WO2013151361A1 - 신규 바실러스 서브틸리스 - Google Patents

신규 바실러스 서브틸리스 Download PDF

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WO2013151361A1
WO2013151361A1 PCT/KR2013/002826 KR2013002826W WO2013151361A1 WO 2013151361 A1 WO2013151361 A1 WO 2013151361A1 KR 2013002826 W KR2013002826 W KR 2013002826W WO 2013151361 A1 WO2013151361 A1 WO 2013151361A1
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cjmpb150
strain
bacillus subtilis
feed
culture
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English (en)
French (fr)
Korean (ko)
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백승희
양시용
우서형
서효실
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씨제이제일제당(주)
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Priority to JP2015504497A priority Critical patent/JP5872104B2/ja
Publication of WO2013151361A1 publication Critical patent/WO2013151361A1/ko
Priority to PH12014502242A priority patent/PH12014502242A1/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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • 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

Definitions

  • the present invention relates to a novel Bacillus subtilis CJMPB150 (KCCM11268P) strain and a probiotic agent comprising the same.
  • feed grains contain a large number of indigestible carbohydrates, namely cellulose (Cellulose), hemicelluloses (Lignin) and lignin (Lignin).
  • Indigestible carbohydrates inhibit digestion by altering the functioning of the digestive organs, causing stool or reddening in livestock.
  • various feed enzymes for the purpose of digesting indigestible carbohydrates have been widely used.
  • Cellulase which can hydrolyze cellulose, Xylan, which is a major component of hemicellulose, and Xylanase, which break down lignin associated with it, are widely used as an enzyme for feed addition, and livestock of grain feed Increasing the intestinal availability of nutrients (Beguin and Aubert, FEMS Microbiol. Rev. 13: 25-58, 1994), as well as promoting the composting of livestock manure (Kim Tae-il et al., Korean Journal of Microbiology) 35 (4): 277-282, 1999).
  • Mannanase is an enzyme that breaks down mannan-containing substances, which are the major components of hemicellulose. Soy protein is used as feed for livestock such as pigs and chickens, and the metase breaks down the galactan (Galactan) in carbohydrates, an energy source for soybean meal, so that it can be easily metabolized by unit animals.
  • galactan galactan
  • Probiotics is a concept that includes microorganisms that help balance intestinal microflora, microorganisms with antimicrobial and / or enzymatic activity and the products they produce (Fuller, R. J Appl Bacteriol. 66 (5): 365-378, 1989).
  • Korean Patent No. 10-0318554 discloses Bacillus sp . 79-23 strain producing neutral cellulase
  • Korean Patent No. 10-1062309 discloses Bacillus richeni secreting cellulase and xylanase.
  • Bacillus licheniformis DK42 strain KACC 91410P is disclosed.
  • Korean Patent No. 10-0426930 discloses Bacillus amyloliquefaciens B4 strain KCTC 18083P having amylase, protease, cellulase, and lipase enzyme activity.
  • An object of the present invention is to provide a Bacillus subtilis CJMPB150 (KCCM11268P) strain excellent in complex digestive enzyme production capacity, acid resistance and bile resistance.
  • an object of the present invention is to provide a probiotic formulation, feed additives and feed comprising the Bacillus subtilis CJMPB150 (KCCM11268P) strain or its culture.
  • One aspect of the present invention provides a novel Bacillus subtilis CJMPB150 (KCCM11268P) strain excellent in complex digestive enzyme production ability, acid resistance and bile resistance.
  • it provides a culture of the Bacillus subtilis CJMPB150 (KCCM11268P) strain.
  • a Bacillus subtilis CJMPB150 (KCCM11268P) strain or a culture of the strain is provided, which provides a probiotics formulation.
  • a feed additive comprising the probiotic formulation.
  • a feed comprising the feed additive.
  • the present invention improves the digestibility of livestock by providing probiotics, feed additives and feed comprising a new Bacillus subtilis CJMPB150 (KCCM11268P) strain or its culture having excellent complex digestive enzyme production ability, acid resistance and bile resistance.
  • KCCM11268P Bacillus subtilis CJMPB150
  • KCCM11268P Bacillus subtilis CJMPB150
  • it has the effect of contributing to the development of livestock industry by maintaining the intestinal microbial balance and / or antimicrobial activity.
  • Bacillus subtilis of the present invention Bacillus subtillis ) CJMPB150 (KCCM11268P) An electron micrograph of the strain.
  • Figure 2 is a graph showing the growth of the candidate strains of Example 1 over time.
  • FIG. 3 is a photograph showing the digestive enzyme activity of Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention.
  • FIG. 4 is a graph showing survival rate when Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention is treated with artificial gastric juice or artificial bile.
  • Example 5 is a graph showing the digestibility of the feed according to Example 6.
  • Figure 6 is a photograph showing the presence or absence of hemolytic Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention.
  • Figure 7 shows the 16s rDNA base sequence of Bacillus subtilis CJMPB150 (KCCM11268P) of the present invention.
  • One aspect of the present invention provides a novel isolated Bacillus subtilis CJMPB150 (KCCM11268P) (hereinafter 'CJMPB150') strain.
  • the CJMPB150 was deposited on March 22, 2012 at the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under the accession number KCCM11268P.
  • the CJMPB150 has a complex digestive enzyme production capacity.
  • Digestive enzyme having the production capacity of the CJMPB150 includes cellulase (Cellulase), xylanase (Xylanase) and mannanase (Mannanase), selected from the group consisting of Protease (Protease), Amylase (Lipase) and Lipase (Lipase) It may further comprise one or more.
  • the CJMPB150 may more preferably produce all of cellulase, xylanase, mannanase, protease, amylase and lipase.
  • the CJMPB150 has excellent heat resistance.
  • the CJMPB150 may be incubated at 37 ° C. and 200 rpm for 24 hours, and then endogenous spore formation rate after heat treatment at 95 ° C. for 10 minutes may be 80% or more, and more preferably 85% or more. Can be.
  • the endogenous spore formation rate after incubation for 48 hours at 37 °C, 200 rpm condition, heat treatment at 95 °C 10 minutes may be preferably 100%.
  • the endogenous spores are resistant to extreme environments such as UV, low temperature, drying and / or high pressure, as well as high temperature conditions, and the higher the endogenous spore formation rate, the higher the survival rate of the strain.
  • the CJMPB150 has excellent acid resistance.
  • the CJMPB150 has a survival rate after incubation for 3 hours in a medium containing artificial gastric juice prepared by adding 1% (w / v) of pepsin (Pepsin) to a solution adjusted to pH 2.5. It may be more than%.
  • the CJMPB150 has excellent bile resistance.
  • the CJMPB150 may have a survival rate of preferably 80% or more after incubation for 3 hours in a medium containing artificial bile containing 1% (w / v) pancreatin.
  • the novel isolated strain CJMPB150 of the present invention can be cultured through a conventional method for culturing Bacillus strains.
  • the CJMPB150 is preferably obtained by incubating for 12 hours to 4 days at a culture temperature in the range of 20 to 40 °C.
  • the culture is a concept including a culture medium or culture medium in which the CJMPB150 strain is cultured, and the resultant cultured in the culture medium or culture solution, and the culture may or may not include the CJMPB150 strain.
  • the culture is not particularly limited in its dosage form, and may be a dosage form commonly used in the art.
  • the culture may be a liquid or a solid, and may be in a circular state of the culture or in a concentrated form or in a dried form thereof.
  • a natural medium or a synthetic medium can be used as a medium for culturing the CJMPB150 of the present invention.
  • the carbon source of the medium is not particularly limited, and any known in the art may be used.
  • Non-limiting examples of the carbon source include glucose, sucrose, dextrin, glycerol or starch. These can be used individually or in mixture of 2 or more types.
  • the nitrogen source of the medium is not particularly limited, and any known in the art may be used.
  • Non-limiting examples of such nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate and other organic or inorganic nitrogen containing compounds. These can be used individually or in mixture of 2 or more types.
  • the inorganic salt contained in the medium is not particularly limited and may be any known in the art.
  • Non-limiting examples of the inorganic salts include magnesium, manganese, calcium, iron, potassium and the like. These can be used individually or in mixture of 2 or more types.
  • the medium for culturing the CJMPB150 of the present invention may be added with amino acids, vitamins, nucleic acids, and / or other components that may generally be included in the culture medium.
  • the culture solution of the CJMPB150 strain of the present invention may be a culture stock solution, and may be a solution from which the culture supernatant is removed from the culture stock solution and / or a solution thereof.
  • the culture solution may contain the CJMPB150 strain.
  • the composition of the culture medium is not particularly limited, and may include not only components known to be required for culturing Bacillus strains, but also components that synergistically act on the growth of Bacillus, and the composition thereof is conventional in the art. It can be easily chosen by one with knowledge.
  • the culture solution may be in a liquid state or a dry state.
  • the method of drying the culture solution is not particularly limited and may be a method commonly used in the art.
  • Non-limiting examples of the drying method include ventilation drying, natural drying, spray drying or freeze drying. These may be used alone or in combination of two or more methods.
  • a probiotic formulation comprising the CJMPB150 strain or a culture of the strain.
  • Probiotics means a microorganism or a component that exhibits a beneficial effect on the health of the host, such as humans and animals, and settles on the gut wall of the host intestine and serves to inhibit the settlement of other harmful bacteria and the growth of pathogens.
  • the beneficial digestive enzymes produced by probiotics are known to facilitate the absorption and utilization of nutrients.
  • Probiotic formulations of the present invention may be one comprising the CJMPB150 strain and / or culture of the strain.
  • Probiotics of the present invention may preferably comprise the CJMPB150 strain 5 x 10 4 to 5 x 10 10 CFU / ml, more preferably 1 x 10 6 to 1 x 10 9 CFU / ml have.
  • the probiotic formulation may further include a pharmaceutically acceptable carrier, and may be provided in combination with the carrier.
  • the pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound.
  • carriers usable in probiotic formulations that are formulated as liquid solutions are suitable for sterilization and living bodies, such as saline solution, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution or glycerol. Can be. These can be used individually or in mixture of 2 or more types, and can add other conventional additives, such as antioxidant, buffer, and / or bacteriostatic agent, as needed.
  • Diluents, dispersants, surfactants, binders and / or lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Formulations for oral administration comprising the probiotic formulations of the present invention as an active ingredient are not particularly limited and may be used as formulations known in the art.
  • Non-limiting examples of such oral dosage forms include tablets, troches, lozenges, aqueous or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like.
  • lactose For formulation into such tablets or capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax and the like, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • binders such as cellulose or gelatin
  • excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch
  • stearic acid Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax and the like
  • the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • a feed additive comprising the probiotic formulation.
  • Probiotic formulations comprising the CJMPB150 strain of the present invention and / or cultures thereof may be separately prepared in the form of feed additives and mixed in the livestock feed, or may be mixed in such a manner as to directly add the probiotic preparations during feed preparation.
  • the form of the feed additive is not particularly limited and may be preferably in the liquid or dry state, more preferably in the form of dry powder.
  • the drying method of the dry powdered probiotic formulation is not particularly limited and may be a method known in the art.
  • Non-limiting examples of the drying method include ventilation drying, natural drying, spray drying or freeze drying. These may be used alone or in combination of two or more methods.
  • the feed additive may further include other additives that may increase the preservation of the feed in addition to the probiotic formulation including the CJMPB150 of the present invention and / or its culture.
  • Additives that may be additionally included in the feed additives of the present invention are not particularly limited and may be those known in the art.
  • Non-limiting examples of the additives include, but are not limited to, binders, emulsifiers, preservatives, and the like, which prevent degradation of feed additives; Amino acids, vitamins, enzymes, flavors, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc., which increase their utility. It may include. These can be used individually or in mixture of 2 or more types.
  • the probiotic formulations or feed additives of the present invention may be administered to animals alone or in combination with other feed additives in an edible carrier. It may also be administered directly to top dressings, livestock feed, oral formulations separate from the feed, or in combination with other ingredients.
  • a feed comprising the feed additive.
  • the feed of the present invention may include the feed additive preferably 0.05 to 10 parts by weight based on 100 parts by weight of feed, more preferably 0.1 to 1 parts by weight. Within this range, it is possible to effectively increase the digestibility of the livestock, thereby increasing the efficiency of the feed.
  • the ingredient of the feed of the present invention is not particularly limited and may be known in the art.
  • Non-limiting examples of the feed ingredients include, but are not limited to, cereals, fruits, food processing by-products, algae, fibres, oils, starches, gourds, grain by-products, etc .
  • Animals include proteins, inorganic substances, fats and oils, minerals, fats and oils, single cell proteins, zooplankton, fish meal and the like. These can be used individually or in mixture of 2 or more types.
  • Livestocks that can use the probiotic formulations, feed additives or feed comprising the feed additives of the present invention include, for example, edible cows, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, and the like.
  • livestock and poultry such as chicks, chickens, domestic chickens, roosters, ducks, geese, turkeys, quails and the like.
  • Bacillus subtilis Bacillus subtilis (Bacillus subtilis Isolation of CJMBP150 Strain
  • Meju and various enteric samples which are traditional fermented foods, were obtained, and the obtained samples were gradually diluted and incubated in BHI solid medium (Difco, USA) added with 3% sodium chloride (Difco, USA) for 24 hours at 37 ° C.
  • the enzyme activity evaluation of the three protease, cellulase, and amylase was performed on the isolated enteric-derived bacteria. Enzyme activity evaluation was carried out using the medium containing the substrate for each enzyme was measured the enzyme activity capacity according to the degree of clear zone (clear zone) formation.
  • the culture supernatant of the first selected strain was cultured in BHI liquid medium for 24 hours and 48 hours, and centrifuged at 4 ° C. and 13,000 rpm for 5 minutes, and the final supernatant was used as a crude enzyme solution for enzyme activity analysis. As described below, the degree of substrate degradation was determined using a medium containing respective substrates for each enzyme.
  • YM Yeast extract 3g / L, Malt extract 3g / L, Peptone 5g / L, Dextrose 10g / L, Agar 20g / L) with 2% skim milk (Skim milk, Sigma, USA); Difco, USA, 'YM medium') media were prepared. 1.5 ⁇ l of the crude enzyme solution collected above was dispensed into the substrate medium, and then reacted at 30 ° C. for 15 hours, and then enzyme activity was measured according to the degree of formation of the transparent ring.
  • YM medium to which 1% CMC (carboxyl methyl cellulose) substrate was added was prepared.
  • 1.5 ⁇ l of the obtained crude enzyme was dispensed into the substrate medium, and then reacted at 37 ° C. for 15 hours, and then stained with 0.2% Congo red aqueous solution for 30 minutes, and decolorized with 1M aqueous NaCl solution.
  • the activity of the enzyme was measured according to the degree of formation of the transparent ring formed by decomposition of the substrate around the crude enzyme solution.
  • YM medium was prepared to which 1% soluble starch substrate was added. 1.5 ⁇ l of the coenzyme solution collected above was dispensed into the substrate medium, followed by reaction at 37 ° C. for 15 hours. After dyeing with an aqueous solution containing 0.1% and 2% of I 2 and KI, respectively, the activity of the enzyme was measured according to the degree of formation of a transparent ring formed by decomposition of the substrate around the crude enzyme solution.
  • Table 1 below shows the digestive enzyme productivity of the secondary selection strain.
  • the two candidate strains excellent in complex digestive enzyme productivity were evaluated for growth. Each strain was incubated at 37 ° C., 200 rpm in BHI liquid medium for 15 hours. 0.1% of the culture was inoculated in 100 mL of BHI liquid medium, and cultured at 37 ° C. for 10 hours and 24 hours, and each culture was plated in BHI solid medium to measure the number of bacteria.
  • CJMPB150 150 strains having excellent complex digestive enzyme production ability and growth ability were finally selected and named as CJMPB150.
  • CJMPB150 selected as an excellent digestive enzyme activity strain
  • the activity of xylanase, mannanase, and lipase in addition to protease, cellulase and amylase was evaluated.
  • CJMPB150 strain was cultured in a BHI liquid medium for 24 hours, culture supernatant was collected, centrifuged at 13,000 rpm for 5 minutes at 4 ° C, and the final supernatant was used as crude enzyme solution for enzyme activity analysis.
  • the medium containing each substrate was used to determine the degree of substrate degradation.
  • YM medium was prepared with 1% xylan added. 1.5 ⁇ l of the crude enzyme solution was dispensed into the substrate medium, reacted at 37 ° C. for 15 hours, and then stained with 0.2% aqueous solution of Congo Red for 30 minutes, and decolorized with 1M aqueous NaCl solution. Then, the enzyme activity was measured according to the degree of transparent ring formation.
  • YM medium was prepared to which 1% tricaprylin was added. 1.5 ⁇ l of the crude enzyme solution was dispensed into the substrate medium, followed by reaction at 37 ° C. for 15 hours.
  • Substrate medium Yeast extract 3g / l, Peptone 5g / l, KH 2 PO 4 1g / l, Agar 20g / l, pH 5
  • 1% mannan logust bean gum, sigma, USA
  • CJMPB150 secreted all six digestive enzymes such as xylanase, mannanase, and lipase in addition to protease, cellulase, and amylase to excellent levels.
  • CJMPB150 strain finally selected as an excellent strain of complex digestive enzyme activity
  • morphological and biochemical investigations were performed primarily.
  • the morphological characteristics were confirmed to be Gram-positive bacillus (FIG. 1), and the sugar fermentation pattern of CJMPB150 strain was analyzed by API 50 CHB system (biomerieux, France) to analyze biochemical properties.
  • Table 3 shows the results of analyzing the sugar release pattern of the CJMPB150 strain.
  • Sequencing was performed to amplify the gene of 16s rDNA using PCR premix (Bionia, Korea) and universal primer 27F (5'AGAGTTTGATCCTGGCTCAG3 ': SEQ ID NO: 2) and 1492R (5'GGTTACCTTGTTACGACTT 3': SEQ ID NO: 3).
  • the total reaction solution was adjusted to 20 ⁇ l, and a total of 30 times of 94 ° C 1 minute, 56 ° C 1 minute, and 72 ° C 1 minute was repeated, and the amplified DNA sequences were analyzed.
  • the base sequence of the analyzed 16s rDNA of the CJMPB150 strain is shown in SEQ ID NO: 1 (FIG. 7).
  • Bacillus subtilis CJMPB150 a novel microorganism of the present invention identified by the above method, was dated March 22, 2012, Korean Culture Center of Microorganisms, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul. Deposited as KCCM11268P.
  • Bacillus forms endogenous spores to survive when stressed, such as depletion of one or more of the essential nutrients. Endogenous spores are resistant to extreme environments such as ultraviolet light, high temperature, low temperature drying and high pressure, so the formation of endogenous spores is very important in maintaining the viability of Bacillus. For this reason, Bacillus subtilis CJMPB150 was cultured for a long time to confirm the endogenous spore formation ability.
  • Table 4 shows the measurement results of the endogenous spores.
  • Bacillus subtilis CJMPB150 of the present invention has a high endogenous spore forming ability when cultured for more than 24 hours, it is determined that it can maintain a high survival rate in the digestive organs of animals when used as probiotics.
  • Probiotic strains must be resistant to strong acidic gastric juice and bile in order to survive and reach the intestine, the final target site. For this reason, in order to confirm the application of Bacillus subtilis CJMPB150 as a probiotic strain of the present invention, acid resistance and bile resistance were confirmed.
  • Artificial gastric juice was prepared by adding 1% (w / v) of pepsin (Pepsin; Sigma, USA) to a 0.05M sodium phosphate solution adjusted to pH 2.5 using HCl.
  • the CJMPB150 strain was incubated at 37 ° C. and 200 rpm for 24 hours in BHI liquid medium, and then centrifuged at 13,000 rpm for 5 minutes to discard the supernatant and the cells were recovered. Incubated. After incubation, the BHI medium was plated to measure the number of bacteria to determine acid resistance and resistance to artificial gastric juice.
  • CJMPB150 strain incubated for 3 hours in the artificial gastric juice of (1) was centrifuged at 13,000 rpm for 10 minutes to discard the supernatant and the cells were recovered, and the artificial bile was added in the same amount as the supernatant and incubated at 37 ° C for 24 hours. . After incubation, the BHI medium was plated to measure the number of bacteria to determine acid resistance and resistance to artificial bile.
  • CJMPB150 was treated with artificial gastric juice at pH 2.5 for 2 hours, and the survival rate was about 83%, and 83% survival rate was maintained even after 24 hours of artificial bile treatment. .
  • Bacillus subtilis CJMPB150 of the present invention showed a high survival rate even after a chain treatment of artificial gastric juice and artificial bile was confirmed that it can be usefully used as a probiotic strain.
  • the digestibility of feed was investigated to determine whether Bacillus subtilis CJMPB150, which has high ability to produce complex digestive enzymes, could increase digestibility under the same conditions as intestinal gut.
  • Standard feed (50% corn, 30% soybean meal, 10% wheat, 5% rice bran and 5% brew Bacillus subtilis CJMPB150 was mixed with 0.1 g of 10 g of the feed prepared by mixing, added to 125 ml of phosphate buffer having a pH of 6.0, and titrated to pH 2 using 3M HCl. 2 ml of pepsin (20 mg / ml) was added thereto and reacted at 200 rpm and 38 ° C. for 2 hours.
  • digested paper (Whatman No. 541) was used to filter out the remaining feed and dried in an 135 ° C. oven. Its dry weight was measured to calculate the digestibility of the feed according to Equation 1 below.
  • Feed digestibility (%) ⁇ (dry weight before reaction-dry weight after reaction) / dry weight before reaction ⁇ X 100
  • the measured feed digestibility is shown in Table 5 and FIG.
  • ⁇ -hemolysis is the action of producing phospholipid enzymes in harmful bacteria to hydrolyze erythrocytes by hydrolyzing phospholipids supplied by red blood cells.
  • TSA Bacillus subtilis CJMPB150
  • sheep blood serum, Kisan Biotech, Korea

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CN108841747A (zh) * 2018-06-25 2018-11-20 青岛农业大学 产蛋白酶的枯草芽孢杆菌及使用方法
CN111172077A (zh) * 2020-02-14 2020-05-19 广东中科无抗养殖科技有限公司 一种调节生猪肠道菌群的微生物制剂及制备方法
CN113061555A (zh) * 2021-04-20 2021-07-02 江南大学 一株产纤维素酶的芽孢杆菌菌株筛选及其应用
US11173184B2 (en) * 2016-05-31 2021-11-16 Evonik Operations Gmbh Bacillus subtilis strain with probiotic activity
CN113862196A (zh) * 2021-11-03 2021-12-31 中谷生物科技(大连)有限公司 一株枯草芽孢杆菌sd-kc-001及其应用
CN114395506A (zh) * 2022-01-12 2022-04-26 福建省农业科学院农业生物资源研究所 一种耐高温产纤维素酶枯草芽胞杆菌及其培养方法和应用
CN114642239A (zh) * 2022-03-25 2022-06-21 播恩集团股份有限公司 一种提升ddgs营养品质的方法及其应用

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KR101661745B1 (ko) * 2014-09-15 2016-10-04 녹십자수의약품(주) 효소 생성능을 갖는 신규한 바실러스 서브틸리스 gcb-13-001 균주 및 이를 이용한 클로스트리듐 속 세균 독소 예방 또는 치료용 조성물
KR101985513B1 (ko) * 2015-12-11 2019-06-03 주식회사 템셀코리아 음식물 쓰레기 분해 소멸용 조성물
AU2019361501A1 (en) * 2018-10-18 2021-05-27 Sds Biotech K.K. Plant residue-decomposing agent using liquid culture of Bacillus pumilus KS-C4 strain
KR101985515B1 (ko) * 2019-03-08 2019-06-03 주식회사 템셀코리아 음식물 쓰레기 분해 소멸용 조성물
KR101985514B1 (ko) * 2019-03-08 2019-06-03 주식회사 템셀코리아 음식물 쓰레기 분해 소멸용 조성물
KR101985516B1 (ko) * 2019-03-08 2019-06-03 주식회사 템셀코리아 음식물 쓰레기 분해 소멸용 조성물
KR102176078B1 (ko) * 2020-06-09 2020-11-09 이정복 유기물 중 탄수화물과 단백질 및 섬유소에 대한 분해능을 갖는 신규 미생물 바실러스 서브틸리스 bs300 균주를 포함하여 배양하는 고체배양 방법
KR102466220B1 (ko) 2021-09-24 2022-11-11 롯데제과 주식회사 글루텐 분해를 통해 빵의 물리·화학적 소화율을 높여주는 락티플랜티바실러스 플랜타럼 lrcc 5318 균주 및 이의 용도

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KR20160007792A (ko) * 2014-06-30 2016-01-21 서울대학교산학협력단 바실러스 서브틸리스를 이용한 가축의 장관벽 보호 방법
US11173184B2 (en) * 2016-05-31 2021-11-16 Evonik Operations Gmbh Bacillus subtilis strain with probiotic activity
CN108841747A (zh) * 2018-06-25 2018-11-20 青岛农业大学 产蛋白酶的枯草芽孢杆菌及使用方法
CN108841747B (zh) * 2018-06-25 2020-08-14 青岛农业大学 产蛋白酶的枯草芽孢杆菌及使用方法
CN111172077A (zh) * 2020-02-14 2020-05-19 广东中科无抗养殖科技有限公司 一种调节生猪肠道菌群的微生物制剂及制备方法
CN113061555A (zh) * 2021-04-20 2021-07-02 江南大学 一株产纤维素酶的芽孢杆菌菌株筛选及其应用
CN113862196A (zh) * 2021-11-03 2021-12-31 中谷生物科技(大连)有限公司 一株枯草芽孢杆菌sd-kc-001及其应用
CN113862196B (zh) * 2021-11-03 2023-09-08 中谷生物科技(大连)有限公司 一株枯草芽孢杆菌sd-kc-001及其应用
CN114395506A (zh) * 2022-01-12 2022-04-26 福建省农业科学院农业生物资源研究所 一种耐高温产纤维素酶枯草芽胞杆菌及其培养方法和应用
CN114395506B (zh) * 2022-01-12 2023-05-02 福建省农业科学院农业生物资源研究所 一种耐高温产纤维素酶枯草芽胞杆菌及其培养方法和应用
CN114642239A (zh) * 2022-03-25 2022-06-21 播恩集团股份有限公司 一种提升ddgs营养品质的方法及其应用
CN114642239B (zh) * 2022-03-25 2023-02-03 播恩集团股份有限公司 一种提升ddgs营养品质的方法及其应用

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