KR20120088437A - Probiotics agent against vibrio sp - Google Patents

Abstract

PURPOSE: A probiotic for biological prevention against vibrio sp. microbe is provided to control quorum sensing of Vibrio harveyi and to effectively block pathogen. CONSTITUTION: A probiotic for biological prevention against vibrio sp. microbe contains Bacillus sp. CJS-26(KCCM 11144P), culture thereof, concentrate thereof, a dried product thereof. A feed additive used for farming fishes or crustaceans contains the strain, culture, concentrate, or dried product. An antibacterial agent contains the strain, culture, concentrate, or dried product thereof. A method for preventing diseases caused by pathogen in animals except for human body comprises a step of administering the strain, culture, concentrate, or dried product thereof to animals.

Description

Probiotics Agent Against Vibrio sp.

The present invention is Vibrio genus microorganisms ( Vibrio The present invention relates to a probiotic for biological control against sp.), specifically, a newly isolated Bacillus strain which degrades the quorum sensing signal factor of the pathogenic bacterium Vibrio and inhibits the production of a biofilm, a culture obtained by culturing the strain, Probiotic preparations, feed additives, antimicrobial agents or water-improving agents comprising the concentrates or their dried products and the strains, cultures, concentrates or dried products, and methods for farming fish or shellfish using the strains, cultures, concentrates or dried products, the strains, A method of inhibiting quorum sensing signal degradation and biofilm of Vibrio Havey by administering a culture, concentrate or dry matter to aquaculture, or adding the strain, culture, concentrate or dry matter to prevent bacterial diseases in animals other than humans or water Into the strain, culture, concentrate or Relates to a method of treating dried matter to produce modified water.

Recently, as the world's population has increased and living standards have improved, interest in improving diets has been increasing in advanced countries such as the United States, Europe and Japan. As a result, the demand for aquatic products continues to increase, leading to a surge in fish production. Aquaculture is therefore a major economic tool in many countries, and disease outbreaks in farmed fish have a major impact on economic development (Jose Luis Balcazar et al. Veter. Microbio. 114 (2006): 173-186; Laurent venrschuere et al. Microbio. Mot. Biol. Rev. Dec. 2000.655-671).

Vibrio bacteria, one of the causes of bacterial diseases in fish farms and shrimp farms, are known to cause not only primary problems due to their own toxicity but also secondary problems caused by biofilms formed by Vibrio bacteria. . In other words, Vibrio bacteria synthesize low-molecular chemical signal factors known as autoinducers or AIs or pheromone to secrete them outside the cell, and detect their concentration when the concentration of the signal factor is above a certain level. It recognizes the density of surrounding Vibrio bacteria and induces the expression of specific genes, thereby showing various physiological activities such as symbiosis, pathogenesis, competition, antibiotic production, migration ability, spore formation, and biofilm formation (Miller, MB). et al. Annu. Rev. Microbiol. 55 (2001), 165-199). As such, the process of maintaining the population density of the microorganisms at a constant level and accompanying a series of phenomena controlling gene expression is called quorum sensing, which is present in gram negative and positive bacteria, It is known to be used to infect a host while avoiding the host's defenses by promoting gene expression of the various elements necessary for infection only when the bacteria are of sufficient density to induce all bacteria to infect the host at once. (Dangl. JL et al., Nature. 411 (2001) 826-833).

Biofilm formation is known as a representative symptom caused by quorum sensing. When a low concentration of microorganisms settle and form colonies and reaches a certain number of populations, specific genes are expressed by quorum sensing signal mucus The polymer polysaccharide forms a structured biofilm, accounting for 65% of the symptoms of bacterial infection (Kjelleberg, S. et al. Curr Opin Microbiol. 5 (3), 2002, 254-258). The biofilm is formed on the surface of the host to cause infectious diseases of the host, and in particular, the fish supply nutrients and oxygen for the growth of the causative microorganism, while inhibiting the penetration of antibiotics and preventing the access of the immune system. It is known to reduce the effectiveness of disinfectants and probiotics, as well as a major cause of antibiotic resistance (Hall-Stoodley, L. et al., Nat Rev Microbiol. 2 (2), 2004, 95-108). ).

Autoinducer-1 (AI-1) and autoinducer-2 (AI-2) are known as chemical signal factors secreted from microorganisms in the early stages of biofilm formation. The autoderiver-1 is an AHL-based compound produced by the AHL synthetic protein of the LUX-1 group, and has various lengths and oxidations to homoserine lactones having a common structure. Acyl residues having degrees and saturation are combined to form N-acylhomoserine lactone (AHLs), which is a signal factor of Gram-negative bacteria. In addition, autoderivers-1 are Pseudomonas, Burkholderia, Yersinia, Aeromonas, Agrobacterium, Serratia, Erwinia, Erwinia, It has been reported that it is a strain-specific substance secreted from more than 70 kinds of Gram-negative bacteria such as Citrobacter, Enterobacter, Proteus, Chromobacterium, and Rhizobium genus (Citrobacter, Enterobacter, Proteus, Chromobacterium) Taga, ME et al. Proc. Natl. Acad. Sci. USA (2003). In addition, autoderiver-2 is a compound produced by a protein expressed from the luxS gene, has a furanosyl borate diester structure, and has a wide range of signals present in both Gram-positive and negative bacteria. It has been reported to be a universal signal (Bassler, BL et al. Mol. Microbiol. 9 (1993) 773-786). The luxS gene is E. coli as well as Salmonella typhimurium ( S. typhimurium ), Haemophilus influenzae ( H. influenzae ), H. pylori ( H. pylori ), B. subtilis ( B. subtilis) ), B. burgdoferi , N. meningitides , N. meningitides , C. jejuni , V. cholerae , Mycobacterium tuberculosis (M. tuberculosis), Enterococcus faecalis (E. faecalis), Streptococcus pies come Ness (S. pyogenes), Streptococcus pneumoniae in (S. pneumoniae), Staphylococcus aureus (S. aureus) , C. perfrengens , C. difficile , K. pneumoniae , and other bacteria, and luminescence in Vibrio Havey As well as pathogenic, motility, biofilm formation And expression of genes required for antibiotic production (Miller, MB et al. Annu. Rev. Microbiol. 55 (2001), 165-199).

Meanwhile, the mortality rate of rainbow trout infected with fish pathogenic microorganism V. anguillarum was 80 to 100%, but one of the compounds that inhibits quorum sensing by regulating AHL, a quorum sensing signal molecule. As the mortality rate of quorum-sensitized treatments with furanone was reported to be reduced to 4-40% (Rasch, M. et al. Syst. Appl. Microbiol. 27 (2004), 350-359 In order to reduce the damage caused by microbial infection, studies have been actively conducted.

For example, U. S. Patent No. 641685 discloses a patent for an antimicrobial composition comprising a phototherapeutic photosensitive agent linked to a polypeptide having a binding affinity for a magnetic derivative (AI-2). A medium for killing the bacteria by binding to the same is disclosed. US Patent No. 6,637,533 discloses a mixture of a magnetic derivative and a compound in contact with a luminescent bacterial cell in response to the magnetic derivative, and only contacting the bacterial cell with the magnetic derivative. A method for screening a compound that modulates the activity of the magnetic inducer in comparison with the degree of luminescence in the present invention is disclosed. US Patent Publication No. 2004-0009160 discloses quorum sensing controlled by signal molecules such as HSL and AHL of pathogens. Furanone is disclosed to suppress the quorum, and Korea Patent Publication No. 2008-0060777 discloses a quorum sensing antagonist An antimicrobial amide derivative that functions as a method and a method for preventing the formation of a biofilm using the same is disclosed. Korean Patent Registration No. 832565 discloses an antimicrobial furanone derivative that functions as a quorum sensing antagonist and a method for preventing the formation of a biofilm using the same. , Korean Patent Registration Nos. 841289, 841294 and 841333 disclose antimicrobial homoserine lactone derivatives having antimicrobial properties and functioning as quorum sensing antagonists, methods for removing microorganisms using the same, and methods for preventing biofilm formation using the same have.

On the other hand, as the demand for eco-friendly aquaculture grows, probiotics are receiving high attention as immunostimulators. Probiotics were defined in 1989 by R. Fuller as "probiotics that benefit host animals by improving the intestinal microflora of host animals." Strains used as probiotics include bacteria such as Lactobacillus, Enterococcus, Bifidobacterium and Bacillus, yeasts including Saccharomyces, and fungi including Aspergillus. 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. Probiotics should be non-pathogenic, easy to in vitro proliferation, fast growth in vivo, acid and bile resistant. In addition, the activity by the feed ingredients should not be inhibited, the effect should be maintained at room temperature, and convenient to mix with the feed.

Efficacy of such probiotics, adhered to the intestinal mucosa with strong adhesion, competitively dropping pathogens that cause intestinal disease to be excreted outside, and quickly recover the destruction of the enterobacterial flora following antibiotic administration. 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 in feeding and improve the productivity of livestock. In addition, probiotics in aquaculture are either added to feed or incidentally administered to water. Recently, active research is being conducted on the improvement of disease due to the enhancement of immunity against pathogenicity and the improvement of water quality by feeding probiotics to fish and shrimp (Jiqiu Li et al. Aquaculture 291 (2009) 35-40).

Probiotics used as biocontrol agents in aquaculture include lactobacillus and enterococcus such as lactic acid bacteria and Bacillus. Among them, Bacillus is a Gram-positive bacillus that forms endogenous spores and has a unique form among bacteria used as probiotics. . Bacillus subtilis Among these Bacillus (B. subtilis), Bacillus cereus (B. cereus), Bacillus core figures lance (B. coagulans), Bacillus claw when (B. clausii), Bacillus MEGATHERIUM (B. megaterium) , B. licheniformis is used as a probiotic. 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): 61-368). These Bacillus are used for human health supplements and animal growth promoters, to promote the growth of fish and shrimp farming and to increase disease resistance, and it has been reported that Bacillus not only improves water quality and promotes growth of shrimp, but also reduces vibrio. (Dalmin, G. et al. Indian J. Exp. Biol 39 (2001) 939-942; Wang, YB et al. Fish. Sci. 71 (2005) 1034-1039).

The inventors of the present invention separated probiotics excellent in the production of antibacterial and digestive enzymes against pathogenic bacteria in question in fish and shrimp, and confirmed their morphological, biochemical and genetic properties, and the probiotics were excellent in heat resistance. It was confirmed that it is Bacillus. In particular, the Bacillus controls Vibrio Havey, which is a problem in aquaculture, and inhibits physiological activities (symbiosis, pathogenesis, competition, biofilm formation, etc.) that occur during quorum sensing by degrading quorum sensing signal factor, autoderiver-2. .

The present inventors separated the novel Bacillus species (Bacillus sp . The present invention was completed by confirming that the biofilm formation was suppressed by vibrio havey inhibition and quorum sensing control, and can be used as a probiotic for aquaculture fish and shellfish.

It is an object of the present invention to provide a novel isolated Bacillus strain that degrades the quorum sensing signal factor of vibrio, a pathogenic bacterium, and inhibits biofilm production.

Another object of the present invention is to provide a culture solution obtained by culturing the strain, a concentrate of the culture solution or a dried product thereof.

Still another object of the present invention is to provide a probiotic formulation comprising the above strain, culture, concentrate or dried product.

Still another object of the present invention is to provide a feed additive comprising the above strain, culture, concentrate or dried product.

Still another object of the present invention is to provide a method for farming fish or shellfish using the strain, culture, concentrate or dried product.

Still another object of the present invention is to provide an antimicrobial agent comprising the strain, culture, concentrate or dried product.

Still another object of the present invention is to provide a method for inhibiting quorum sensing signal degradation and biofilm of Vibrio Havey by administering the strain, culture, concentrate or dried product to the farm.

Still another object of the present invention is to provide a method for preventing diseases caused by pathogens of animals other than humans by adding the above strains, cultures, concentrates or dried products.

Still another object of the present invention is to provide a water-quality improving agent comprising the strain, culture, concentrate or dried product.

Still another object of the present invention is to provide a method of preparing modified water by treating the strain, culture, concentrate or dried product with water.

An object of the present invention is to provide a new isolated Bacillus strain CJS-26 ( Bacillus sp., CJS-26) (KCCM11144P). The strain can degrade the quorum sensing signal factor of Vibrio Havey and inhibit biofilm production.

Specifically, samples from seawater were collected, mainly from shrimp farms in Ganghwa-do, and cultured in BHI solid medium containing 3% sodium chloride, and then grouped according to colony observation to isolate the strains. Among the isolates, strains with excellent antimicrobial activity against aerobic monas salmononeda, vibrio habay, vibrio anguillalum, edward ciella tadda, streptococcus inier and vibrio hemoratiticus Primary selection. In addition, six kinds of strains excellent in the activity of useful digestive enzymes such as protease, cellulase, amylase, and lyphase among the first selection strains having excellent antibacterial activity were selected secondarily. Bacillus sp. CJS-26, a strain that degrades the signal factor of quorum sensing of Vibrio Havey in the second selection strain and inhibits biofilm formation, was finally selected.

The morphological characteristics of Bacillus sp. CJS-26 were Gram-positive bacillus, and showed 16% homology with Bacillus sp. In this regard, the inventors of the newly isolated Bacillus sp. CJS-26 were added to the Korea Spawn Association (3F, Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea). Deposited as " Bacillus sp. CJS-26" on the 14th day (KCCM11144P).

In another aspect of the present invention, there is provided a culture of the strain, a concentrate of the culture or a dried product of the culture. Specifically, the culture medium of the present invention means a culture medium for culturing Bacillus strain CJS-26, preferably a culture medium containing the strain.

In the present invention, the culture medium refers to a medium containing nutrients necessary for growing animal cells, plant cells, bacteria, and the like, and the culture medium means cultured by inoculating a strain into a liquid medium. The culture solution may include a strain, or a culture filtrate in which the bacteria are inoculated, and then the bacteria are inoculated, and the strain is removed. The concentrate of the culture medium refers to the concentration of the culture solution, and the dried product of the culture solution means that the water of the culture solution is removed. Drying methods may be, but not limited to, ventilation drying, natural drying, spray drying and freeze drying.

In another aspect of the present invention, there is provided a probiotic formulation comprising the newly isolated Bacillus sp. Strain or a culture solution of the strain, a concentrate thereof or a dried product thereof as an active ingredient, and a pharmaceutically acceptable carrier.

As used herein, 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 pharmaceutically acceptable carriers in compositions formulated as liquid solutions include sterile and physiologically compatible carriers, including saline solution, sterile water, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, 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. In addition, diluents, semiacids, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

Probiotics settle on the gut wall of the intestine, preventing harmful bacteria from settling and inhibiting the growth of pathogens. In addition, the beneficial digestive enzymes produced by probiotics facilitate the absorption and utilization of nutrients, improving feed demand.

The composition of the present invention comprises 5 x 10 ⁴ to 5 x 10 ¹⁰ CFU / ml, preferably 1 x 10 ⁶ to 1 x 10 ⁹ CFU / ml Bacillus sp. CJS-26.

Oral dosage forms containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. 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.

In another aspect, the present invention relates to a feed additive comprising the newly isolated Bacillus sp. Strain or a culture solution of the strain, a concentrate thereof, or a dried product thereof.

Typically, Bacillus spp. Form endogenous spores and have very stable heat resistance. Therefore, the newly isolated Bacillus spp. CJS-26 may be prepared by separately mixing the feed additives in the form of feed additives or by adding them directly when preparing the feed. Bacillus sp. CJS-26 in the feed of the present invention may be liquid or dried, 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 sp. CJS-26 of the present invention may be mixed in the form of powder at a component ratio of 0.05 to 10% by weight, preferably 0.1% to 1% by weight of the feed weight. In addition, the aquaculture feed may further include conventional additives to increase the shelf life of the feed in addition to Bacillus sp. CJS-26 of the present invention.

In the present invention, the feed containing Bacillus sp. CJS-26 is a vegetable, and there are grains, fruits, food processing by-products, algae, fibres, 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.

Probiotic formulations comprising Bacillus spp. CJS-26 in the present invention include binders, emulsifiers, preservatives, and the like, added to prevent deterioration of quality, and include amino acids, vitamins, enzymes, and flavoring agents added to feeds to increase their effectiveness. , Nonprotein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, and the like. In addition, the feed mixture may further include, but is not limited thereto.

In still another aspect of the present invention, there is provided a method of farming a fish or shellfish, comprising treating the newly isolated Bacillus sp. Strain or the culture solution, the concentrate or dried product thereof, with a fish or shellfish farm.

As described above, the newly isolated Bacillus sp. CJS-26 of the present invention can inhibit symbiosis, pathogenesis, competition, and biofilm formation with a wide range of antibacterial activity, so that the representative pathogenic bacteria of cultured fishes can be obtained by using the strain. It is possible to obtain the effect of preventing the disease caused by the, and can safely fish or shellfish.

In still another aspect of the present invention, there is provided an antimicrobial agent comprising the newly isolated Bacillus sp. Strain or the culture solution of the strain, the concentrate thereof or the dried product thereof, and further, the newly isolated Bacillus sp. Strain or the culture solution of the strain, A method of inhibiting the formation of a biofilm as a contaminant in a fish or shellfish farm using a concentrate or a dried product thereof, or by using the newly isolated Bacillus sp. Strain or a culture solution of the strain, a concentrate thereof or a dried product thereof, except for humans It provides a method for preventing diseases caused by pathogenic bacteria in animals.

In the present invention, the term "prevention" means any action that inhibits or delays the onset of the disease by administration of the composition.

The newly isolated Bacillus sp. CJS-26 decomposes the autoderiver-2, a quorum-sensing signal factor of Vibrio Havey, and inhibits the formation of biofilm, thus controlling quorum sensing of Vibrio Havey, thereby promoting symbiosis, pathogenesis, competition, bio By inhibiting film formation, diseases caused by Vibrio Havey can be prevented. In addition, the newly isolated Bacillus sp. CJS-26 has antimicrobial activity against six representative pathogenic bacteria of the aquaculture fish described above, and it was confirmed that it is not hemolytic in blood agar medium. It can prevent the disease caused.

Pathogens refer to the disease-causing organisms. In particular, pathogens that can be prevented include Aeromonas Salmonesida. salmonicida , Vibrio harveyi ), Vibrio angelalium ( Vibrio anguillarum ), Edwardsiella tarda ), Streptococcus iniae) and V. H. ET Mora kusu (Vibrio hemolyticus ) and the like, and most preferably can prevent Vibrio Havey.

In still another aspect of the present invention, there is provided a water quality improving agent comprising the newly isolated Bacillus sp. Strain or a culture solution of the strain, a concentrate thereof, or a dried product thereof.

The novel isolated Bacillus sp. CJS-26 of the present invention inhibits the biofilm formed by Vibrio Havey and reduces ammonia present in the aquaculture environment. The water improver of the present invention may be prepared separately from the newly isolated Bacillus sp. CJS-26 in the form of a water improver, or directly sprayed with the strain and / or probiotic agent. Bacillus sp. CJS-26 in the water improver of the present invention may be liquid or dried, preferably in the form of a dried powder.

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. In addition, diluents, semiacids, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

The water quality improver can be used to improve the water quality of the farm, specifically, the water quality improver can be added to the farm before or during the farming, preferably by adding it to the farm before leaving it for a period of time. By inhibiting the quorum sensing signal factor using the newly isolated Bacillus spp. CJS-26 of the present invention, biofilm production is suppressed, and the water quality improvement agent is added at least once in the middle of culture, thereby suppressing further production of biofilm. .

In still another aspect of the present invention, there is provided a method of preparing modified water by removing pathogens, the method comprising treating the newly isolated Bacillus sp. Strain or the culture solution, the concentrate thereof or the dried product thereof with water. . The modified water may be used in aquaculture farms, of course, may be used as drinking water when raising livestock or as drinking water for humans. At this time, the newly isolated Bacillus sp. Strain or the culture solution of the strain, the concentrate or dried water thereof is preferably treated with water, the step of water purification is carried out by a known method.

Newly Isolated Bacillus Species of the Invention CJS-26 controls Vibrio Havey's quorum sensing, which is a problem in aquaculture, and can effectively block the physiological activities (symbiosis, pathogenesis, competition, biofilm formation, etc.) and pathogenicity caused by quorum sensing. It can be widely used in a variety of products that can control bacterial infectious diseases, including feed additives for fish farming, probiotics, water quality improvers and the like.

1 is a Bacillus species of the present invention It is a graph showing the resolution of autoinducer-2, a quorum sensing signal molecule, in Vibrio Habei of CJS-26 strain.
2 is a Bacillus species of the present invention The figure shows the evaluation of inhibition of biofilm formation by Vibrio habei when CJS-26 strain was incubated for 30 hours.
3 shows the present invention Bacillus species The figure shows the hemolytic presence of CJS-26 strain.
4 is the present invention Bacillus species A graph showing the ammonia availability of the CJS-26 strain.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Bacillus  Bell ( Bacillus sp .) CJS Isolation of -26 Strains

Example  1-1: Sample acquisition and strain isolation

Considering the characteristics of strains of probiotics with high environmental and host specificities, the present inventors secured samples from seawater centering on shrimp farms with low domestic disease occurrence and high productivity. The obtained sample was diluted in stages and plated in BHI agar (Difco, USA) to which 3% sodium chloride was added and incubated for 24 hours at 37 ° C. Strains isolated from each sample were separated by grouping according to colony observation. 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.

Example  1-2: Selection of strains with excellent antimicrobial activity

Aeromonas Salmonici for the primary selection of strains with antimicrobial activity against representative pathogenic bacteria in question salmonicida), Vibrio Harvey (Vibrio harveyi), anggwil raryum Vibrio (Vibrio anguillarum ), Edwardsiella tarda ), Vibrio hemoraiticus ( Vibrio hemolyticus ) was evaluated for 6 species.

Antimicrobial evaluation was made through analysis of growth inhibition of pathogenic bacteria. Specifically, 150 ml of 0.7 ml of agar (W / V) and 3 ml of the above six pathogenic bacteria shaking cultures (OD ₆₀₀ = 2.0) were respectively mixed and dispensed onto BHI medium to prepare top-agar. 10 μl of the strain culture solution selected on the prepared top-agar was incubated at 30 ° C. for 18 hours, and the presence or absence of a clear zone formed around the selected strain was observed (Table 1).

Antimicrobial Activity of Primary Starter strains One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 AS + - + - - + - - + - + + + + - - - - SI + - + - - - + - - - + + + + + - - - ET - - - - - + - - - - + + - + - - - - VH - - - - - - - - - - + + - + - - - - VP - - - - - - - - - - + + - + - - - - VA + - + - - + - - - - + - - - - - - - 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 AS - - - - + - - + + - - - - + - - + - SI - - - - + - - + + - - - - - - + + - ET - - - - + - - + + - - - - - - - - - VH - - - - + - - + - - + - - - - - - - VP - - - - - - - + - - + - - - - - - - VA - - - - - - - + - - - - - - - - - - 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 AS - - - - - - - - - - - - - - - + + SI - + - - - - - - - - - - - - - + + ET - - - - - - - - - - - - - - - - - VH - - - - - - - - - - - - - - - + - VP - - - - - - - - - - - - - - - + - VA - - - - - - - - - - - - - - - - -

-: No activity, +; Active

AS: Aeromonas Salmonesida salmonicida );

SI: Streptococcus iniae );

ET: Edwardsiella tarda );

VH: Vibrio harveyi );

VP: Vibrio Hemoratiticus hemolyticus );

VA: Vibrio Angelarium anguillarum )

As shown in Table 1, among the 53 strains, six strains showing antimicrobial activity against fish farming pathogens in total 20 species (strain numbers 1, 3, 6, 7, 9, 11, 12, 13, 14, 15) , 23, 26, 27, 29, 32, 34, 35, 38, 52, and 53), of which six strains (strain numbers 7, 9, 15, 32, 34, and 38) had one fish pathogenicity. Antimicrobial activity was shown only against bacteria, but the remaining 14 strains (strain numbers 1, 3, 6, 11, 12, 13, 14, 23, 26, 27, 29, 35, 52 and 53) It showed complex antimicrobial activity against pathogenic bacteria. Thus, 14 strains showing antimicrobial activity were selected first.

Example  1-3: Selection of strains with excellent enzyme activity

Example  1-3-1: Coenzyme solution  Collection

Among 14 strains showing the antimicrobial activity, the selected strains were selected to have a complex digestive enzyme activity.

Specifically, 14 strains showing complex antimicrobial activity selected in Example 1-2 were cultured in BHI medium for 8 and 24 hours, respectively, and the cultures were centrifuged at 4 ° C. and 13,000 rpm for 5 minutes, respectively. Culture supernatant of was obtained. Using the obtained culture supernatant as a coenzyme fraction for enzyme activity analysis, the activity of protease, cellulase, amylase and lyase contained therein was measured as follows.

Example  1-3-2: Protease Activity

YM (Difco, USA) medium (substrate medium) containing 2% skim milk (Skimma, Sigma, USA) as a substrate of the protease was prepared, and each of the substrate medium obtained in Example 1-3-1 The crude enzyme fraction of was added, and then reacted at 30 ° C. for 2 hours. After the reaction was completed, the formation area of the clear zone generated by decomposing the substrate contained in the medium by the protease included in each coenzyme fraction was measured to determine the activity of the protease contained in each coenzyme fraction. Calculated.

Example  1-3-3: Cellulase  activation

A YM medium (substrate medium) containing 1% CMC (carboxyl methyl cellulose) as a substrate of cellulase was prepared, and each coenzyme fraction obtained in Example 1-3-1 was added to the substrate medium, followed by 30 ° C. The reaction was carried out for 2 hours at. After the reaction was completed, the cellulase included in each coenzyme fraction was measured to determine the area of the transparent ring formed by decomposition of the substrate contained in the medium, thereby calculating the activity of the cellulase contained in each coenzyme fraction.

Example  1-3-4: Amylase  activation

A YM medium (substrate medium) containing 1% soluble starch as a substrate of amylase was prepared, and each coenzyme fraction obtained in Example 1-3-1 was added to the substrate medium, and then at 30 ° C. The reaction was carried out for 2 hours. After the reaction was completed, the activity of amylase contained in each crude enzyme fraction was calculated by measuring the formation area of the transparent ring formed by decomposing the substrate contained in the medium by amylase included in each crude enzyme fraction.

Example  1-3-5: Rye Phase  activation

A YM medium (substrate medium) containing 1% tricaptylin as a substrate of lyphases was prepared, and each coenzyme fraction obtained in Example 1-3-1 was added to the substrate medium, and then 30 The reaction was carried out at 2 ° C. for 2 hours. After the reaction was completed, the area of the transparent ring formed by decomposing the substrate contained in the medium by the lipases contained in the respective coenzyme fractions was measured to calculate the activity of the lipases contained in the respective coenzyme fractions.

Example  1-3-6: Comparison of Enzyme Activity of Selected Strains

By comparing the activities of the enzymes respectively calculated in Examples 1-3-2 to 1-3-5, six strains (strain Nos. 11, 12, 14, 23, 26 and 52) were selected secondarily (Table 2). The six selected strains were named CJS11, CJS12, CJS114, CJS26, CJS59 and CJS83, respectively.

 Enzyme Activity of Second Screening Strains enzyme Protease Amylase Cellulase Rye Phase Strain Culture Time 8 hr 24 hr 8 hr 24 hr 8 hr 24 hr 8 hr 24 hr 11 +++ +++ + ++ +++ ++ - - 12 +++ ++ +++ ++ +++ ++ - + 14 ++ + + ++ +++ +++ - +++ 23 +++ ++ + ++ +++ ++ - - 26 ++ +++ + +++ +++ +++ - +++ 52 +++ ++ + + +++ +++ - ++

-: No activity, +; Active, ++: excellent activity, +++: very active

Example  2: Quorum sensing  Selection of strains showing control and biofilm inhibitory effects

Six strains excellent in antimicrobial activity and enzyme activity selected in Example 1 were decomposed to decompose the autoinductor that induces quorum sensing, and to select a strain that can effectively inhibit the formation of biofilm.

Example  2-1: Quorum sensing  Selection of strains showing control effect

Vibrio , a standard strain of quorum sensing in response to autoderivers-1 and autoderivers-2 Among harveyi) BB120 (ATCC BAA-1116 , USA) and the parties may use a mutant strain of Vibrio Harvey BB170 able to respond only to the derivative 2, in Example 1 the strains of the six kinds of starting a high antimicrobial activity and enzyme activity in In Vibrio Havey, a strain was selected that could inhibit the activity of autoinductor-2, which induces quorum sensing. At this time, the method for selecting a strain that can inhibit the activity for autoderiver-2 was carried out by applying the fact that when the Vibrio Habei BB120 and Vibrio Habei BB170 is in close proximity, it emits itself by the quorum sensing reaction.

First, a quorum sensing evaluation medium (AB medium) capable of culturing the two Vibrio habayes was prepared with the composition shown in Table 3 below.

Quorum Sensing Evaluation Badge (AB Badge) Sodium Chloride (Nacl) 17.5 g Magnesium Sulfate (MgSO ₄ ) 12.3 g Casamino acid 2.0 g 1M Potassium Phosphate 10 ml 0.1M L-arginine 10 ml Glycerol 10 ml

Then, Vibrio Habei BB120 was inoculated with 0.5% (v / v) of the AB medium, incubated for 12 hours at 30 ° C. and 200 rpm, and Vibrio Habei BB170 was inoculated with 0.02% (v / v) of the AB medium, The cultures were incubated for 3 hours at 30 ° C. and 200 rpm. The six strains selected in Example 1 were inoculated with 0.1% (v / v) in BHI medium, and incubated for 8 hours.

After the incubation was completed, each culture was centrifuged at 4 ° C. and 13,000 rpm, and the supernatant thereof was filtered with a 0.45 μm filter to obtain an active fraction of each strain.

Vibrio Havey to evaluate whether six probiotic selection strains have the resolution of autoinducer-2 (AHL), a quorum-sensing signaling molecule from Vibrio Havey The supernatant of BB120 culture and the culture supernatant of probiotic selection strain were mixed 1: 1 and reacted for 3 hours. After 1 hour addition to Vibrio Habei BB120 incubated for 3 hours was further incubated for 4 hours at 30 ℃, 200 rpm to measure the degree of luminescence by Luminomete.

As a result, the six strains significantly degraded Vibrio Habei autoderiver-2 (AHL) ( p <0.001), and among these three strains (CJS11, CJS26 and CJS83), the most autolytic derivatives (AHL) degraded most. ) Was selected ( FIG. 1 ). 1 is a Bacillus species of the present invention It is a graph showing the resolution of autoinducer-2, a quorum sensing signal molecule, in Vibrio Habei of CJS-26 strain.

Example  2-2: Vibrio In habei  Selection of strain inhibiting biofilm formation by

In order to select a probiotic strain that inhibits the formation of a biofilm formed by Vibrio harveyi , an evaluation of the strain having excellent quorum sensing control effect was conducted. The group (control) and the treatment group in which the biofilm was formed by culturing Vibrio Havey were treated with each probiotic selection strain, and the three repeated evaluations were performed as follows.

200 μl of Vibrio Habei was inoculated into 3 ml of LB medium (Difco, USA) to which 3% sodium chloride was added, and the cells were incubated at 30 ° C. for 6 hours. Thereafter, 200 μl of each probiotic selection strain was added thereto, followed by further incubation for 24 hours. The culture medium was removed for a total of 30 hours, and stained with 0.4% crystal violet for 10 minutes to confirm biofilm formation. As shown in Table 4, the Vibrio habei formed a purple band-shaped biofilm stained with crystal violet, while probiotic selection strains inhibited biofilm formation (Fig. 2). 2 is a Bacillus species of the present invention The figure shows the evaluation of inhibition of biofilm formation by Vibrio habei when CJS-26 strain was incubated for 30 hours.

Evaluation of inhibition of biofilm activity at 30 hours of incubation vibrio Habei +++ CJS114 + CJS26 - CJS53 +

Example  3: Identification, Physiology and Biochemical Characterization of Selected Strains

Example  3-1: Strain Identification

Among the selected strains, CJS26 strains excellent in quorum sensing control and biofilm formation inhibitory ability were finally selected and analyzed. The strains were identified by physiological, biochemical and molecular lineage methods. The morphological characteristics were identified as Gram-positive bacillus and identified as a novel microorganism having 99% homology with Bacillus sp. By 16s rDNA gene sequencing. The base sequence of the analyzed 16s rDNA of the isolated strain is shown in SEQ ID NO: 1.

The sequencing was performed by gene amplification of 16s rDNA using PCR premix (Bionia, Korea) and universal primers 27F and 1492R having the following sequencing.

27F: 5'-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 2)

1492R: 5'-GGYTACCTTGTTACGACTT-3 '(SEQ ID NO: 3)

In the amplification of the gene, the total reaction solution was adjusted to 20 μl and repeated 30 times at 94 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1 minute to analyze the amplified DNA sequences. The novel microorganism of the present invention identified by the above method was deposited in the Bacillus sp. CJS26 on December 14, 2010 to the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul). (Accession No. KCCM11144P).

Example  3-2: Investigation of physiological and biochemical properties

In order to analyze the biochemical characteristics of the strain of the invention, the sugar fermentation pattern of the strain was analyzed by API 50 CHB system (Korea Microorganism Conservation Center, Korea) (Table 5).

Sugar fermentation pattern analysis results Control - Esculline + Glycerol + Salicine + Erythritol - Cellobiose + D-Arabinose _ Maltose + L-Arabinose + Lactose + Ribose + Melibiose _ D-Xylose + Saccharose + L-Xylose _ Trehalose + Adonitol _ Inuline _ β-methyl-gyroside
(βMethyl-xyloside) _ Merezitose _ Galactose _ D_Raffinose + D-Glucose + Amidon + D_Fructose + Glycogene + D-Mannose + Xylitol _ L-Sorbose _ βGentiobiose _ Rhamnose _ D-Turanose _ Dulcitol _ D-Lyxose _ Inositol + D-Tagatose _ Mannitol + D-Fucose _ Sorbitol + L-Fucose _ αmethyl-dimannoside
(αMethyl-Dmannoside) _ D-Arabitol _ α-methyl-glucoside
(αMethyl-glucoside) + L-Arabitol _ N Acetyl Glucosamine
( N Acetyl glucosamine) _ Gluconate _ Amygdaline + 2 ceto-gluconate _ Arbutine + 5 ceto-gluconate _

+, Positive; -, voice

Example  4: safety and usability

Example  4-1: hemolytic (β- hemolysis )

β-hemolysis is the action of producing phospholipid enzymes in harmful bacteria to hydrolyze erythrocytes by hydrolyzing phospholipids supplied by red blood cells.

In order to determine the hemolytic properties of the selected bacteria, TSA (Difco, USA) containing 5% sheep blood (sheep blood, Kisan Biotech, Korea) was prepared and linearly inoculated the Bacillus strain CJS26 of the present invention on the prepared blood agar medium. After (streaking), incubation at 37 ℃ for 24 hours to check the hemolysis. At this time, hemolysis was determined to be hemolyzed when the cultured strain showed white or yellow color, and not hemolyzed when the cultured strain did not show color (refer to FIG. 3). 3 shows the present invention It is a photograph showing the presence or absence of hemolytic activity of Bacillus CJS-26 strain. As shown in Figure 3, the Bacillus CJS-26 strain of the present invention was confirmed that does not show hemolytic properties.

Example  4-2: Ammonia Solubility

In order to confirm ammonia availability of the Bacillus CJS-26 strain of the present invention, a medium containing ammonia was prepared as shown in Table 6 below. Bacillus spp. In the prepared medium CJS-26 was inoculated at 0.1% and then cultured at 30 ° C. and 200 rpm for 6 hours, 12 hours, and 24 hours. The collected culture solution sample was quantified by measuring ammonia present in the supernatant of the medium obtained by centrifuging at 13,000 rpm for 5 minutes at 4 ° C. (see FIG. 4).

Ammonia Availability Evaluation Medium Composition Ammonium Sulfate ((NH ₄ ) ₂ SO ₄ ) 0.5 g / L Disodium Phosphate (Na ₂ HPO ₄ ) 13.5 g / L Potassium Phosphate (K ₂ HPO ₄ ) 0.7 g / L Magnesium sulfide (MgSO ₄ ㆍ 7H ₂ O) 0.1 g / L Calcium chloride (CaCl ₂ ) 0.18 g / L Sodium bicarbonate (NaHCO ₃ ) 0.5 g / L Ferric chloride (Fecl ₃ ㆍ 6H ₂ O) 0.014 g / L Glucose 0.5 g / L

4 is the present invention A graph showing the ammonia availability of Bacillus CJS-26 strain. As shown in Figure 4, the initial ammonia concentration in the medium was 400 ppm, Bacillus spp. Ammonia was not detected at 30 hours of incubation as the CJS-26 strain grew and used ammonia. The fact that no ammonia was detected in the medium can prevent water pollution due to ammonia using the Bacillus CJS-26 strain of the present invention, as well as by reducing ammonia which causes stress on fish or shellfish. This means that the quality of fish or crustaceans can be improved.

Based on these results, Bacillus spp. CJS-26 strains can act in combination with quorum sensing control and antimicrobial activity of pathogenic bacteria including Vibrio habay, promoting digestibility of fish by production of complex digestive enzymes, improving weight gain, reducing feces and improving water quality.

Example  5: Probiotics  Efficacy Evaluation

When the strain of the present invention is added to the feed to feed the fish, in order to see if it acts as a probiotics, after mixing the strain of the present invention into the feed for 8 weeks to flounder fry, weight gain, daily growth rate, feed Conversion efficiency, protein conversion efficiency and survival rate were measured.

Specifically, the basic feed was prepared as shown in Table 7 so that the same energy content as the crude protein of 38%. The strain of the present invention was added to the basic feed (control) without added probiotics at a concentration of 0.5%, and the energy was equalized by reducing the amount of cellulase of the basic feed by the amount of probiotics added.

Basic feed composition table Raw material name % White fish meal 36.0 Soybean meal 12.0 Corn gluten meal 8.0 Wheat flour 30.4 Squid liver oil 5.0 Soy bean meal 5.0 CMC 1.0 Cellulose 0.5 Mineral premix 1.0 Vitamin premix 1.0

The initial average weight of the flounder fry used for the test was 25 g, and randomly selected 25 animals were placed in a 150 L round PP bath. The breeding water was adjusted to a flow rate of 2 to 3 L / min using sand-filtered seawater, and airstones were installed in all test tanks to maintain dissolved oxygen and smooth breeding water. Photoperiod was maintained at 12L: 12D condition using fluorescent lamps, and the breeding water temperature depended on natural water temperature in the range of 21 ~ 29 ℃ during the whole experiment. Experimental feed was fed two times a day, full stomach. Growth rate was measured every three weeks, and all experimental fish were fasted for 24 hours before measurement.

The Bacillus CJS 26 strain showed higher growth rate of about 16% compared to the control group, and showed higher trend in feed conversion efficiency and protein conversion efficiency than the control. Survival was 21% higher than that of Daebo (Table 8). Therefore, the strain of the present invention is effective for promoting the digestibility of fish and improving the weight gain.

Evaluation of Efficacy in Feeding Probiotics in Olive Flounder Control Test Final average weight (g) 50.6 ± 1.7 ^a 55.1 ± 2.5 ^b % Increase 103.03 ± 5.6 ^a 121.16 ± 18.3 ^b Daily growth rate (%) 1.42 ± 0.06 ^a 1.58 ± 0.12 ^b Survival rate (%) 77.3 ± 15.1 ^a 98.7 ± 2.3 ^b Feed conversion efficiency 1.59 ± 0.15 ^b 1.24 ± 0.05 ^b Protein Conversion Efficiency 1.10 ± 0.1 ^a 1.30 ± 0.1 ^b

Korea Microorganism Conservation Center (overseas) KCCM11144P 20101214

Claims (13)

Bacillus strain CJS-26 ( Bacillus sp. CJS-26) (KCCM11144P).
The Bacillus strain of claim 1 CJS-26 (Bacillus sp., CJS-26) culture broth of (KCCM11144P), its concentrated liquid or a dry product.
Probiotic formulation comprising the strain of claim 1 or the culture solution of claim 2, its concentrate or dried product as an active ingredient
A feed additive comprising the strain of claim 1, the culture solution of claim 2, a concentrate thereof, or a dried product thereof as an active ingredient.
The feed additive according to claim 4, which is used in aquaculture of fish or crustaceans.
A fish or shellfish aquaculture method comprising treating a strain of claim 1 or a culture of claim 2, a concentrate thereof or a dried product thereof in a farm of fish or a shellfish.
An antimicrobial agent comprising the strain of claim 1 or the culture solution of claim 2, a concentrate thereof, or a dried product thereof.
(a) farming fish or crustaceans on the farm; And
(b) A method for decomposing a quorum sensing signal factor of Vibrio havey, comprising administering the strain of claim 1 or the culture solution of claim 2, a concentrate thereof, or a dried product thereof to the aquaculture farm, and inhibiting the biofilm.
The method of claim 8, wherein steps (a) and (b) are performed sequentially, simultaneously or in reverse order.
A method for preventing a disease caused by pathogens in animals other than humans, comprising adding the strain of claim 1 or the culture solution of claim 2, a concentrate thereof, or a dried product thereof.
The method of claim 10, wherein the pathogen is aeromonas salmonicida ( Aeromonas salmonicida), Vibrio Havey ( Vibrio harveyi ), Vibrio angelalium ( Vibrio anguillarum ), Edwardsiella tarda ), Streptococcus iniae ) and Vibrio hemolyticus .
The water quality improving agent containing the strain of Claim 1 or the culture solution of Claim 2, its concentrate, or its dried substance as an active ingredient.
A process for producing modified water, comprising the step of treating the strain of claim 1 or the culture solution of claim 2, a concentrate thereof, or a dried product thereof with water.

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