KR101230813B1 - Probiotics Agent Against Saprolegnia sp. - Google Patents

Abstract

The present invention relates to probiotics for biological control against microorganisms of the genus Saprolegnia sp. Specifically, it inhibits the growth of the pathogenic fungus Saprolegnia sp. And produces sideropores . Newly isolated Bacillus strains, cultures obtained by culturing the strains, probiotic preparations, feed additives, antimicrobial agents, antifungal or water-enhancing agents, including the strains, the concentrates or dried products thereof and the strains, cultures, concentrates or dried products, and the strains , A method of farming fish or shellfish using culture, concentrate or dried product, adding the strain, culture, concentrate or dried product to prevent aquatic fungal diseases in animals other than humans or the strain, culture, concentrate in water Or to a method of producing modified water by treating dry matter.

Description

Probiotics Agent Against Saprolegnia sp.

The present invention relates to probiotics for biological control against microorganisms of the genus Saprolegnia sp. Specifically, it inhibits the growth of the pathogenic fungus Saprolegnia sp. And produces sideropores . Newly isolated Bacillus strains, cultures obtained by culturing the strains, probiotic preparations, feed additives, antimicrobial agents, antifungal or water-enhancing agents, including the strains, the concentrates or dried products thereof and the strains, cultures, concentrates or dried products, and the strains , A method of farming fish or shellfish using culture, concentrate or dried product, adding the strain, culture, concentrate or dried product to prevent aquatic fungal diseases in animals other than humans or the strain, culture, concentrate in water Or to a method of producing modified water by treating dry matter.

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).

The causes of aquaculture diseases are transmitted from infected fish, or from the introduction of pathogens in aquatic water environment. In addition, it is caused by contamination of the feed supplied or due to poor management of the fish farming environment. Infectious diseases of fish are divided into fungal, pathogenic bacterial and viral diseases. Among them, fungal diseases are caused by the aquatic fungus Saprolegnia sp. ( Focus on Diagnosis and Treatment of Fish Diseases. Ministry of Maritime Affairs and Fisheries. 2001). Saffronegnia is a representative species of water fungus that grows in freshwater ecosystems and causes fungal infections of freshwater fish. In fish, saffronegnia invades epithelial tissue, usually starting from the head or fins and spreading to the surface of the entire body, causing serious disease in the farm. Although it is primarily infected by saffronegnia, it has been reported that the primary infection is caused by parasites such as viruses and bacteria, and then secondary infection through the damaged site (Aquatic). fungi and fish production in Egypt, II- in vivo studies).

In order to prevent the damage caused by the saprolegnia, synthetic antifungal agents (for example, malachite green agents, etc.) have been used so far, but these synthetic antifungal agents are not easily biodegraded and tend to accumulate in the environment, thereby deteriorating the environment. As well as causing the side effects, there are problems such as large and small side effects, resistant strains are generated, and there is a continuous effort to limit the use thereof and to develop a formulation that can replace it (Ebele M. et al. African Journal of Biotechnology Vol. 8 (24), 7130-7132, 2009). As part of this effort, US Patent No. 6160023 discloses a method for treating aquatic organisms infected with fungal infections, protozoa infections and bacterial gill diseases using a substance that releases bronopol of aquatic organisms infected with molds and the like. A method for disinfecting a water tank and a fishing device using bronopol is disclosed, and Korean Patent No. 082500 discloses a fungal disease treatment agent for fish containing sweet flag (sweet flag. Acorus calamus) extract as an active ingredient. 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. The 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 intestinal flora caused by antibiotics. 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. Therefore, 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 has been conducted on disease improvement due to increased immunity to pathogenicity and improved water quality for 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 at low pH of the gastric 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).

On the other hand, siderophore is a type of antibiotic produced by microorganisms including pathogenic bacteria to compete with other microorganisms for rapid growth and proliferation in a given environment. It transports into cells using a transport system, and the iron ions (Fe3 +) are used to grow microorganisms. Microorganisms produce sideropores in order to competitively use iron ions (Fe ^{3 +} ), and the growth of other microorganisms may be suppressed by many microorganisms that produce sideropores. Research into the use of microorganisms producing sideropores for biocontrol is being actively conducted. Since the 1970s, the disease suppression in soils that plague the land reported by the antagonism of microorganisms in the soil as part of a biological pest control methods genus Pseudomonas (Pseudomonas A study on the control method using competitive antagonism that inhibits the growth of phytopathogens by siderophore, an iron-specific binding substance secreted by sp.), etc. (Woo Sang-min and Kim Sang-dal, Kor. J. Microbiol.Biotechnol.Vol. 36 (4): 326-335 (2008)). So far known, sideropores are metabolites that are soluble, have a low molecular weight of less than 1 kDa, and have good affinity with iron ions. Sideropores are classified into catechol type and hydroxamate type according to functional groups that bind iron ions, and active research is being conducted on the sideropores and probiotics producing them. As a part of this research, US Patent No. 6667667 discloses a method of inhibiting the growth of L. lactis by using a siderophore produced in Bifidobacteria, and the Korean Patent No. No. 037043 uses a novel antagonistic strain Pseudomonas fluorescein LS20, which produces sideropores, and a biological control method for inhibiting the growth of the plant root pathogen, Fusarium solani, by using the pseudomonas fluorescein LS20 and the sideropores. A method for treating a disease derived from a pathogenic fungus of a plant is disclosed, and Korean Patent No. 0747700 discloses a red pepper containing Bacillus subtilis producing sideropores and sideropores produced therefrom as an active ingredient. Phytohtora capsici ) or tomato Fusarium oxysporum control composition is disclosed .

As a result of patent analysis, the technology mainly focused on the biological control effect of pathogenic microorganisms and molds using microorganisms with excellent sideropore production, and the technology for inhibiting fish pathogenic microorganisms using probiotic Bacillus is reported. It has not been done.

The inventors of the present invention separated probiotics excellent in the production of antibacterial and digestive enzymes against pathogenic bacteria which are a problem in fish and shrimp, and confirmed their morphological, biochemical and genetic properties. As a result, the probiotics have excellent heat resistance. It was confirmed that it is Bacillus, it was confirmed that the Bacillus is excellent in the inhibition and inhibition of the sideropore productivity and aquatic fungus sapregegnia.

Therefore, the present inventors inhibit the newly isolated Bacillus species aquatic fungus saffronegnia, and has excellent siderophore productivity and competent antagonism against pathogenic bacteria as it is excellent in capturing iron, cultured fish and crustaceans It has been confirmed that the present invention can be used as a probiotic formulation for the present invention and has been completed.

It is an object of the present invention to provide novel isolated Bacillus strains that produce saprolegnia growth and produce sideropores.

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 or antifungal agent comprising the strain, culture, concentrate or dried product.

Another object of the present invention is to provide a method for preventing aquatic fungal diseases by adding the strain, culture, concentrate or dried product.

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.

In one embodiment of the present invention, Bacillus sp. Strain CJP-14 ( Bacillus sp., CJP-14) (KCCM11143P) is provided. The strain produces sideropores and may inhibit sapregnia growth.

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 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 strains of the first selection strains having excellent antibacterial activity were excellent in the activity of useful digestive enzymes such as protease, cellulase, amylase, and lyphase.

Among the second selected strains, the inhibitory effect of sapregegnia, a pathogenic fungus that is a problem in freshwater fishes, is excellent, and the sideropore productivity is excellent, so the ability to capture iron (Fe3 +) increases the growth of other pathogenic bacteria. The Bacillus spp. CJP-14 was finally selected which could inhibit competitive antagonism.

The morphological characteristics of Bacillus spp. CJP-14 were Gram-positive bacillus. The 16s rDNA sequence analysis showed 99% homology with Bacillus sp. The results show that the 16s rDNA sequence of Bacillus subtilis AH18, which produces a conventional siderophore, Bacillus Compared with the results of the subtilis strain CICC 10088 and 98% homology (see Republic of Korea Patent Registration No. 0817700), it can be seen that the strain of the present invention is a new strain that is not known in the prior art. In addition, the strain of the present invention can produce not only siderophore, but also cellulase, protease, amylase, lyphase, and the like, and the Bacillus subtilis AH18 can produce cellulase and auxin in addition to siderophore. Since it can only be, once again confirmed that the strain of the present invention is a new strain that is not known in the past.

The present inventors have found that "Bacillus, Bacillus species of the CJP-14 a new separation as of December 14, 2010 the corporation Korea Seed Association (Seoul Seodaemun Hongje 1 Dong 361-221 Yoo - Lim Bldg, my material Korea Culture Center of Microorganisms) sp. CJP-14 "(KCCM11143P).

In another aspect of the present invention, there is provided a culture solution, a concentrate thereof, or a dried product thereof, wherein the newly isolated Bacillus sp. Strain is cultured. Specifically, the culture medium of the present invention means a culture medium of the newly isolated Bacillus sp. Strain CJP-14, and preferably, the 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 be one containing a strain, or a culture filtrate in which the strain is inoculated, and then cultured by inoculation. The concentrated solution of the culture medium refers to the concentration of the culture solution, and the dried material 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.

The newly isolated Bacillus sp. Strain, which is an active ingredient included in the probiotics of the present invention, is settled on the digestive tract wall of cultured fish or crustaceans, preventing harmful bacteria from settling and inhibiting the growth of pathogens. In addition, the beneficial digestive enzymes produced from the strains facilitate the absorption and utilization of nutrients, thereby improving feed rate requirements.

The formulations of the invention comprise 5 × 10 ⁴ to 5 × 10 ¹⁰ CFU / ml, preferably from 1 × 10 ⁶ to 1 × 10 ⁹ CFU / ml of Bacillus sp. CJP-14.

The formulations of the present invention may preferably be prepared in oral dosage forms, for example tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or It can be formulated in soft capsules, syrups or elixirs. 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 include a liquid carrier such as fatty oil in addition to the above-mentioned materials.

In still another aspect of the present invention, there is provided 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. CJP-14 may be prepared separately in the form of feed additives and mixed with the feed, or directly added during feed preparation. Bacillus sp. CJP-14 in the feed of the present invention may be liquid or dried, preferably in the form of a dried powder. Drying methods include, but are not limited to, air drying, natural drying, spray drying and freeze drying. Bacillus sp. CJP-14 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. In addition, the aquaculture feed may further include conventional additives to increase the shelf life of the feed in addition to the Bacillus species CJP-14 of the present invention.

In the present invention, the feed containing Bacillus spp. CJP-14 includes grains, 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.

The probiotic agent containing Bacillus spp. CJP-14 in the present invention includes a binder, an emulsifier, a preservative, and the like, which are added to prevent quality deterioration. , 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. CJP-14 of the present invention can play a competitive antagonism that inhibits the growth of other pathogenic bacteria with a wide range of antimicrobial activity, so that the representative pathogenic bacteria of cultured fish 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 antibacterial or antifungal agent comprising the newly isolated Bacillus sp. Strain or the culture solution of the strain, the concentrate thereof or the dried product thereof.

The present invention also provides a method for preventing aquatic fungal diseases of animals other than humans by using the newly isolated Bacillus sp. Strain or the culture solution of the strain, the concentrate thereof or the dried product thereof.

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

The newly isolated Bacillus sp. CJP-14 of the present invention has antimicrobial activity against six representative pathogenic bacteria of the aquaculture fish described above, and grows other pathogenic bacteria according to its ability to capture iron by producing sideropores. Competitive antagonism. In addition, since it was confirmed that it can inhibit the growth of pathogenic aquatic fungus sapregegnia, it is possible to prevent diseases caused by the representative pathogenic bacteria of the aquaculture fish described above using the new isolated Bacillus sp. Strain of the present invention. .

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. CJP-14 of the present invention can be used to reduce the nitrous acid content present in the aquaculture environment.

The water quality improver of the present invention may be prepared separately from the newly isolated Bacillus sp. CJP-14 in the form of a water quality improver, or may be directly sprayed with the newly isolated Bacillus sp. Strain or the culture solution of the strain, its concentrate or dried product thereof. Bacillus sp. CJP-14 in the water conditioner of the present invention may be liquid or dried, preferably in the form of a dried powder.

Acceptable carriers for the preparation of water quality are sterile and biocompatible, and include saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and one or more of these components. It can be used, and other conventional additives such as antioxidants, buffers, bacteriostatics can be added as necessary. 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. In addition, by producing enough siderophore from the newly isolated Bacillus sp. CJP-14 of the present invention, it has excellent iron trapping ability, inhibits the growth of other pathogens, and inhibits the growth of saffronegnia, thereby improving the water quality in the middle of culture. The agent is added one or more times to inhibit further growth of saffronegnia.

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 Since CJP-14 has excellent antifungal effect against aquatic fungus saffronegnia and can produce sideropores, CJP-14 can control a variety of fungal and bacterial infectious diseases against aquatic fungus saffronegnia. It can be widely used in products such as aquaculture feed additives, probiotics, water quality improvers and the like.

1 is a Bacillus species of the present invention It is a photograph showing the result of the inhibition of growth of aquatic fungus Sproegnia of CJP-14 strain.
Figure 2 shows the bacterium CJP-14 strain of the present invention and vibrio habei ( V. harveyi) fish farming pathogenic microorganisms VH1, VH2, VH3), Vibrio cholera ( V. cholerae , VC), Vibrio blniificus ( V. vulnificus , VV), antibacterial by siderophore formation to aeromonas Salmonicida ( A.salmonicida , AS) It is a photograph showing the result of evaluation of activity in CAS medium.
FIG. 3 is a cross- Bacillus species It is a photograph showing the hemolytic presence of CJP-14 strain.
4 is the present invention Bacillus species Graph showing nitrous acid availability of CJP-14 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 Isolation of CJP-14 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 step-diluted and incubated in BHI agar (Difco, USA) to which 3% sodium chloride was added and then incubated at 37 ° C. for 24 hours. 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: antibacterial and Antifungal  Selection of good active strains

Aeromonas Salmonesida for the primary selection of strains with antimicrobial activity against representative pathogenic bacteria in fish farming salmonicida , Vibrio harveyi ), Vibrio angelalium ( 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. Top-Agar was prepared by mixing 3 ml of 0.7% agar (W / V) and 150 µl of shaking cultures of the six pathogenic bacteria (OD ₆₀₀ = 2.0), respectively, and dispensing on BHI medium. 10 μl of the prepared strain culture solution was dropped on the prepared top-agar and cultured at 30 ° C. for 18 hours to observe the presence of a clear zone formed around the selected strain. Of the 53 strains, 19 strains showing antimicrobial activity against 6 fish farming bacteria were selected primarily (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 Edward Siella tarda );

VH: Vibrio harveyi );

VP: Vibrio Hemoratiticus hemolyticus );

VA: Vibrio Angelarium anguillarum ).

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

Example  1-3-1: Coenzyme solution  Collection

In order to select strains with complex digestive enzyme activity, 39 supernatant strains were cultured in BHI medium for 8 hours, 24 hours and 48 hours, and centrifuged at 4 ° C and 13,000 rpm for 5 minutes. Then, the final supernatant 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.

Example  1-3-2: Protease ( Protease ) activation

YM (Difco, USA) medium containing 2% skim milk (skim milk, Sigma, USA) was prepared. After dispensing 3 μl of the crude enzyme solution collected into the substrate medium, the reaction was carried out at 30 ° C. for 2 hours, and the enzyme activity was measured according to the degree of clear zone formation.

Example  1-3-3: Cellulase Activity

YM medium with 1% CMC (carboxyl methyl cellulose) substrate was added. 3 μl of the coenzyme solution collected above was dispensed into the substrate medium, and then reacted at 30 ° C. for 2 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.

Example  1-3-4: Amil Reyes ( Amylase ) activation

YM medium was prepared to which 1% soluble starch substrate was added. 3 µl of the crude enzyme solution collected above was dispensed into the substrate medium, and then reacted at 30 ° C for 2 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.

Example  1-3-5: Lipase activity

YM medium with 1% tricaptylin was prepared. After dispensing 3 μl of the crude enzyme solution collected into the substrate medium, the enzyme activity was measured according to the degree of clear zone formation after 2 hours of reaction at 30 ° C.

Of the 19 strains with excellent antimicrobial activity, 6 strains were selected to produce digestive enzymes of protease, cellulase, amylase and lipase as shown in Table 2 below (Table 2). )

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

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

Example  2: inhibit freshwater fish pathogenic fungi in vitro  exam

Antibacterial and antifungal, activity inhibitory effect of the pathogenic fungus of four legs Pro California with a mixed enzyme productivity, problems in freshwater fish with an excellent four candidate strains in Measured in vitro .

Saprolegnia was used by standing incubation at 30 ° C. for 72 hours in a PDA (Difco, USA) solid medium. Saprolegnia cultured in solid medium was cut into 5 mm x 5 mm (horizontal x vertical) sizes and inoculated in the center of new PDA solid medium and incubated for 24 hours. Four activated candidate strains were dispensed into sterile paper disks by 20 µl, and then inoculated into solid medium containing sapregegnia and incubated at 30 ° C. for 48 hours. As shown in Table 3, the presence or absence of the growth zone (clear zone) formed around the candidate strain was observed (Fig. 1).

Saprolegnia sp. Growth Inhibition Assessment Candidate strain Growth inhibition ring formation CJP11 ++ CJP12 + CJP14 +++ CJP26 +++

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

Four candidate strains were cultured in BHI liquid medium at 37 ° C. and 200 rpm for 8 hours in order to evaluate the inhibitory and inhibitory effect of sapregnia activity under liquid medium conditions. The culture 24 well plate (Falcon, USA) in ^{10 8 CFU, 10 7 CFU,} 10 6 CFU, 10 5 CFU, 10 4 and diluted with CFU, 10 ² concentration in the four profile leg California was added to each well To 1 ml final. 0.5 ml of PDB (Difco, USA) liquid medium containing 0.5 g / L of peptone and 20 g / L of yeast extract was added, and then cultured at 25 ° C. for 48 hours. Experimental results confirmed the ability to inhibit the fungal activity through the degree of solidification, two candidate strains inhibited the growth of sapregnia as shown in Table 4 below.

Saprolegnia growth inhibition evaluation of two candidate strains Test bacteria 10 ⁸ 10 ⁷ 10 ⁶ 10 ⁵ 10 ⁴ CJP14 +++ +++ ++ - - CJP26 +++ + + - -

+, Positive; -, voice

Example  3: Side rope  Selection of strains with high productivity

In order to confirm the sideropore productivity of the four candidate strains excellent in antibacterial, antifungal, and complex enzyme production, the evaluation was performed by CAS test method. CAS medium was prepared as follows.

Siderope productivity evaluation medium composition Fe solution (1 mM FeCl ₃ 6H ₂ O in 10 mM HCl) 1.5 ml CAS solution (0.121 g CAS in 100 ml distilled water) 7.5 ml HDTMA (0.0219 g HDTMA in 50 mL distilled water) 50 ml pH 6.5 piperazine buffer
(4.307g piperazine in 30ml distilled water) 30 ml Distilled water 100 ml (the rest)

Inoculated with 0.1% of each of the four selected bacteria in BHI liquid medium, and incubated at 37 ℃, 200 rpm for 18 hours. In addition, the pathogenic microorganisms Vibrio havey ( V. harveyi ), Vibrio cholera ( V. cholerae ), Vibrio vulnificus , Aeromonas Salmonicida ( A. salmonicida ) 0.1% of each was inoculated and incubated at 30 ° C. and 200 rpm for 18 hours. Each culture supernatant was taken, filtered through a 0.45 μm filter, and tested with the filtered culture supernatant. After a hole of 0.5 cm in diameter was made in CAS agar, 50 μl of each filtered culture supernatant was reacted at 37 ° C. for 8 hours. As shown in FIG. 2, the production of orange rings formed around holes during the use of iron was observed. As shown in Table 4, Bacillus sp. CJP-14 strain having a better sideropore productivity than the four pathogenic microorganisms was finally selected (Table 6).

Siderope Productivity Assessment Candidate strain Sidelopor
Productivity Fish Pathogenic Microorganisms Sidelopor
Productivity CJP11 ++ Vibrio Habei ++ CJP12 + Vibrio cholera ++ CJP14 +++ Vibrio Blinpicus ++ CJP26 ++ Aeromonas Salmonesida +

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

Example  4: Identification, Physiology and Biochemical Characterization of Selected Strains

Example  4-1: Strain Identification

Among the selected strains, CJP-14 strains excellent in suppressing sapregegnia activity and sideropore productivity were 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. In sequencing, gene amplification of 16s rDNA was performed using PCR premix (Bionia, Korea) and universal primers 27F and 492R having the following sequencing. 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.

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

92R: 5'-GGYTACCTTGTTACGACTT-3 '(SEQ ID NO: 3)

Novel microorganisms of the present invention identified in the above manner was found on December 14, 2010 in the Korean spawn association (361-221, Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea). Bacillus sp. CJP-14 "(KCCM11143P).

Example  4-2: 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 7).

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 - β genthiobiose
(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  5: stability and usability

Example  5-1: hemolytic (β- hemolysis )

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

To determine the hemolytic properties of the selected bacteria, TSA (Difco, USA) containing 5% sheep blood (sheep blood, Kisan Biotech, Korea) is prepared. After linear inoculation (streaking) in the prepared blood agar medium, and cultured at 37 ℃ for 24 hours to confirm the hemolysis as shown in Figure 3 it was confirmed that hemolysis did not show.

Example  5-2: nitrite availability

In order to confirm the nitrite availability of the strain of the invention was prepared a medium containing nitrite as shown in Table 6 below. Bacillus spp. In the prepared medium CJP-14 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 the nitrous acid present in the supernatant of the medium obtained by centrifuging for 5 minutes at 4 ℃, 13,000rpm.

 Composition of nitrous acid availability evaluation medium Nitrite (NaNO ₂ ) 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 hydrogen carbonate (NaHCO ₃₎ 0.5 g / L Ferric chloride (Fecl ₃ ㆍ 6H ₂ O) 0.014 g / L Glucose 0.5 g / L

The initial nitrite concentration in the medium was about 10 mg, and about 90% of the nitrite was consumed at 30 hours of cultivation as the Bacillus sp. CJP-14 strain was grown and nitrite was used (see FIG. 4).

Based on these results, Bacillus spp. CJP-14 strains showed aquatic fungus sapregegran inhibition, sideropore productivity and antimicrobial activity.

Example  6: freshwater fish pathogenic fungi Saffronegnia  Efficacy evaluation

in In vitro , efficacy evaluation of saffronegnia against Bacillus spp. CJP-14 strain, which has the effect of inhibiting the activity of pathogenic fungi saffronegnia, and having excellent sideropore productivity, was performed. Therefore, the inventors investigated the percentage of incidence after infection of saffronegnia in the presence of the strain of the present invention in rainbow trout fertilized eggs.

Specifically, after counting about 500 fertilized eggs of rainbow trout in a beaker containing sterilized fresh water, the strain of the present invention and saffronegna bathed for 30 minutes a day for 3 days in the chemical solution of the concentration shown in Table 9 , The rate of draft (%) was measured as follows. Sodium chloride and formalin were used as a control.

% Proposition = (number of eggs / number of fertilized eggs used) X 100

As shown in Table 9, the control rate was 0.5%, and 52.3% of sodium chloride and 19.5% of formalin were used as antifungal substitutes. When the selected Bacillus spp. CJP-14 was treated with 1 × 10 ⁸ CFU or more, the efficacy was higher than that of bathing sodium chloride and formalin. Therefore, the present invention shows that the strain of the present invention is effective for the inhibition of sapregnia, an aquatic fungus of freshwater fish.

Efficacy evaluation on saffronegnia Treatment density Fertilized eggs % Draft Bacillus spp. CJP14 1 x 10 ⁹ CFU 300 54.7 1 x 10 ⁸ CFU 300 53.2 1 x 10 ⁷ CFU 300 40.5 Sodium chloride 10 ppm 300 52.3 Formalin (37% formaldehyde solution) 120 ppm 300 19.5 Control - 300 0.5

Example  7: 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 formulated as shown in Table 10 so as to have 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 the measurement (see Table 11).

Evaluation of Efficacy in Feeding Probiotics in Olive Flounder Control Test Final average weight (g) 50.6 ± 1.7 ^a 57.4 ± 3.8 ^b % Increase 103.03 ± 5.6 ^a 130.05 ± 9.3 ^b Daily growth rate (%) 1.42 ± 0.06 ^a 1.67 ± 0.08 ^b Survival rate (%) 77.3 ± 15.1 ^a 97.3 ± 2.3 ^b Feed conversion efficiency 15.9 ± 0.15 ^a 1.16 ± 0.05 ^b Protein Conversion Efficiency 1.10 ± 0.1 ^a 1.40 ± 0.0 ^b

As shown in Table 11, the addition of Bacillus sp. CJP 14 strain showed a high growth rate of about 13% compared to the control, and also showed a higher tendency in feed conversion and protein conversion efficiency than the control. Survival rate was about 25% higher than that of Daebo.

Based on these results, the Bacillus spp. CJP-14 strain has excellent complex enzyme production, and thus, it can be found that the bacterium CJP-14 strain can act in combination with promoting fish digestibility, improving weight gain, reducing excreta, and improving water quality.

Korea Microorganism Conservation Center (overseas) KCCM11143P 20101214

Claims (11)

Bacillus strain CJP-14 ( Bacillus sp., CJP-14) (KCCM11143P).
The culture solution of Bacillus strain CJP-14 of claim 1 ( Bacillus sp., CJP-14) (KCCM11143P), its concentrate or dried product thereof.
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 or 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.
The antimicrobial agent or antifungal agent containing the strain of Claim 1, or the culture solution of Claim 2, its concentrate, or its dry matter.
A method for preventing an infectious disease of Saprolegnia sp. Microorganisms of 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.
delete Water quality improvement 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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