WO2013151363A1 - 신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스 - Google Patents

신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스 Download PDF

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WO2013151363A1
WO2013151363A1 PCT/KR2013/002828 KR2013002828W WO2013151363A1 WO 2013151363 A1 WO2013151363 A1 WO 2013151363A1 KR 2013002828 W KR2013002828 W KR 2013002828W WO 2013151363 A1 WO2013151363 A1 WO 2013151363A1
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cjmpb361
bacillus licheniformis
cells
culture
bacillus
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PCT/KR2013/002828
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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 IN2396KON2014 priority Critical patent/IN2014KN02396A/en
Priority to JP2015504499A priority patent/JP6117336B2/ja
Publication of WO2013151363A1 publication Critical patent/WO2013151363A1/ko
Priority to PH12014502244A priority patent/PH12014502244A1/en

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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
    • 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
    • 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
    • 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
    • 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
    • C12R2001/10—Bacillus licheniformis

Definitions

  • the present invention relates to novel probiotics and their use.
  • Probiotics are of the highest interest as dual immunopotentiators. Probiotics have an etymological meaning as opposed to antibiotics (antibiotics), meaning antibiotics, and are defined as microbial agents or microbial components that help balance the microorganisms in the intestines, and are commonly referred to as lactic acid bacteria such as Lactobacillus and Bifidus. Representative. In addition, probiotics are classified as GRAS (Generally Recognized As Safe). They do not contain toxic genes for humans and animals and do not produce pathogenic substances. And it should be a microorganism confirmed the improvement effect on the livestock productivity.
  • GRAS Generally Recognized As Safe
  • the efficacy of probiotics adhered to the intestinal mucosa with strong adhesion, competitively dropping pathogens that cause intestinal diseases, and excreted out of the gut flora. It also inhibits the prevention and growth of pathogenic microorganisms, promotes the optimal environment and growth for the growth of enteric beneficial bacteria, produces lactic acid or acetic acid, which is a major component of intestinal organic acids, and lowers intestinal pH. Suppresses the development of. By this complex action, it is possible to maintain normal flora when administered and improve the productivity of livestock.
  • Probiotics mainly used are lactic acid bacteria such as Lactobacillus and Enterococcus and Bacillus.
  • Bacillus is a Gram-positive bacillus that forms endogenous spores and has a unique form among bacteria used as probiotics. Bacillus has better heat resistance than Lactobacillus, which does not form endospores. It also survives the low pH of the stomach wall, so most of the bacteria administered can reach the small intestine. (Barbosa, TM et al . Appl. Environ . Microbiol . 71 (2005) 968-978; Spinosa, MR et al . Res. Microbiol . 151 (2000) 361-368).
  • Korean Patent Publication No. 10-2011-035554 discloses a novel mixed strain of Bacillus CMB L1 and Lactobacillus CMB201 and anticancer using the same And microbial preparations having an immuno-enhancing food composition and antimicrobial activity are disclosed, and Korean Patent No. 10-0977407 discloses various activities of neutrophils, which are the main phagocytic cells of animals, and nonspecific defense against the challenge of pathogenic bacteria.
  • Animal enhancers, vaccine adjuvant additives, adjuvant and feed additives are disclosed , which contain a lysate extract of Zygosaccharomyces bailii , which enhances potency . Studies on the immunopotency of Bacillus have been insufficient.
  • the present inventors separated the probiotics for digestive enzyme production and lactic acid production from doenjang, a traditional fermented food of Korea, and confirmed their morphological, biochemical and genetic properties, and found that the probiotics were Bacillus having excellent heat resistance. It was confirmed that the Bacillus is excellent in myeloid cell proliferation effect in bone marrow transplanted mice as well as in the efficacy of immunity to increase splenocytes, bone marrow cells of immune mice, and vaccine adjuvant to increase antibody production.
  • Another object of the present invention is to provide a culture selected from the group consisting of the culture solution of Bacillus licheniformis CJMPB361, its concentrate and dried product thereof.
  • Another object of the present invention is to provide a probiotic formulation comprising the Bacillus licheniformis CJMPB361 or the culture.
  • Another object of the present invention is to provide a feed additive comprising the probiotic formulation.
  • Another object of the present invention is to provide a feed comprising the feed additive.
  • Still another object of the present invention is to provide an agent or vaccine adjuvant for immunosupplement comprising the probiotic agent.
  • Another object of the present invention is to provide a health functional food for enhancing immunity containing the probiotic agent.
  • Bacillus licheniformis CJMPB361 of the present invention is obtained by separating from the traditional Korean doenjang.
  • the morphological feature of the strain of the present invention is Gram-positive bacilli, and has the 16s rDNA nucleotide sequence of SEQ ID NO: 1. Analysis of the nucleotide sequence showed 99% homology with Bacillus licheniformis . Accordingly, the present inventors deposited the newly isolated Bacillus Richenformis CJMPB361 at the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea) on March 22, 2012 with the accession number KCCM11269P.
  • BHI Brain heart infusion
  • the culture supernatant of the strain has a particularly strong immunopotentiating effect on mouse B cells by increasing the activity of non-T cells, mouse bone marrow, and peripheral blood of the mouse spleen, the molecular weight of the culture supernatant 1,000 KDa or more When the fractions were injected with the antigen, it showed a side effect of antibody production by the antigen.
  • a culture selected from the group consisting of a culture of a novel isolated strain of the present invention, a concentrate thereof, and a dried product thereof.
  • the novel isolated strain of the present invention can be cultured through a conventional method for culturing Bacillus strains.
  • the medium may be natural or synthetic medium.
  • the carbon source of the medium for example, glucose, sucrose, dextrin, glycerol, starch and the like can be used, and as the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate and other organic or inorganic Nitrogen containing compounds may be used, but are not limited to these components.
  • Inorganic salts contained in the medium include magnesium. Manganese, calcium. Iron, potassium and the like can be used, but are not limited thereto.
  • culture temperature conditions of the newly isolated strain of the present application can be incubated for 4 days at 12 hours in the temperature range of 20 ⁇ 40 °C.
  • the culture solution of the newly isolated strain may be a culture stock solution including the cells, and may also be a culture cell from which the culture supernatant is removed or concentrated.
  • the composition of the culture solution may further include not only components necessary for normal Bacillus culture, but also components that synergistically act on the growth of Bacillus, and thus the composition may be easily selected by those skilled in the art. Can be.
  • the state of the strain may be a liquid state or dry state
  • the drying method is possible, but not limited to ventilation drying, natural drying, spray drying and freeze drying.
  • a probiotic formulation comprising a novel isolated strain or culture of the present invention as an active ingredient.
  • Probiotics settle on the gut wall of the intestine, preventing harmful bacteria from settling and inhibiting the growth of pathogens.
  • beneficial digestive enzymes produced by probiotics facilitate the absorption and utilization of nutrients, improving feed demand.
  • Probiotic formulations of the present invention comprise Bacillus licheniforms CJMPB361 at 5 x 10 4 to 5 x 10 10 CFU / ml, preferably 1 x 10 6 to 1 x 10 9 CFU / ml Bacillus licheniformes CJMPB361 Include.
  • Probiotic formulations of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier to provide food and feed additives.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in probiotic formulations formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, buffered saline, albumin injectable solutions, dextrose solutions, maltodextrin solutions, glycerol and these components.
  • sterile and biocompatible include saline, sterile water, buffered saline, albumin injectable solutions, dextrose solutions, maltodextrin solutions, glycerol and these components.
  • One or more components can be mixed and used, and other conventional additives, such as an antioxidant, a buffer solution, and bacteriostatic agents, can be added as needed.
  • diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • binders emulsifiers and preservatives added to prevent the degradation of probiotics formulations, and amino acids, vitamins, enzymes, flavors, nonprotein nitrogen compounds, silicates, buffers, etc., which are added to feeds to increase their effectiveness.
  • Extractant oligosaccharide, and the like.
  • the feed mixture may further include, but is not limited thereto.
  • Formulations for oral administration comprising the probiotic formulation of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs It can be formulated.
  • lactose For formulation into tablets and 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 Lubricants such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be included, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • the present invention relates to a feed additive comprising the probiotic formulation as an active ingredient.
  • Bacillus spp. Form endogenous spores and have very stable heat resistance. Therefore, the newly isolated Bacillus licheniformis CJMPB361 may be prepared separately in the form of feed additives and mixed in the feed, or directly added during the preparation of the feed.
  • Bacillus licheniformis CJMPB361 in the feed of the present invention may be a liquid or dry state, preferably in the form of a dry powder. Drying methods may be, but not limited to, ventilation drying, natural drying, spray drying and freeze drying.
  • Bacillus licheniformis CJMPB361 of the present invention may be mixed in the form of a powder of 0.05 to 10% by weight, preferably 0.1 to 1% by weight of the feed weight.
  • the feed may further include conventional additives that can increase the shelf life of the feed in addition to Bacillus Richenformis CJMPB361 of the present invention.
  • the feed additive of the present invention may be provided in a form selected from the group consisting of concentrates, powders and granules.
  • the concentrate may comprise from 20% to 90% by weight of the Bacillus Richenomyces CJMPB361 strain, its culture, or its dried product, or probiotic formulation, according to the present invention in the total weight of the feed additive.
  • Feed additives of the present invention can be used by adding to the animal feed by dipping, spraying or mixing.
  • Animals to which the feed additive of the present invention may be used include, for example, livestock such as pigs, cattle, sheep, goats, horses, rabbits, dogs, cats, and poultry such as chickens, ducks, geese, turkeys, quails, and the like. Including but not limited to.
  • the composition is mixed with the animal feed in an amount of 0.05 to 0.5% by weight on a dry weight basis.
  • % means weight percent unless otherwise defined.
  • Another aspect is to provide a feed comprising a feed additive of the present invention.
  • the feed containing Bacillus lychenimoform CJMPB361 includes grains, fruit fruits, food processing by-products, algae, fiber, oils, starches, gourds, grain by-products, and the like. Proteins, minerals, fats, oils, minerals, fats, unicellular proteins, zooplankton, fish meal, and the like are not limited thereto.
  • Still another object of the present invention is to provide an immuno-enhancing preparation or vaccine-assisted preparation comprising the novel strain or culture solution, its concentrate or dried product thereof as an active ingredient.
  • the Bacillus licheniformis CJMPB361 Bacillus licheniformis, CJMPB361; Accession No. KCCM11269P
  • its culture, its concentrate or its dried product contains a fraction of 100 to 300 kDa or a fraction of 1,000 kDa or more having immunopotentiating activity.
  • the culture supernatant of Bacillus licheniformis CJMPB361 was isolated by molecular weight and then measured for lymphocyte proliferation activity.
  • the growth of mouse splenocytes was excellent at a molecular weight of 1,000 kDa or more and 100 to 300 kDa (FIG. 3).
  • B cells showed proliferative responses depending on the culture supernatant concentrations, and increased the proliferative responses of non-T and non-B cells in addition to B cells.
  • the effect of proliferation was higher at molecular weights of 1,000 kDa and above (FIGS. 5 and 6).
  • the culture supernatant of 1,000 kDa or more has an adjuvant effect on the antibody production by the antigen.
  • Immunity-enhancing formulations and vaccine adjuvant formulations of the present invention comprises 0.1 to 50% by weight of the strain or strain culture or concentrate or dried product thereof relative to the total weight of the composition.
  • the immunopotentiator of the present invention can be used as a prophylactic or therapeutic adjuvant of immunodeficiency that may occur in animals or humans.
  • the vaccine adjuvant of the present invention may be used as an adjuvant for inducing a systemic immune response to an antigen by administering the antigen together with an antigen in an animal body.
  • the antigen can be used in combination with one or two or more antigens selected from the group consisting of fungi, actinomycetes, bacteria, viruses, protozoa, components of these microorganisms, tissues, cellular components, antigenic proteins and antigenic peptides.
  • the preparation of the pharmaceutical composition of the immunoadjuvant preparation and the vaccine adjuvant containing the strain or culture solution of the present invention, the concentrate thereof or the dried product thereof may further include appropriate carriers, excipients and diluents commonly used.
  • Adjuvant preparations or vaccine adjuvant according to the present invention in the form of a dispersant, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods
  • Carriers, excipients and diluents which may be used in a composition comprising a culture, a concentrate thereof, or a dried product thereof may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyridone, water and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations are prepared by mixing at least one excipient with the culture solution, concentrate thereof, or dried product thereof.
  • Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may include various excipients in addition to commonly used simple diluents such as water, liquid, and paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used.
  • base of the suppository utopsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Preferred dosages of the immunoadjuvant and vaccine adjuvant of the present invention vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. However, for the desired effect, it is advisable to administer the immunoadjuvant and vaccine adjuvant of the present invention at 0.001 to 100 mg / kg per day, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • Immunostimulating agents and vaccine adjuvant of the present invention can be administered to a variety of routes to livestock, poultry, humans, mammals.
  • the mode of administration may be administered by oral, intramuscular, subcutaneous, intravenous, injection.
  • Examples of the food to which the probiotic agent or the immunopotentiating agent of the present invention may be added include various foods, beverages, gums, teas, and health functional foods.
  • the amount of a novel strain or probiotic formulation or an immune enhancing formulation of the present invention in food or beverage may be added at 0.01 to 50% by weight of the total food weight, the health beverage composition is 0.05 to 5% by weight based on 100 ml Can be added.
  • the health functional food of the present invention includes the form of tablets, capsules, liquids and the like.
  • the health beverage composition of the present invention has no particular limitation except for containing the novel strain of the present invention, the probiotic agent, or the immuno-enhancing agent as essential ingredients in the ratios indicated above, and various flavoring agents or Natural carbohydrates and the like may be included as additional components.
  • the natural carbohydrates are conventional sugars such as glucose, fructose, maltose, sucrose, textine, sclotextrin and the like and sugar alcohols such as xylitol, sorbitol and erythritol.
  • natural flavoring agents such as taurine and stevia extract, and synthetic flavoring agents such as saccharin and aspartame may be used.
  • Probiotics formulations or immunopotentiating formulations of the present invention other than the above are various nutrients, vitamins, minerals (electrolytes), flavors, coloring agents and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
  • the extract of the present invention may contain natural fruit juices, fruit juice drinks, pulp for the manufacture of vegetable drinks. These components can be used independently or in combination.
  • the newly isolated Bacillus licheniformis CJMPB361 is excellent in the production of digestive enzymes and lactic acid, such as amylase, cellulose, mannaise, and xylanase, and has excellent adjuvant efficacy in producing animals and immune enhancing activity and antibodies. In addition, it has an effect of inducing an enhanced bone marrow graft proliferative response in leukemia patients.
  • the newly isolated Bacillus licheniformis CJMPB361 can be used not only as a probiotic, but also as an animal immune enhancing agent and a vaccine adjuvant.
  • 1 is a photograph of the strain taken by electron microscopy of Bacillus Richenomyces CJMPB361 strain of the present invention.
  • FIG. 2 shows the present invention The figure shows the presence or absence of hemolytic activity of Bacillus licheniformis CJMPB361.
  • the culture supernatant of Bacillus licheniformis CJMPB361 is a graph showing the mouse lymphocyte proliferation reaction according to the molecular weight fraction.
  • Figure 4 is a graph showing the proliferation of mouse splenocytes (lymphocytes), bone marrow cells, thymic cells according to the molecular weight fraction of the culture supernatant of Bacillus licheniformis CJMPB361 of the present invention.
  • Figure 5 is a graph showing the growth of mouse spleen B cells in vitro conditions according to the molecular weight fraction of the culture supernatant of Bacillus licheniformis CJMPB361 of the present invention.
  • Figure 6 is a FACS results showing the mouse lymphocyte proliferation response in vivo when the culture supernatant of the fraction of 1,000 kDa or more of the Bacillus licheniformis CJMPB361 of the present invention administered to the abdominal cavity.
  • a isotype control, B-E is 0.67 mg, 1.35 mg.
  • FIG. 7 is a graph evaluating the effects of antibody production upon administration of the culture supernatant of at least 1,000 kDa of Bacillus licheniformis CJMPB361 of the present invention.
  • FIG. 8 is a graph showing the mortality after evaluating the effect of the cultured supernatant of Bacillus licheniformis CJMPB361 over 1,000 kDa on cell proliferation after bone marrow transplantation.
  • A a graph evaluated in a mouse irradiated with 7.5Gy
  • B a graph evaluated in a mouse irradiated with 9Gy.
  • Doenjang was collected between 70 and 80 days, a traditional Korean food, and the obtained samples were diluted in stages and incubated for 24 hours at 37 ° C after smearing in BHI solid medium (Difco, USA) to which 3% sodium chloride was added. The predominant strain was isolated from the sample. The selected colonies were separated by pure culture by transferring them to a new medium over three times, and the pure cultured cells were stored in the medium added with 20% glycerol and stored below 70 ° C.
  • Bacillus licheniformis CJMPB361 Bacillus licheniformis CJMPB dated March 22, 2012
  • KCCM11269P Bacillus licheniformis CJMPB361
  • the culture medium was used as a coenzyme solution for enzyme activity analysis, and the degree of substrate degradation was determined using a medium containing respective substrates for each enzyme as follows.
  • YM medium to which 1% CMC (carboxyl methyl cellulose) substrate was added was prepared. 2 ⁇ l of the coenzyme solution collected above was dispensed into the substrate medium, reacted at 37 ° C. for 15 hours, and then stained with 0.2% congo red aqueous solution for 30 minutes, and decolorized with 1M NaCl aqueous solution. The activity of the enzyme was measured according to the degree of formation of a clear zone resulting from decomposition of the substrate around the crude enzyme solution.
  • YM medium was prepared to which 1% soluble starch substrate was added. 2 ⁇ l of the crude enzyme solution collected above was dispensed into the substrate medium, and then reacted 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 the clear zone resulting from decomposition of the substrate around the crude enzyme solution.
  • YM medium was prepared with 1% xylan added. 2 ⁇ l of the culture solution was dispensed into the substrate medium, and after reaction at 37 ° C. for 15 hours, 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 clear ring formation.
  • a substrate medium (Yeast extract 3g / L, Peptone 5g / L, KH 2 PO 4 1g / L, Agar 20g / L, pH5) to which 1% manna (logust bean gum, sigma, USA) was added was prepared. 2 ⁇ l of the culture solution was dispensed into the substrate medium, followed by reaction at 37 ° C. for 15 hours, and the activity of the enzyme was measured according to the degree of transparent ring formation.
  • the newly isolated strain Bacillus licheniformis CJMPB361 had a digestive enzyme activity against cellulose, amylase, xylanase, mannase as shown in Table 2. In particular, the activity against xylase and mannaise was high.
  • Lactic acid conversion rate (lactic acid production rate / initial sugar concentration) x 100
  • the strain was found to convert 93.5% of lactic acid.
  • the paper reported that lactic acid conversion of lactic acid-producing Bacillus polyfermentus was 60.7% ( Kim, K. M et al. J. Microbiol. Biotechnol., 19 (2009) 1013-1018 ).
  • the isolated strain Bacillus licheniformis CJMPB361 was found to convert more lactic acid. Therefore, it was found that Bacillus licheniformis CJMPB361, the isolated strain, has the characteristic of producing lactic acid.
  • 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 rays, high temperature, low temperature drying and high pressure, so the formation of endospores is important in maintaining the viability of Bacillus. Therefore, Bacillus licheniformis CJMPB361 was incubated for a long time to confirm the endogenous spore forming ability.
  • the BHI liquid medium was inoculated with 0.1% of the bacteria, and cultured at 37 ° C. and 200 rpm for 24 hours and 48 hours.
  • the total bacterial count was measured by smearing the culture medium for each time period in the BHI solid medium, and the endogenous spores were measured by smearing the culture solution heat-treated at 95 ° C. for 10 minutes in the BHI solid medium.
  • Bacillus licheniformis CJMPB361 As shown in Table 3, when Bacillus licheniformis CJMPB361 was incubated for 24 hours, about 0.3% formed endogenous spores, and when cultured for 48 hours, 78% endogenous spores were formed.
  • Bacillus licheniformis CJMPB-361 of the present invention has a high endogenous sporulation ability when cultured for more than 48 hours can be a characteristic that can maintain a high survival rate when used as probiotics.
  • ⁇ -hemolysis is the action of lysing red blood cells by producing phospholipid enzymes in harmful bacteria and hydrolyzing phospholipids supplied by red blood cells.
  • BALB / c mice (Orient Co., South Korea) between 6 and 7 weeks of age were sacrificed through a respiratory anesthesia and spleens were collected and lymphocytes were liberated by gently compressing the spleen into two sterile slide glasses.
  • the isolated lymphocytes were washed three times with RPMI 1640 (Invitrogen, USA), a cell culture medium.
  • RPMI 1640 Invitrogen, USA
  • the red blood cells in the cell suspension were removed and suspended in RPMI 1640 medium containing 10% fetal bovine serum at 2 ⁇ 10 6 cells / ml.
  • the suspended lymphocytes were dispensed into 100 ⁇ l aliquots into 96 well plates, and 100 ⁇ l of the prepared samples were added.
  • the plates were incubated for 2 days at 37 ° C. in a 5% CO 2 incubator, followed by 6 hours of incubation with 10 ⁇ l of tritiated thymidine (3H TdR) (New England Nuclear, Boston).
  • the cultured cells were adsorbed onto a glass fiber filter and dried at room temperature for 24 hours, and then Melt-on scintillator sheet (MeltLexTM A, Wallac) was placed on a filtermat and dissolved for 1 to 4 minutes on a thermostat.
  • Isolation of the bone marrow cells was performed by cutting both ends of the thighs of the BALB / c mouse with scissors and inserting a 10 ml syringe needle containing RPMI1640 medium into the thighs to release the bone marrow cells.
  • the thymic cells were separated from the thymus by the same procedure as the splenocytes.
  • the proliferative response of bone marrow cells and thymic cells proceeded in the same manner as the proliferative response test of the splenocytes.
  • B-cells and non-B cells were isolated from mouse spleen lymphocytes in order to know what molecular weight material in the culture supernatant of Bacillus licheniformis CJMPB361 fractionated by molecular weight induces the proliferation of splenocytes.
  • the ratio of T cells, B cells, non-T cells, and non-B cells in normal mouse splenocytes was analyzed by FACS and the results are shown in Table 4.
  • B cells were isolated by magnetic cell selection method and analyzed by FACS.
  • Bacillus licheniformis CJMPB361 of three fractionated molecular weight was added to the isolated B cells and the non-B cells and cultured in the same manner as the proliferation of the splenocytes.
  • the molecular weight of 1,000 kDa and the molecular weight of 100- In the 300 kDa fraction the proliferation of B cells was observed, and the proliferation of non-T and non-B cells was increased in addition to B cells.
  • the effect of proliferation was higher at molecular weights of 1,000 kDa and above (FIG. 5).
  • mice were slaughtered to separate the spleen. The isolated spleen was weighed and the change in splenic lymphocyte count was measured by FACS. Administration of the adjuvant 0.67 mg, 1.35 mg, 2.7 mg and 5.4 mg into the mouse abdominal cavity increased the spleen size to 15%, 20%, 98% and 51%, respectively.
  • B cells in the 0.67 mg group did not change, but the percentage of B cells in the 1.35 mg group increased from 41% to 45.3%, 47.6% in the 2.7 mg group and 49.7% in the 5.4 mg group. It showed an aspect. Therefore, B cells were shown to exhibit a proliferative response depending on the concentration of the culture supernatant of more than 1,000 kDa fraction of Bacillus licheniformis CJMPB361 (Fig. 6).
  • mice were divided into three groups, and administered with cultured supernatants of at least 1,000 kDa of ovalbumin (OVA) and Bacillus licheniformis CJMPB361 as follows.
  • Group A was divided into physiological saline, B group with subcutaneous injection of 100 ⁇ g OVA and C group with subcutaneous injection of 100 ⁇ g OVA subcutaneously every 2 days after intraperitoneal injection of fractionated culture supernatant of the strain. .
  • Immunization was injected subcutaneously with 100 ⁇ l (1 mg / ml) of the antigen, OVA, and 100 ⁇ l (2.7 mg) of the culture supernatant fractionated over 1,000 kDa of the strain.
  • OVA the antigen
  • serum was isolated.
  • Specific antibodies to OVA were measured by ELISA method.
  • OVA was diluted to a concentration of 2 ⁇ g / ml in the coating buffer (0.03M Na 2 CO 3 , 0.068M NaHCO 3 , pH9.4 ⁇ 9.8) and added to 100 ⁇ l to 96 well plate and reacted at 4 °C for 15 hours. I was.
  • treatment with physiological saline only did not measure specific antibodies to OVA.
  • Treatment with OVA alone produced specific antibodies to IBA, but showed low antibody titers.
  • the antibody value was significantly increased compared to the treatment group administered with OVA alone. Therefore, more than 1,000 kDa of culture supernatant fraction of the strain means that the adjuvant (adjuvant) effect on the antibody production by the antigen.
  • the same type of bone marrow cells were transplanted into the irradiated mice, and the survival rate was compared by administering or not administering the culture supernatant fractionated 100 kDa or more of the strain.
  • BALB / c mice were irradiated with 7.5 Gy or 9 Gy, and the irradiated mice were divided into 3 groups, respectively.
  • Group 1 was administered saline, and group 2 was transplanted with bone marrow cells (1 ⁇ 10 7 cells / mouse) obtained from homogenous normal mice.
  • Group 3 transplanted the same bone marrow cells in the same manner and received 2.7 mg of the above cultured supernatant of 1,000 kDa or more of the strain intraperitoneally.
  • mice Both physiological saline and bone marrow cells were administered to the mouse tail vein, and the culture supernatant fractionated above 1,000 kDa was administered intraperitoneally. All of these administrations were performed within 2 hours after irradiation, and the survival rate of mice after 30 days was observed. As shown in FIG. 8, all mice died between 8 and 11 days in the saline-administered group irradiated with 7.5 Gy of radiation dose, and 66% survival rate in the group transplanted with bone marrow cells alone. In contrast, 100% of the bone marrow cells and the culture supernatants treated with the fractions of the strain above 1,000 kDa survived 100% (FIG. 8A).
  • the present invention is a newly isolated Bacillus Richenformis CJMPB361 is excellent in the production of digestive enzymes and lactic acid, such as amylase, cellulase, mannaise, lyphases, excellent immune boosting activity of animals and ancillary efficacy of producing antibodies, Efficacy in inducing an increase in myeloid graft cell proliferative response in leukemia patients. Therefore, the newly isolated Bacillus licheniformis CJMPB361 can be used not only as a probiotic, but also as an animal immune enhancing agent and a vaccine adjuvant.

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