WO2013151364A1 - 신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스 - Google Patents

신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스 Download PDF

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WO2013151364A1
WO2013151364A1 PCT/KR2013/002829 KR2013002829W WO2013151364A1 WO 2013151364 A1 WO2013151364 A1 WO 2013151364A1 KR 2013002829 W KR2013002829 W KR 2013002829W WO 2013151364 A1 WO2013151364 A1 WO 2013151364A1
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cjmpb283
bacillus licheniformis
ammonia
strains
culture
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PCT/KR2013/002829
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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 JP2015504500A priority Critical patent/JP6129294B2/ja
Publication of WO2013151364A1 publication Critical patent/WO2013151364A1/ko
Priority to PH12014502245A priority patent/PH12014502245B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • 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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/22—Nature of the water, waste water, sewage or sludge to be treated from the processing of animals, e.g. poultry, fish, or parts thereof
    • 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.
  • antibiotics In general, antibiotics, antibacterial agents, and chemicals are used to improve disease and water quality in farmed fish, but their use is limited due to the increase of resistant bacteria, residual body, and water pollution. I'm doing it.
  • development of alternatives such as organic acids, probiotics, non-specific immunopotentiators, and natural substances is urgently progressed to fundamentally block the use of antibiotics (diagnosis of fish diseases). And treatment measures, Ministry of Oceans and Fisheries, 2001).
  • Probiotics has an etymological meaning that is opposed to antibiotics (antibiotics), which means antibiotics, defined as a microbial agent or microbial component that helps balance microorganisms in the intestine, and is called a lactic acid bacterium, such as Lactobacillus and Bifidus.
  • antibiotics defined as a microbial agent or microbial component that helps balance microorganisms in the intestine, and is called a lactic acid bacterium, such as Lactobacillus and Bifidus.
  • the fungus species is representative.
  • Bacillus which is used as a probiotic, is a gram-positive bacillus that forms endogenous spores and has a unique form among bacteria used as probiotics. Bacillus is characterized by excellent heat resistance than lactobacillus that 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 Laid-Open Publication No. 2007-0036016 discloses a method for preventing or mitigating the accumulation of nitrite in the aquatic environment using nitrite-oxidizing bacteria.
  • the nitrite-oxidizing bacteria are Nitrococcus and Nitrospira .
  • Korean Patent Laid-Open Publication No. 2010-0040960 discloses a method for removing carbon and nitrogen contaminants from wastewater using heterotrophic ammonia oxidizing bacteria, and the heterotrophic ammonia oxidizing bacteria disclosed in the literature are Bacillus pseudopymus NH-. 2 ( Bacillus pseudofimus NH-2), Arthrobacter globiformis WR-2.
  • Nitrococcus and nitrospy disclosed in the above documents cannot be used as probiotics because they are not GRAS strains, and because they belong to archaea, they are very difficult to produce and thus cannot be used industrially.
  • Bacillus Pseudomusus WR-2 also known as GRAS strain, is a strain that has not been registered as a probiotic.
  • the present inventors completed the present invention by separating the strains having digestive enzyme activity in Korean doenjang, which is a traditional fermented food in Korea, and oxidizing ammonia and nitrite, and confirming the morphological, biochemical and genetic properties of the strain.
  • Another object of the present invention is to provide a culture selected from the group consisting of the culture solution of Bacillus licheniformis CJMPB283, the concentrate thereof and the dried product thereof.
  • 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 aquatic aquaculture improving agent comprising Bacillus licheniformis CJMPB283, its culture, its concentrate or its dried product.
  • Still another object of the present invention is to provide a method for improving the quality of the farm by spraying the water improver before or at the farming stage.
  • a novel Bacillus licheniformis CJMPB283 which produces a digestive enzyme and has an ammonia and nitrite oxidizing ability.
  • Bacillus licheniformis CJMPB283 of the present invention is obtained by separating from the traditional Korean food meju.
  • the morphological feature of the strain of the present invention is Gram-positive bacilli (FIG. 6) and has the 16s rDNA nucleotide sequence of SEQ ID NO: 1. Analysis of the base sequence showed 97% homology with Bacillus licheniformis . Accordingly, the present inventors deposited the newly isolated Bacillus Richenformis CJMPB283 to Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) on March 22, 2012 with the accession number KCCM11270P.
  • the isolate was excellent in useful digestive enzyme activities such as amylase, cellulose, and protease, and has the characteristic of oxidizing ammonia and nitrite.
  • growth is possible under anaerobic and anaerobic conditions, and because it is flame resistant to 10% sodium chloride, it is possible to grow in various water environments.
  • the control of ammonia in the flounder tank was excellent and the mortality was low.
  • 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.
  • the inorganic salt included in the medium magnesium, manganese, calcium, iron, phosphorus, etc. may be used, but is not limited thereto.
  • culture temperature conditions of the novel isolated strain of the present application can be cultured in 12 hours to 4 days 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 of the invention or a culture thereof.
  • 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.
  • the probiotic formulation of the present invention comprises Bacillus licheniforms CJMPB283 of 5 x 10 4 to 5 x 10 10 CFU / ml, preferably Bacillus licheniforms CJMPB283 of 1 x 10 6 to 1 x 10 9 CFU / ml It includes.
  • the probiotic formulation of the present invention may further include 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 for probiotics formulated as liquid solutions include sterile and physiologically compatible saline, sterile water, buffered saline, albumin injectable solutions, dextrose solutions, maltodextrin solutions, glycerol and one of these components. More than one component can be mixed and used, and other conventional additives, such as antioxidant, buffer, and bacteriostatic agent, can be added as needed.
  • Diluents, dispersants, surfactants, binders and 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.
  • binders, emulsifiers, preservatives, and the like added to prevent deterioration of the quality of the probiotic formulations in the present invention, and amino acids, vitamins, enzymes, flavors, nonprotein nitrogen compounds, and silicates added to the feed to increase the efficacy.
  • the feed mixture may further include, but is not limited thereto.
  • Formulations for oral administration containing the probiotics of the present invention as an active ingredient can be formulated into tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, soft capsules.
  • 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, magnesium stearate And lubricating agents such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the aforementioned substances.
  • a liquid carrier such as fatty oil in addition to the aforementioned substances.
  • the present invention relates to aquaculture feed additive comprising the probiotic formulation as an active ingredient.
  • Bacillus forms endogenous spores and has a very stable characteristic of heat. Therefore, the newly isolated Bacillus licheniformis CJMPB283 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 CJMPB283 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 CJMPB283 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 aquaculture feed additives may further include conventional additives to increase the shelf life of the feed in addition to Bacillus Richenomyces CJMPB283 of the present invention.
  • the plants include grains, fruits, food processing by-products, algae, fibres, oils, starches, gourds, grain by-products, and the like, proteins, minerals, oils, Minerals, fats and oils, unicellular proteins, zooplankton, fishmeal, and the like, but are not limited thereto.
  • the aquaculture feed additive of the present invention can be used by adding to the aquaculture feed by dipping, spraying or mixing.
  • Fish species to which the feed additive of the present invention may be added may be used for fish or crustaceans, and more specifically, but not limited thereto, such as farmed fish such as flounder, dom, shrimp, tilapia, salmon, eel, trout, etc. Do not.
  • the feed additive is mixed with the animal feed in an amount of 0.05 to 0.5% by weight on a dry weight basis.
  • the aquaculture modifier for aquaculture including Bacillus Richenomyces CJMPB283 as an active ingredient; And it relates to a method for improving the water quality of the farm by spraying the water improver before or in the farming step in the farm.
  • the novel isolated Bacillus licheniformis CJMPB283 of the present invention can be used to reduce the ammonia and nitrite content present in the aquaculture environment.
  • Fish species of aquaculture farms in which the water quality improving agent of the present invention can be used may be used for fish or crustaceans, and more specifically include, but are not limited to, farmed fish such as flounder, dome, shrimp, tilapia, salmon, eel, trout, and the like.
  • the water quality improver of the present invention may be prepared separately from the newly isolated Bacillus Richenomyces CJMPB283 in the form of a water quality improver, or may be directly sprayed with the strain and / or probiotic agent.
  • Bacillus licheniformis CJMPB283 in the water improver of the present invention may be a liquid or dry state, preferably in the form of a dried powder. Drying methods may be, but not limited to, ventilation drying, natural drying, spray drying and freeze drying.
  • Bacillus licheniformis CJMPB283 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.
  • Acceptable carriers for water quality improvers are sterile and biocompatible materials, and may be used by mixing saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol and one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added. Diluents, dispersants, surfactants, binders and 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.
  • the newly isolated Bacillus licheniformis CJMPB283 has an excellent digestive enzyme activity such as amylase, cellulose, and protease, and has excellent oxidation ability against ammonia and nitrous acid, thereby improving water quality at the stage of farming fish and shrimp. It has the effect to do it.
  • the newly isolated Bacillus licheniformis CJMPB283 can be used not only as a probiotic, but also as a water quality improver.
  • 1 is a figure quantitatively evaluating the consumption of ammonia in the ammonia-containing medium of the first selected strains of the present invention.
  • Figure 3 is a picture evaluating the growth of anaerobic and anaerobic conditions of the secondary screening strains of the present invention.
  • Figure 4 is a figure evaluating the flame resistance of the secondary screened strains of the present invention.
  • FIG. 5 is a diagram evaluating the ammonia reduction effect according to the water quality improvement evaluation in the flounder fry tank of the secondary selection strain of the present invention.
  • Figure 6 is a photograph taken with an electron microscope of the Bacillus Richenomyces CJMPB283 strain of the final selection strain of the present invention.
  • Oxidation evaluation of ammonia and nitrous acid was carried out to select the microorganisms that oxidize ammonia and nitrite, which are the main causes of water pollution.
  • Ion medium with ammonia (NH 4 ) 2 SO 4 4.95g / L, K 2 HPO 4 8.82g / L, MgSO 4 (1M solution) 1.1ml / L, CaCl 2 (1M solution) 0.3 ml / L, FeSO 4 (30 mM solution) 0.5 ml / L, CuSO 4 (50 mM solution) 0.04 ml / L, NaH 2 PO 4 0.7 g / L, 5% (W / V) Na 2 CO 3 anhydrous 12 ml / L) with nitrous acid added (CaCl 2 0.01g / L, MgSO 4 ⁇ 7H 2 O 0.1g / L, EDTA 1.4mg / L, FeSO 4 ⁇ 7H 2 O 5mg / L, H 2 SO 4 0.5 ⁇ l / L, Na 2 MoO 4 2H 2 O 0.05 mg / L, MnCl 2 4H 2 O 0.1 mg / L, CoCl 2 6H
  • the first strain was inoculated in 5 ml of the prepared two media 0.01% incubation at 30 °C for 14 days.
  • the culture solution was centrifuged to collect 1 ml each of the culture supernatant, and then 100 ⁇ l of an indicator (sulanilic acid, N, N-Dimethyl-1-naphylamine) that exhibits color reaction in the presence of nitrous acid was added.
  • the culture supernatant to which the indicator was added was reacted at 25 ° C. for 10 minutes and then observed for color change. When nitrous acid was present or produced, the color of the medium changed to dark purple, and when nitrous acid was not present or consumed, no color change of the medium was observed.
  • Ammonia consumption was quantified in the medium to which ammonia was added to 10 strains of the selected strains in order to evaluate the reduction effect of ammonia oxidation of the first selected strains.
  • the first 10 strains were inoculated in 0.1% BHI liquid medium, and then cultured at 37 ° C. at 200 rpm for 15 hours to activate the strains.
  • Ammonia-containing medium ((NH 4 ) 2 SO 4 0.5g / L, NaH 2 PO 4 13.5g / L, K 2 HPO 4 0.7g / L, MgSO 4 7H 2 O 0.1g / L, CaCl 2 2H 2 O 0.18g / L, NaHCO 3 0.5g / L, FeCl 3 ⁇ 6H 2 O 0.014g / L, glucose 0.5g / L) 1% inoculated and incubated at 200rpm at 37 °C. During 6 hours, 12 hours, 24 hours, 36 hours of culture was collected and centrifuged to recover only the culture supernatant. The residual amount of ammonia in the recovered culture supernatant was quantified.
  • nitrite consumption was quantified by quantifying in the medium to which nitrite was added to the 10 selected strains.
  • the first 10 strains were inoculated in 0.1% BHI liquid medium, and then cultured at 37 ° C. at 200 rpm for 15 hours to activate the strains.
  • Nitrous acid-containing medium NaNO 2 0.5g / L, NaH 2 PO 4 13.5g / L, K 2 HPO 4 0.7g / L, MgSO 4 7H 2 O 0.1g / L, CaCl 2 2H 2 O 0.18g / L , NaHCO 3 0.5g / L, FeCl 3 ⁇ 6H 2 O 0.014g / L, glucose 0.5g / L
  • Nitrous acid-containing medium NaNO 2 0.5g / L, NaH 2 PO 4 13.5g / L, K 2 HPO 4 0.7g / L, MgSO 4 7H 2 O 0.1g / L, CaCl 2 2H 2 O 0.18g / L , NaHCO 3 0.5g / L, FeCl 3 ⁇ 6H 2 O 0.014g / L, glucose
  • nitrite in the culture supernatant was reduced in all 10 selected strains in the nitrite-containing medium, in particular, strains 102, 109, 251, 253 and 268 were 6 hours of initial culture. From the second it was confirmed that the content of nitrite is sharply reduced.
  • the color reaction was observed by adding an indicator indicating the color reaction in the presence of nitrous acid. As a result, dark purple color was observed in the initial medium containing nitrous acid. Did not change. That is, it was found that nitrite was not oxidized because the first selection strain oxidized nitrite.
  • the aquatic environment coexists with aerobic, flue-gas, and anaerobic conditions, and especially anaerobic (a process in which ammonia is oxidized to nitric acid under anaerobic conditions) under anaerobic conditions. Therefore, it is one of the important factors to grow the selection strain under various conditions. Therefore, growth of anaerobic and anaerobic conditions of 10 primary strains was evaluated.
  • strains of the first selected strains were inoculated in BHI liquid medium by 0.1%, followed by incubation for 24 hours under anaerobic and anaerobic conditions at 37 ° C. Most of the selected strains were grown under both anaerobic and anaerobic conditions. Especially, strains 253 and 368 showed excellent growth under anaerobic conditions, and strain 283 showed growth under anaerobic conditions. Was the best (FIG. 3). Accordingly, strains 253, 268, and 283 which were excellent in growth anaerobic and anaerobic conditions were selected.
  • Three candidate strains selected from BHI broth added with 0%, 3%, 5%, and 10% sodium chloride were inoculated by 0.1% and incubated at 37 ° C at 200 rpm. During the cultivation progress, samples were taken at 8, 10 and 22 hours to measure absorbance.
  • Example 3 in vivo Evaluation and final strain selection
  • Bacillus Richenformis CJMPB283 Bacillus Richenformis CJMPB283
  • the novel microorganism of the present invention identified by the above method was deposited as " Bacillus licheniformis CJMPB283" on March 22, 2012 to the Korea Microorganism Conservation Center ( Accession number KCCM11270P).
  • Example 5 Characterization of probiotics of strain B. licheniformis CJMPB283
  • the selected strains were cultured in BHI liquid medium for 24 hours, and then the culture medium was used as a coenzyme solution for enzyme activity analysis.
  • the degree of substrate degradation was determined using a medium containing respective substrates for each enzyme as follows.
  • YM (Difco, USA) medium containing 2% skim milk (skim milk, Sigma, USA) was prepared. 2 ⁇ l of the crude enzyme solution collected above was dispensed into a substrate medium, and then reacted at 37 ° C. for 15 hours, and enzyme activity was measured according to the degree of clear zone formation.
  • YM medium with 1% CMC (carboxyl methyl cellulose) substrate was added. 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. 3 ⁇ l of the coenzyme 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 a clear zone resulting from decomposition of the substrate around the crude enzyme solution.
  • the final selected strain Bacillus licheniformis CJMPB283 had a digestive enzyme activity against protease, amylase, cellulose as shown in Table 3. In particular, the activity against protease was excellent.
  • 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 CJMPB283 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 CJMPB283 As shown in Table 4, when Bacillus licheniformis CJMPB283 was incubated for 24 hours, about 24.5% formed endospores, and when incubated for 48 hours, 100% endospores were formed. Bacillus licheniformis CJMPB283 of the present invention has a high endogenous spore forming ability when cultured for more than 48 hours can be a characteristic that can maintain a high survival rate when used as probiotics.
  • Bacillus licheniformis CJMPB283 of the present invention is excellent in the production of digestive enzymes such as amylase, cellulose, and protease, and has an excellent oxidation effect against ammonia and nitrous acid, probiotics and water quality in the stage of farming fish and shrimp It has the efficacy to improve. Therefore, the newly isolated Bacillus licheniformis CJMPB283 can be used not only as a probiotic, but also as a water quality improver.
PCT/KR2013/002829 2012-04-05 2013-04-04 신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스 WO2013151364A1 (ko)

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JP2015504500A JP6129294B2 (ja) 2012-04-05 2013-04-04 新たに分離したバチルス・リケニフォルミス及びこれを利用したプロバイオティクス
PH12014502245A PH12014502245B1 (en) 2012-04-05 2014-10-03 Novel separated bacillus licheniformis and probiotics using same

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KR1020120035434A KR101370943B1 (ko) 2012-04-05 2012-04-05 신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스
KR10-2012-0035434 2012-04-05

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