, 3.0g of beef extract powder, 5.0g of NaCl, 30.0g of soluble starch, 15.0g of agar and pH 7.8.
The testing method comprises the following steps:
inoculating Bacillus amyloliquefaciens DN-1 selected in example 1 onto LB plate, activating at 37deg.C for 24 hr, inoculating to LB liquid medium, culturing at 37deg.C for 3 hr/min to turbidity, diluting with 0.9% NaCl solution at 10 times gradient, collecting 10 -4 ～10 -6 100. Mu.L of the dilution of (C) was spread on CMC-Na plates, protein medium plates or starch medium plates, and incubated at 37℃for 48 hours.
Clear circles were formed around bacterial colonies having proteolytic or amylolytic capabilities as shown in FIG. 4, and colony diameters and transparent circle diameters were measured with a vernier caliper and recorded, and the results are shown in Table 2.
The method for measuring the cellulose decomposing ability comprises the following steps: selecting a plate for forming clear single colonies, injecting 10mL of Congo red dye liquor, standing for 15min to pour the dye liquor, injecting 10mL of decolorizing liquor, standing for 15min to pour the decolorizing liquor, forming obvious transparent rings around bacterial colonies with cellulose decomposing capacity, measuring the colony diameter D and the transparent ring diameter D by using a vernier caliper, recording, and judging the cellulose decomposing capacity by using D/D generally, wherein the cellulose decomposing capacity is shown in Table 2.
TABLE 2 multiple substance decomposing Capacity of Bacillus amyloliquefaciens DN-1
| Nutrient source variety | Colony diameter d/mm | Diameter D/mm of decomposing ring | D/d |
| Cellulose | 6.02±0.22 | 14.11±0.32 | 2.34 |
| Proteins | 11.81±0.49 | 23.77±0.58 | 2.01 |
| Starch | 6.36±0.11 | 9.47±0.35 | 1.44 |
Example 3 optimal bacteriostatic temperature of Bacillus amyloliquefaciens DN-1
The embodiment provides the optimal antibacterial temperature of bacillus amyloliquefaciens DN-1, and the specific exploration steps are as follows:
diluting 100L to 10 7 After the cfu/mL of pathogenic bacteria liquid is coated on an LB flat plate, 5 holes are punched on the flat plate by a puncher with the diameter of 6mm after the bacteria liquid is dried, wherein 30L of LB liquid culture medium is injected into 1 hole to serve as a control group, the other 4 holes are injected with bacillus amyloliquefaciens DN-1 bacteria liquid, the bacteria liquid is placed in an ultra-clean workbench until the bacteria liquid is completely diffused, the flat plate is respectively placed at 20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃ and 50 ℃ for culturing for 24 hours, and a bacteriostasis zone is measured, and the table 3 is shown.
It should be noted that, at a temperature of 20 ℃,25 ℃,40 ℃, and 45 ℃, streptococcus agalactiae is not a proper growth temperature, and the streptococcus agalactiae grows too slowly or stops growing or is killed, so that the inhibition zone of the streptococcus agalactiae cannot be read.
TABLE 3 antibacterial circle of Bacillus amyloliquefaciens DN-1 at different temperatures
| Temperature/. Degree.C | Streptococcus agalactiae/mm | Coli/mm | Staphylococcus aureus/mm |
| 20 | - | 8.99 | 9.49 |
| 25 | - | 10.65 | 13.31 |
| 30 | 15.16 | 10.78 | 13.14 |
| 35 | 13.54 | 10.24 | 11.82 |
| 40 | - | 8.42 | 12.48 |
| 45 | - | - | 13.14 |
EXAMPLE 4 Co-cultivation of Bacillus amyloliquefaciens DN-1 with pathogenic bacteria in liquid
The present example provides the bacteriostatic ability of bacillus amyloliquefaciens DN-1 in LB broth, and the specific investigation steps are as follows:
activating Staphylococcus aureus, escherichia coli, streptococcus agalactiae, DN-1 in LB nutrient agar medium, inoculating respectively into LB broth medium, culturing at 37deg.C and 160rpm for about 3 hr to turbidity, centrifuging 4 bacteria at 4deg.C and 4000rpm for 6min, and re-suspending with LB broth medium for 2 times, wherein 3 pathogenic bacteria are diluted to 10 with LB broth medium 7 cfu/g is reserved.
The test of mixed culture of pathogenic bacteria and target bacteria is carried out, wherein the test group is that 2.5mL of LB broth culture medium, 25 mu L of staphylococcus aureus bacterial liquid, 25 mu L of escherichia coli bacterial liquid, 25 mu L of streptococcus agalactiae bacterial liquid and 2.5mL of DN-1 bacterial liquid are added into a 1-50 mL centrifuge tube. The control group is that 5mL of LB broth culture medium, 25 mu L of staphylococcus aureus bacterial liquid, 25 mu L of escherichia coli bacterial liquid and 25 mu L of streptococcus agalactiae bacterial liquid are added into a 1-50 mL centrifuge tube, and the culture is carried out for 3-5 hours, and when the bacterial liquid is turbid, the bacterial liquid stops.
Three replicates were arranged for each treatment group. And 3d, detecting the number of pathogenic bacteria, wherein the detection method comprises the following steps: taking a proper amount of a sample to be tested, diluting with 0.9% NaCl solution in a gradient manner by 10 times, selecting 100 mu L of bacterial liquid with a proper gradient, respectively coating the bacterial liquid on a Maiconk agar medium (the escherichia coli is a pink colony), an improved Edwardsiella culture medium (the streptococcus is a blue-green colony), a Baird-Parker agar medium (the staphylococcus aureus is a black colony), inversely culturing for 18-24 hours at 37 ℃, selecting plates with proper colony numbers for counting, and calculating the number of pathogenic bacteria, wherein the inhibition rate of bacillus amyloliquefaciens on the pathogenic bacteria is shown in Table 4.
TABLE 4 inhibition of pathogenic bacteria by Bacillus amyloliquefaciens in LB broth
| Streptococcus agalactiae | Coli bacterium | Staphylococcus aureus | |
| Inhibition rate | 99% or more | 95% or more | - |
Example 5 Bacillus amyloliquefaciens DN-1 used as cow dung regeneration pad for cow dung production
The embodiment provides the bacteriostatic ability of bacillus amyloliquefaciens DN-1 in cow dung, and the specific exploration steps are as follows:
activating Staphylococcus aureus, escherichia coli, streptococcus agalactiae, DN-1 in LB nutrient agar medium, inoculating respectively in LB broth medium, culturing at 37deg.C and 160rpm for about 3 hr to turbidity, centrifuging 4 bacteria at 4deg.C and 4000rpm for 6min, and re-suspending with 0.9% NaCl solution for 2 times, wherein pathogenic bacteria is diluted to 10 with 0.9% NaCl solution 3 cfu/g was used, DN-1 was centrifuged again, and 20mL of the supernatant was discarded and concentrated to 3mL.
20g of cow dung is put into a conical flask, sterilized for 20min at 121 ℃, 3mL of concentrated DN-1 bacterial liquid is added after cooling, 0.2mL of each of three pathogenic bacteria is added, DN-1 bacterial liquid is changed into 0.9% NaCl solution by a control group, other conditions are kept unchanged, 3 treatments are arranged in parallel, and the culture is carried out for 3 days. Taking a proper amount of a sample to be tested, diluting with 0.9% NaCl solution in a gradient manner by 10 times, selecting 100 mu L of bacterial liquid with a proper gradient, respectively coating the bacterial liquid on a Maiconk agar medium (the escherichia coli presents a pink colony), a modified Edwardsiella culture medium (the streptococcus presents a blue-green colony), a Baird-Parker agar medium (the staphylococcus aureus presents a black colony), inversely culturing for 18-24 hours at 37 ℃, selecting a flat plate with a proper colony number for counting, and calculating the pathogenic bacteria inhibition rate based on a control group, wherein the pathogenic bacteria inhibition rate is shown in Table 5.
TABLE 5 inhibition of pathogenic bacteria in cow dung by Bacillus amyloliquefaciens
| Streptococcus agalactiae | Coli bacterium | Staphylococcus aureus | |
| Inhibition rate | 99% or more | More than 85 percent | - |
Example 6 potential of Bacillus amyloliquefaciens DN-1 for cow dung production of cow dung regeneration padding
The embodiment provides the bacteriostatic ability of bacillus amyloliquefaciens DN-1 in cow dung, and the specific exploration steps are as follows:
activating Staphylococcus aureus, streptococcus agalactiae and DN-1 in LB nutrient agar medium, inoculating respectively in LB broth medium, culturing at 37deg.C and 160rpm for about 3 hr until turbidity, regulating concentration of Staphylococcus aureus and Streptococcus agalactiae, and adding into 60g sterilized cow dung to make concentration of Staphylococcus aureus and Streptococcus agalactiae respectively 10 8 cfu/g and 10 7 Taking 30mL of cfu/g and DN-1 bacterial liquid, centrifuging at 4 ℃ and 4000rpm for 6min, discarding supernatant, concentrating 30mL of bacterial liquid to 6mL, adding the concentrated bacterial liquid and pathogenic bacterial liquid into cow dung, replacing DN-1 bacterial liquid by 6mL of LB broth culture medium in a control group, keeping the other conditions consistent, setting three parallel groups in each treatment group, detecting the number of streptococcus agalactiae and staphylococcus aureus after 2d, and calculating the bacteriostasis rate based on the control group, wherein the bacteriostasis rate is shown in figure 5.
The streptococcus agalactiae and staphylococcus aureus in the cow dung of the treatment group are lower than those of the control group, and the reduction of the streptococcus agalactiae and staphylococcus aureus in the cow dung of the treatment group is 83.2% and 28.6% respectively.
Example 7 Bacillus amyloliquefaciens DN-1 used as cow dung regeneration pad for cow dung production
1. The embodiment provides the bacteriostatic ability of bacillus amyloliquefaciens DN-1 in cow dung, and the specific exploration steps are as follows:
400g of fresh cow dung is placed in a container with an effective volume of 1L, a test group is inoculated with microorganism bacteria liquid in a logarithmic phase according to the inoculum size of 3%V/W, a control group is added with sterile water according to 3%V/W, a specific test scheme is shown in a table 6, constant temperature culture is carried out at 30 ℃, the numbers of three pathogenic bacteria are detected by sampling at 0,4,8 and 12d, the residual ratio of the pathogenic bacteria is calculated based on the 0 th day, and the results are shown in a table 7, a table 8 and a table 9.
Table 6 inoculum and volume
TABLE 7 Staphylococcus aureus residual Rate
TABLE 8 Escherichia coli residual Rate
TABLE 9 Streptococcus agalactiae residual Rate
2. The embodiment provides the bacteriostasis capability of bacillus amyloliquefaciens and other strains in cow dung, and the specific exploration steps are as follows:
400g of fresh cow dung is put into a container with an effective volume of 1L, bacillus amyloliquefaciens DN-1 and bacillus (with the preservation number of CGMCC No. 24847) are compounded, an experimental group is connected with a microbial liquid in a logarithmic growth phase according to the inoculation amount of 3%V/W, a control group is added with sterile water according to 3%V/W, the specific test scheme is shown in a table 10, the constant temperature culture is carried out at 30 ℃, the numbers of three pathogenic bacteria are detected by sampling at 0,4 and 8d, and the residual ratio of the pathogenic bacteria is calculated based on the day 0, and the results are shown in a table 11, a table 12 and a table 13.
The bacillus provided by the invention is preserved in China general microbiological culture collection center (CGMCC for short, address: north Chen Xi Lu No. 1, 3 of the area of Charpy, beijing, china academy of sciences of microorganisms, post code 100101) in 2022, 5, 9 days, and is named after classification: bacillus sp, with preservation number of CGMCC No.24847.
Table 10 inoculum and volume
TABLE 11 residual Rate of Staphylococcus aureus
TABLE 12 Escherichia coli residual Rate
TABLE 13 Streptococcus agalactiae residual Rate
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. The bacillus amyloliquefaciens is characterized in that the preservation number is CGMCC No.24846, and the bacillus amyloliquefaciens can be rapidly proliferated in a cow dung environment.
2. A microbial agent comprising the Bacillus amyloliquefaciens of claim 1.
3. A fermentation method, wherein the Bacillus amyloliquefaciens of claim 1 is used as a fermentation medium, and cellulose or starch is used as a carbon source.
4. A bacillus amyloliquefaciens fermentation broth or fermentation supernatant, characterized in that the bacillus amyloliquefaciens fermentation broth is obtained by fermentation according to the fermentation method of claim 3; the bacillus amyloliquefaciens fermentation supernatant is obtained by centrifuging and filtering bacillus amyloliquefaciens fermentation liquid.
5. Use of the bacillus amyloliquefaciens of claim 1 or the microbial inoculum of claim 2 or the fermentation process of claim 3 or the bacillus amyloliquefaciens fermentation broth or fermentation supernatant of claim 4 for inhibiting bovine mastitis pathogenic bacteria;
the cow mastitis pathogenic bacteria are cow streptococcus agalactiae.
6. The use according to claim 5, wherein the bacillus amyloliquefaciens or the microbial inoculum or the bacillus amyloliquefaciens fermentation liquid or fermentation supernatant has the effect of inhibiting cow mastitis pathogenic bacteria at the environmental temperature of 30-35 ℃.
7. Use of the bacillus amyloliquefaciens of claim 1 or the microbial inoculum of claim 2 or the fermentation method of claim 3 or the bacillus amyloliquefaciens fermentation broth or fermentation supernatant of claim 4 for preparing cow dung regeneration padding.
8. The cow dung regenerated padding is characterized by being capable of inhibiting the increase of mastitis pathogenic bacteria in the cow dung padding; the cow dung regenerated padding contains the bacillus amyloliquefaciens of claim 1 or the microbial inoculum of claim 2 or the bacillus amyloliquefaciens fermentation liquid or fermentation supernatant of claim 4.
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| CN103555613A (en) * | 2013-10-15 | 2014-02-05 | 广西科技大学 | Bacillus subtilis having antagonistic effect on Streptococcus agalactiae and application thereof |
| CN108676756A (en) * | 2018-06-04 | 2018-10-19 | 中国水产科学研究院珠江水产研究所 | Bei Laisi bacillus and its application as aquatic pathogenic bacterium inhibitor |
| CN110150478A (en) * | 2019-06-28 | 2019-08-23 | 青岛宝创生物科技有限公司 | A kind of feed addictive and the preparation method and application thereof reducing mastitis for milk cows disease incidence |
| CN115322921A (en) * | 2022-06-20 | 2022-11-11 | 中国农业大学 | Bacillus having antagonistic action on pathogenic bacteria of cow mastitis and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103555613A (en) * | 2013-10-15 | 2014-02-05 | 广西科技大学 | Bacillus subtilis having antagonistic effect on Streptococcus agalactiae and application thereof |
| CN108676756A (en) * | 2018-06-04 | 2018-10-19 | 中国水产科学研究院珠江水产研究所 | Bei Laisi bacillus and its application as aquatic pathogenic bacterium inhibitor |
| CN110150478A (en) * | 2019-06-28 | 2019-08-23 | 青岛宝创生物科技有限公司 | A kind of feed addictive and the preparation method and application thereof reducing mastitis for milk cows disease incidence |
| CN115322921A (en) * | 2022-06-20 | 2022-11-11 | 中国农业大学 | Bacillus having antagonistic action on pathogenic bacteria of cow mastitis and application thereof |
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