WO2020179999A1 - Bacillus subtilis cjbs303 et composition le comprenant - Google Patents

Bacillus subtilis cjbs303 et composition le comprenant Download PDF

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Publication number
WO2020179999A1
WO2020179999A1 PCT/KR2020/000133 KR2020000133W WO2020179999A1 WO 2020179999 A1 WO2020179999 A1 WO 2020179999A1 KR 2020000133 W KR2020000133 W KR 2020000133W WO 2020179999 A1 WO2020179999 A1 WO 2020179999A1
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WIPO (PCT)
Prior art keywords
strain
bacillus subtilis
cjbs303
composition
microbial preparation
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PCT/KR2020/000133
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English (en)
Korean (ko)
Inventor
김유진
박민아
오은선
우서형
Original Assignee
씨제이제일제당(주)
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Priority claimed from KR1020200000489A external-priority patent/KR102299266B1/ko
Application filed by 씨제이제일제당(주) filed Critical 씨제이제일제당(주)
Priority to CN202080002103.5A priority Critical patent/CN112074598B/zh
Priority to EP20767389.8A priority patent/EP3766960A4/fr
Priority to CA3095265A priority patent/CA3095265C/fr
Priority to US17/043,463 priority patent/US20230095303A1/en
Priority to BR112020022140-6A priority patent/BR112020022140A2/pt
Priority to JP2020552202A priority patent/JP7068490B2/ja
Publication of WO2020179999A1 publication Critical patent/WO2020179999A1/fr
Priority to PH12020551799A priority patent/PH12020551799A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
    • B01D53/84—Biological processes
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

Definitions

  • the present application relates to Bacillus subtilis CJBS303 having an effect of reducing odor derived from livestock manure and a composition comprising the same.
  • odors occurring in livestock farms are a cause of environmental pollution, as well as an increase in civil complaints and diseases in neighboring villages, and have become a major social problem.
  • the main cause of odor in livestock farms is gas generated by anaerobic fermentation during the production of livestock manure and storage and treatment.
  • the Ministry of Environment and Korea Handon Association designated 12 odor substances, including ammonia, methyl mercaptan, and hydrogen sulfide, and in 2010, the final 22 designated odor substances and emission standards were established.
  • the Korea Handy Pig Association has suggested the standard for odor management of livestock farms, and as one of these, the dilution factor of the complex odor inside the farm is 1000 times. It is demanding the following management.
  • the term “complex odor” refers to a odor that causes discomfort and disgust by stimulating a person's sense of smell while having two or more odor substances in combination.
  • Ammonia (NH 3 ), which is one of the representative harmful gases and odorous substances in pig houses, is an irritating, colorless, fluid gas that can be detected even at a relatively low concentration.
  • Ammonia is known to be produced in the process of amino acid deamination, and other sources of ammonia include urea and nitric acid.
  • Ammonia gas irritates the moist body tissues, sore eyes of workers and congestion of pigs' eyes. According to the study data on the effect of pig productivity according to the concentration of ammonia in the pig house, when ammonia is 50 ppm or more, about 12% of the piglets are delayed in growth, and at 100-150 ppm, 30% of pigs inhibit growth and show symptoms of respiratory disease. .
  • Another major malodorous gas is volatile sulfur compounds, such as hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH). These volatile sulfur compounds are produced in the process of reducing sulfates and metabolizing amino acids containing sulfur.
  • Hydrogen sulfide is a toxic gas, and is heavier than air and is known to be laid on the surface of pig slurries.It causes headaches, dizziness, and nausea of workers, and in pigs, it causes loss of appetite and respiratory diseases.
  • odor gases from livestock farms include indole and phenols such as indole, scatol, and p-cresol, and volatile fatty acids (VOCs) such as propionic acid, butyric acid, and acetic acid. Development of technology to reduce the generation of these odor gases is required. It is a necessary situation.
  • Patent Document 1 Korean Patent Registration No. 10-1229865
  • One object of the present application is to provide a Bacillus subtilis CJBS303 strain deposited under accession number KCCM12435P.
  • Another object of the present application is to provide a composition comprising the Bacillus subtilis CJBS303 strain, a culture of the strain, a concentrate of the culture, or a dried product of the concentrate.
  • Another object of the present application is to provide a feed composition comprising the composition.
  • Another object of the present application is to provide a microbial preparation for removing odor from livestock houses including the Bacillus subtilis CJBS303 strain.
  • Another object of the present application is to provide a method for reducing odor of individual manure, comprising administering the Bacillus subtilis CJBS303 strain, the composition, or the microbial agent to the individual.
  • Another object of the present application is the step of culturing the Bacillus subtilis CJBS303 ( Bacillus subtilis CJBS303) strain deposited as KCCM12435P; And mixing the recovered strain with an additive; to provide a method for producing a microbial preparation comprising.
  • the present application provides a Bacillus subtilis CJBS303 strain deposited with accession number KCCM12435P.
  • the strain may be one having an effect of reducing odor derived from livestock manure.
  • the strain may reduce the level of at least one malodorous gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indole, phenols, and volatile fatty acids (VOCs).
  • malodor derived from livestock manure may be used interchangeably with “malodor derived from livestock manure", or "malodor derived from livestock manure”.
  • the malodor may be generated from malodorous gas derived from livestock manure, for example, may be generated from malodorous gas caused by anaerobic fermentation during the processing of livestock manure.
  • the CJBS303 strain having the highest ammonia consumption rate was selected from a total of 456 strains isolated from samples such as doenjang, red pepper paste, and meju, which are Korean traditional pastes. 16s rDNA sequencing was performed on the selected strain to confirm the nucleotide sequence of SEQ ID NO: 1, which showed 99% homology with Bacillus subtilis. Therefore, the strain was named Bacillus subtilis CJBS303, and the Bacillus subtilis CJBS303 strain was deposited with the Korea Microbial Conservation Center on February 13, 2019, and the accession number KCCM12435P was given.
  • the Bacillus subtilis CJBS303 strain may reduce the level of odor gas derived from livestock manure.
  • the odor gas is ammonia; Hydrogen sulfide; Methyl mercaptan; Indole and phenols such as indole, scatol, and p-cresol; And volatile fatty acids (VOCs) such as propionic acid, butyric acid, and acetic acid.
  • VOCs volatile fatty acids
  • it may be at least one odor gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indole, phenols, and volatile fatty acids. .
  • the addition of the Bacillus subtilis CJBS303 strain reduced the level of ammonia derived from swine feces slurry, and the feeding of the strain also reduced the level of ammonia and hydrogen sulfide derived from manure. Accordingly, the present application can provide a new strain having an effect of reducing odor derived from livestock manure.
  • the present application provides a composition comprising the Bacillus subtilis CJBS303 strain, a culture of the strain, a concentrate of the culture, or a dried product of the concentrate.
  • Bacillus subtilis CJBS303 strain is as described above.
  • the culture of the Bacillus subtilis CJBS303 strain is prepared by inoculating the strain in a microorganism culture medium and culturing microorganisms known in the art (eg, stationary culture, stirring culture). I can.
  • concentrates of the strain culture, and dried products thereof can also be easily prepared according to a method of treating, concentrating or drying microorganisms or cultures known in the art.
  • feeding of the Bacillus subtilis CJBS303 strain reduces the levels of ammonia and hydrogen sulfide derived from manure, thereby contributing to the improvement of the livestock environment and the productivity of livestock.
  • the Bacillus subtilis CJBS303 strain, a culture product of the strain, a concentrate of the culture, or a dried product of the concentrate may be included in the composition and used as a feed additive.
  • composition of the present application may be prepared in powder or granular form, and if necessary, organic acids such as citric acid, humic acid, adipic acid, lactic acid, malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate, , Polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and other natural antioxidants, or any one or more of them may be further included.
  • organic acids such as citric acid, humic acid, adipic acid, lactic acid, malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate, , Polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and other natural antioxidants
  • composition of the present application includes grains such as crushed or crushed wheat, oats, barley, corn and rice; Vegetable protein feeds such as feeds based on rape, soybeans, and sunflowers; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include a dry ingredient composed of sugar and dairy products, for example, various milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.
  • composition of the present application may be administered to an animal alone or may be administered in combination with other feed additives in an edible carrier.
  • the composition may be easily administered to animals as a top dressing, or directly mixed with the feed or in an oral formulation separate from the feed.
  • a pharmaceutically acceptable edible carrier as well known in the art.
  • Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol.
  • a solid carrier the composition may be a tablet, capsule, powder, troche or sugar-containing tablet, or a top dressing in a microdispersible form.
  • a liquid carrier is used, the composition may be a gelatin soft capsule, or a formulation of a syrup or suspension, emulsion, or solution.
  • composition of the present application may contain, for example, a preservative, a stabilizer, a wetting or emulsifying agent, a solution accelerator, a cryoprotectant, or an excipient.
  • the cryoprotectant may be one or more selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder, and starch.
  • the preservative, stabilizer, or excipient may be included in the composition in an effective amount sufficient to reduce deterioration of the Bacillus subtilis CJBS303 strain contained in the composition.
  • the cryoprotectant may be included in the composition in an effective amount sufficient to reduce the degradation of the Bacillus subtilis CJBS303 strain contained in the composition when the composition is in a dried state.
  • the composition may be used by salivating, spraying or mixing and adding to animal feed.
  • composition of the present application can be applied to a plurality of animal diets, including mammals and poultry.
  • mammals and poultry As the mammal, it can be used for pigs, cows, sheep, goats, laboratory animals, or pets (eg, dogs, cats), and can also be used for chickens, turkeys, ducks, geese, pheasants, and quails as the poultry. have.
  • the present application provides a feed composition comprising the composition in another aspect.
  • the feed composition of the present application may be formulated in a conventional feed form, and may include a common feed ingredient.
  • feed composition refers to food fed to animals.
  • the feed composition refers to a material that supplies organic or inorganic nutrients necessary for maintaining animal life or producing meat, milk, and the like.
  • the feed composition may additionally contain nutrients necessary for maintaining animal life or producing meat, milk, and the like.
  • the content of the composition including the Bacillus subtilis CJBS303 strain, the culture of the strain, the concentrate of the culture, or a dried product of the concentrate contained in the feed composition of the present application is the type and age of the applied livestock, the application type, It can be appropriately adjusted according to the desired effect, for example, 0.01 to 1% (w/w), 0.01 to 0.5% (w/w), or 0.15 to 0.5% (w/w).
  • the feed composition of the present application may include the Bacillus subtilis CJBS303 strain in an amount of 1.0 ⁇ 10 7 to 1.0 ⁇ 10 11 cfu/kg based on the total weight of the composition.
  • the content of the strain is 1.0 ⁇ 10 7 to 5.0 ⁇ 10 10 , 1.0 ⁇ 10 7 to 1.0 ⁇ 10 10 , 1.0 ⁇ 10 7 to 5.0 ⁇ 10 9 , 1.0 ⁇ 10 7 to 1.0 ⁇ 10 9 , 1.0 ⁇ 10 7 to 5.0 ⁇ 10 8 , 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 , 1.0 ⁇ 10 7 to 5.0 ⁇ 10 7 , 1.0 ⁇ 10 8 to 5.0 ⁇ 10 10 , 1.0 ⁇ 10 8 to 1.0 ⁇ 10 10 , 1.0 ⁇ 10 8 to 5.0 ⁇ 10 9 , 1.0 ⁇ 10 8 to 1.0 ⁇ 10 9 , 1.0 ⁇ 10 8 to 5.0 ⁇ 10 8 , 1.0 ⁇ 10 9 to 5.0 ⁇ 10 10 , 1.0 ⁇ 10 9 to 1.0 ⁇ 10 10 , or 1.0 ⁇ 10 9 to 5.0 ⁇ 10 9
  • the feed composition of the present application further includes organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid for administration; Phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate; One or more of natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, and tannic acid can be mixed and used, and other conventional additives such as anti-influenza agents, buffers, and bacteriostatic agents are added as needed. can do.
  • organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid for administration
  • Phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate
  • One or more of natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, and tannic acid can be mixed and used, and other conventional additives such as anti-influenza agents, buffers, and
  • the feed composition of the present application may be formulated as an injectable formulation such as an aqueous solution, suspension, emulsion, capsule, granule or tablet by additionally adding a diluent, a dispersant, a surfactant, a binder or a lubricant.
  • the feed composition of the present application includes various auxiliary agents such as amino acids, inorganic salts, vitamins, antioxidants, antifungal agents, and antibacterial agents as auxiliary ingredients, and vegetable protein feed such as crushed or crushed wheat, barley, corn, blood meal, meat meal, fish meal.
  • the main ingredients such as animal protein feed, animal fat and vegetable fat, it can be used together with nutritional supplements, growth promoters, digestion and absorption promoters, and disease prevention agents.
  • the feed composition of the present application may be mixed with livestock feed in an amount of about 10 to 500 g per 1 kg, for example, 10 to 100 g, based on dry weight, and supplied as a mash after thoroughly mixing, or an additional processing process Through the palletization, expansion or extrusion process can be carried out.
  • the present application provides a microbial preparation for removing odor from livestock houses including the Bacillus subtilis CJBS303 strain.
  • feeding of the Bacillus subtilis CJBS303 strain was able to reduce the levels of ammonia and hydrogen sulfide derived from manure, and significantly reduced ammonia, hydrogen sulfide, methylmercaptan, volatile organic compounds, and complex odors in the shed.
  • the strain of the present application can be utilized as a microbial preparation for removing odor in livestock houses.
  • the microbial preparation of the present application may be to reduce the level of at least one malodorous gas selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, volatile fatty acids, and combinations thereof.
  • the microbial preparation of the present application may be in the form of a solid or liquid prepared according to a method known in the art.
  • the microbial preparation of the present application is, for example, in a solid form, after attaching the strain to a carrier, dried so that the moisture content is 0.1 to 10% by weight based on the total weight of the microbial preparation, and in the form of beads It can be commercialized or pulverized to be commercialized in powder form.
  • At least one selected from the group consisting of powdery clay, activated carbon, coke, volcanic ash, and combustion ash may be used, and as the clay, zeolite, vermiculite, diatomaceous earth, kaolin, pottery earth, feldspar, and clay And one or more selected from the group consisting of talc may be used, but is not limited thereto.
  • an excipient may be added to the microbial preparation in the solid form. In the case of the excipient, amino acids, vitamin C, vitamin E, chitosan, and glucose may be used alone or in combination of two or more, but are not limited thereto.
  • the microbial preparation of the present application when the microbial preparation of the present application is, for example, in a liquid form, it may be prepared by mixing the culture of the strain and mixing glucose or glycerin to stabilize the microorganism. Based on the total weight of the microbial preparation, it may be prepared so that the final concentration of the culture is 5 to 40% by weight, for example, it may be prepared so that the final concentration is 10 to 20% by weight, but is not limited thereto.
  • the present application provides a method for reducing odor of individual manure, comprising administering the Bacillus subtilis CJBS303 strain, the composition, or a microorganism preparation to the individual.
  • the term "individual” refers to an object to reduce the odor of manure, and more specifically, may be a non-human animal.
  • the non-human animal may include mammals, and poultry, and the mammal may include pigs, cows, sheep, goats, dogs, cats, etc., and may include laboratory rodents or pets.
  • the poultry may include, but is not limited to, chicken, turkey, duck, goose, pheasant, and quail.
  • the present application is a step of culturing a Bacillus subtilis CJBS303 strain deposited as KCCM12435P; And it provides a method for producing a microbial preparation comprising; and mixing the recovered strain with an additive.
  • the cultivation of the strain may be performed according to a method known in the art, and the cultivation method may include one or more cultivation selected from the group consisting of batch, continuous, and fed-batch culture.
  • the medium used for the cultivation may be a medium capable of satisfying the requirements of specific microorganisms.
  • the medium may be a medium selected from the group consisting of a carbon source, a nitrogen source, a trace element component, and a combination thereof.
  • the carbon source may be a carbon source selected from the group consisting of carbohydrates, fats, fatty acids, alcohols, organic acids, and combinations thereof.
  • the carbohydrate may be glucose, sucrose, lactose, fructose, maltose, starch, cellulose, and combinations thereof.
  • the fat may be soybean oil, sunflower oil, pajama oil, coconut oil, and combinations thereof.
  • the fatty acid may be palmitic acid, stearic acid, linoleic acid, or a combination thereof.
  • the alcohol may be glycerol or ethanol.
  • the organic acid may include acetic acid.
  • the nitrogen source may include an organic nitrogen source, an inorganic nitrogen source, or a combination thereof.
  • the organic nitrogen source may be selected from the group consisting of peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), soybean meal, and combinations thereof.
  • the inorganic nitrogen source may be selected from the group consisting of urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, and combinations thereof.
  • the medium may include one selected from the group consisting of phosphorus, metal salts, amino acids, vitamins, precursors, and combinations thereof.
  • the source of phosphorus may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt corresponding thereto.
  • the metal salt may be magnesium sulfate or iron sulfate.
  • the medium or individual components constituting the same may be added in batch, continuous or fed-batch culture.
  • the method of preparing the microbial preparation may further include recovering the strain from the culture obtained by culturing the strain after the step of culturing the strain. Recovery of the strain may be performed by a method known in the art, for example, may be performed by a method of recovering the precipitate generated after centrifugation of the culture.
  • the additive may be a cryoprotectant, and after the step of mixing the recovered strain with the additive, it may further include a step of freeze-drying the obtained mixture.
  • the freeze drying may be performed by a method known in the art.
  • the strain in the freeze-dried microbial preparation obtained in the freeze-drying step may be in a viable state.
  • the freeze-dried microbial preparation may include a cryoprotectant in an effective amount sufficient to reduce the degradation of the strain contained in the formulation, thereby reducing the degradation of the viable strain.
  • the microbial preparation may include a Bacillus subtilis CJBS303 strain of 1.0 ⁇ 10 7 to 1.0 ⁇ 10 11 cfu/kg based on the total weight of the microbial preparation, eg For example, 1.0 ⁇ 10 7 to 5.0 ⁇ 10 10 , 1.0 ⁇ 10 7 to 1.0 ⁇ 10 10 , 1.0 ⁇ 10 7 to 5.0 ⁇ 10 9 , 1.0 ⁇ 10 7 to 1.0 ⁇ 10 9 , 1.0 ⁇ 10 7 to 5.0 ⁇ 10 8 , 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 , 1.0 ⁇ 10 7 to 5.0 ⁇ 10 7 , 1.0 ⁇ 10 8 to 5.0 ⁇ 10 10 , 1.0 ⁇ 10 8 to 1.0 ⁇ 10 10 , 1.0 ⁇ 10 8 to 5.0 ⁇ 10 9 , 1.0 ⁇ 10 8 to 1.0 ⁇ 10 9 , 1.0 ⁇ 10 8 to 5.0 ⁇ 10 8 , 1.0 ⁇ 10 9 to 5.0 ⁇ 10 10 , 1.0 ⁇ 10 9 to 1.0 ⁇ 10 10 , or 1.0 ⁇ 10 9
  • Bacillus subtilis CJBS303 strain according to the present application can effectively reduce the generation of odor gases derived from livestock manure or livestock shed.
  • composition and the microbial preparation containing the strain can not only contribute to the improvement of an animal husbandry environment through reduction of odor, but also improve the production of livestock.
  • FIG. 1 shows a result of evaluating ammonia consumption capacity according to an embodiment, and is a result of visually confirming the color development reaction of a culture solution according to whether or not ammonia is consumed.
  • FIG. 7A to 7E show the effect of reducing odor gas in the pig house according to the feeding of the CJBS303 strain
  • FIG. 7A shows the level of complex odor in the pig house
  • FIG. 7B shows the level of ammonia in the pig house
  • FIG. 7D is a result of confirming the level of volatile fatty acids (VOCs) in the pig house
  • FIG. 7E is a result of confirming the level of methylmercaptan in the pig house over time.
  • VOCs volatile fatty acids
  • Figures 8a to 8d verify the effect of reducing odor gas in the pig house according to the feeding of the CJBS303 strain
  • Figure 8a shows the level of ammonia in the pig house
  • Figure 8b shows the level of hydrogen sulfide in the pig house
  • Figure 8c shows the level of methylmercaptan in the pig house
  • Figure 8d is a result of comparing the level of complex odor in the pig house.
  • Example 1 Isolation and identification of strains having the effect of reducing odor derived from livestock manure
  • the ion medium to which ammonia was added (4.95 g/L of (NH 4 ) 2 SO 4 , 8.82 g/L of K 2 HPO 4 , 1.1 ml/L of 1M MgSO 4 solution, 0.3 ml/L of 1M CaCl 2 solution, 0.5 ml/L of 30 mM FeSO 4 solution, 0.04 ml/L of 50 mM CuSO 4 solution, 0.7 g/L of NaH 2 PO 4 , and 12 ml/L of 5% (W/V) anhydrous Na 2 CO 3 Including), and then inoculating the strains at a concentration of 0.01%, respectively, in the prepared 10 ml of the medium, and cultured at 30°C for 14 days.
  • Ammonia consumption rate (%) (A-B / A) ⁇ 100
  • the ammonia consumption rate of the CJBS303 strain was observed to be the highest at 74.3% among the four strains that were primarily selected. Therefore, CJBS303, which has the highest ammonia consumption rate, was finally selected as a strain having the effect of reducing odor derived from livestock manure.
  • Morphological and biochemical characteristics were analyzed for the CJBS303 strain. Specifically, the morphological characteristics of the strain were analyzed through Gram staining and electron microscopic observation, and the biochemical characteristics of the strain were determined by confirming the sugar fermentation pattern of the strain through the API 50 CHB system (biomerieux Vitek, Inc, France). Confirmed.
  • the CJBS303 strain was a Gram-positive strain, and as shown in FIG. 3, it was a bacillus.
  • the CJBS303 strain exhibited a sugar fermentation pattern similar to Bacillus subtilis.
  • PCR premix (Bionia, Korea), universal primer 27F (5' AGAGTTTGATCMTGGCTCAG 3'), and 1492R (5' GGTTACCTTGTTACGACTT 3') were used to amplify the gene of 16s rDNA.
  • the total reaction solution was set to 20 ⁇ l, and a total of 30 times was repeated under conditions of 94°C for 1 minute, 56°C for 1 minute, and 72°C for 1 minute.
  • the 16s rDNA nucleotide sequence of the CJBS303 strain was the same as SEQ ID NO: 1, which showed 99% homology with Bacillus subtilis.
  • the isolated strain was named'Bacillus subtilis CJBS303' or'CJBS303 strain' (accession number KCCM12435P).
  • the hemolytic properties of the strain were evaluated. Specifically, after linear inoculation (streaking) the CJBS303 strain on blood agar plate medium (sheep blood 5%, Hanil Comed, Korea), it was cultured at 37°C for 24 hours. Then, whether or not hemolysis in the cultured medium was observed with the naked eye.
  • the CJBS303 strain did not show hemolytic properties, and through this, the safety of the Bacillus subtilis CJBS303 strain was confirmed.
  • the CJBS303 strain reduced the total amount of ammonia gas generated by 41% compared to the control without addition, which had a reduction effect of more than 4 times compared to the standard strain.
  • manure from the control and treatment groups was sampled every week to measure the odor gas generated therefrom.
  • manure sampled from each of the control and treatment groups at a certain ratio (600g feces + 1.2L urine diluted 12.5%)
  • it was subdivided into 300ml bottles and cultured for a total of 42 hours at 39°C under aerobic conditions.
  • the amount of gas generated over a total of 4 times was measured using a portable odor gas measuring device (MultiRAE).
  • MultiRAE portable odor gas measuring device
  • the manure cultivation experiment was carried out every time the manure was sampled on a weekly basis, and data up to the 5th week were averaged to calculate the reduction rate compared to the control.
  • the treatment group was observed to have a lower concentration of ammonia and hydrogen sulfide gas compared to the control group from the 2nd week of feeding, and when the concentrations of the gas generated up to the 5th week were averaged and compared, the CJBS303 strain It was confirmed that the reduction rate of odor gas according to the salary was 38.3% for ammonia and 88.9% for hydrogen sulfide.
  • Control CJBS303 0.1% Control CJBS303 0.1% 2nd week 207 140 286 14 Week 3 278 152 166 22 Week 4 212 135 52 20 Week 5 180 113 91 10 Average 219 135 149 16 Reduction rate compared to control 38.30% 88.90%
  • control and treatment were raised in separate pig houses, and in order to exclude the effect of the existing slurry in the pig house, the slurry of the control and treatment pigs was simultaneously emptied before the start of the test and this test was conducted. From 4 days before the start of the test, gas in the pig house was measured at 9 am and 4 pm every day for control and treatment piglets. Ammonia, hydrogen sulfide, complex odor, and volatile fatty acids (VOCs) were measured using a precision odor measuring instrument (Odor Catch, SLC-OP-1350, Science and Technology Analysis Center), and methyl mercaptan was a portable odor gas measuring instrument (MultiRAE). ) was used, and the data measured every day were calculated for each week.
  • VOCs volatile fatty acids
  • Example 4 Evaluation of pig farming productivity according to feeding of CJBS303 strain
  • Example 3 (3) When pigs were fed with a probiotic containing the CJBS303 strain, it was attempted to determine the effect on pig farming productivity. Under the same conditions as in Example 3 (3), a total of 96 heads of 74-day-old finishing pigs were classified into two groups to conduct a feeding test. Before the start of the feeding test and after the end of the test at the 4th week, the average daily gain (ADG) was calculated by measuring the individual's body weight, and the average daily feed intake (ADFI) and feed were measured by measuring the daily feed feed amount and the remaining amount. The efficiency (feed conversion ratio, FCR) was calculated.
  • ADG average daily gain
  • ADFI average daily feed intake
  • FCR feed conversion ratio
  • the treatment group fed with the CJBS303 strain improved the daily feed intake by 7.3% (Duncan, p ⁇ 0.1) and the daily weight gain by 9.0% (Duncan, p ⁇ 0.03) compared to the control group.
  • FCR also improved by 0.03.
  • Example 5 Verification of the effect of reducing odor gas in pig farms according to the feeding of CJBS303 strain
  • the first farm house (A) was selected as a growing pig house (A-1) and a finishing pig house (A-2), and the second farm house (B) was selected as a finishing pig house, and the test subjects and the number of each farm house are shown in Table 5.
  • Both farms were fed a test feed containing 2x10 9 cfu/kg of CJBS303 strain in feed to growing or finishing pigs for 3 weeks, and the reduction rate was calculated by measuring the concentration of odor gas in the pig house before and at the third week of feeding the test feed.
  • Ammonia, hydrogen sulfide, and methyl mercaptan were measured using a portable odor measuring device (MultiRAE), and the complex odor was measured at 9 am using a precision odor measuring device (Odor Catch, SLC-OP-1350, Science and Technology Analysis Center). It was measured at.

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Abstract

La présente invention concerne une souche de Bacillus subtilis CJBS303 et son utilisation , et fournit une souche ayant pour effet de réduire les odeurs provenant du fumier de bétail, une composition et un agent microbien comprenant la souche, et une composition d'aliment pour animaux comprenant la composition.
PCT/KR2020/000133 2019-03-07 2020-01-03 Bacillus subtilis cjbs303 et composition le comprenant WO2020179999A1 (fr)

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CN202080002103.5A CN112074598B (zh) 2019-03-07 2020-01-03 枯草芽孢杆菌cjbs303以及包括其的组合物
EP20767389.8A EP3766960A4 (fr) 2019-03-07 2020-01-03 Bacillus subtilis cjbs303 et composition le comprenant
CA3095265A CA3095265C (fr) 2019-03-07 2020-01-03 Bacillus subtilis cjbs303 et composition le comprenant
US17/043,463 US20230095303A1 (en) 2019-03-07 2020-01-03 Bacillus subtilis cjbs303 and composition comprising same
BR112020022140-6A BR112020022140A2 (pt) 2019-03-07 2020-01-03 Cepa de bacillus subtilis cjbs303, composição, composição de ração, preparação microbiana para remover odores de gado, método para reduzir odores de esterco de um indivíduo e método para preparar uma preparação microbiana
JP2020552202A JP7068490B2 (ja) 2019-03-07 2020-01-03 枯草菌ｃｊｂｓ３０３、及びそれを含む組成物
PH12020551799A PH12020551799A1 (en) 2019-03-07 2020-10-28 Bacillus subtilis cjbs303 and composition comprising same

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CN114292768A (zh) * 2021-11-16 2022-04-08 山东省科学院生物研究所 一株枯草芽孢杆菌及其应用
WO2022081762A1 (fr) * 2020-10-14 2022-04-21 Dermbiont, Inc. Compositions et méthodes pour améliorer la santé de la peau et pour le traitement et la prévention de maladies, troubles et états associés à des champignons et autres microbes pathogènes
CN115322929A (zh) * 2022-08-10 2022-11-11 青岛农业大学 能降氨除氮的益生菌复合物及其在降解畜禽养殖场污水和粪便中的应用

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CN115322929A (zh) * 2022-08-10 2022-11-11 青岛农业大学 能降氨除氮的益生菌复合物及其在降解畜禽养殖场污水和粪便中的应用

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