KR101616530B1 - Lactococcus lactis KR-W.W-2 as a novel strain with antibacterial activity and use thereof - Google Patents

Abstract

The present invention relates to a lactococcus lactis KR-WW-2 strain of deposit number KCTC 12237BP which is a strain having a novel antibacterial activity, an antibacterial composition and a probiotic composition comprising the above strain or a culture thereof as an active ingredient, A feed additive for fish culturing comprising a probiotic composition as an active ingredient, a feed for fish culturing including the feed additive, a method for preventing diseases caused by pathogenic bacteria of aquaculture including feeding the fish cultured in the feed And a method for preventing infection by pathogenic bacteria in aquaculture, comprising the step of spraying the antimicrobial composition on a fish farm.
The novel Lactococcus lactis KR-WW-2 strain having the antimicrobial activity of the present invention exhibits excellent antibacterial activity against strains of bacterial pathogens such as Streptococcus species, Vibrio angularum and Vibrio vulnificus, Because it has biliary properties, it is a suitable strain as a feed additive. Therefore, it can be added to feed to prevent fish aquatic diseases caused by bacterial pathogens. In addition, since the strain and the culture thereof exhibit an antimicrobial activity against a bacterial pathogenic microorganism of a fish, the microorganism can be sprayed on a farm to prevent infection by fish microbial pathogens of aquaculture.

Description

A novel Lactococcus lactis KR-W.W-2 strain having antimicrobial activity and its use {Lactococcus lactis KR-W.W-2 as a novel strain with antibacterial activity and use thereof}

The present invention relates to a novel antimicrobial activity strain Lactococcus of the deposit number KCTC 12237BP lactis ) KR-WW-2 strain, an antimicrobial composition and a probiotic composition comprising the strain or the culture thereof as an active ingredient, a feed additive for fish culture containing the antimicrobial composition or probiotic composition as an active ingredient, and the feed additive A method for preventing diseases caused by pathogens of aquacultured fish including a step of feeding fish to the fish cultured in the feed, and a step of spraying the antimicrobial composition on a fish farm, Lt; RTI ID = 0.0 > infection. ≪ / RTI >

Seawater farming is carried out in large amounts and at high density, and these fish are exposed to a number of stress and bacterial pathogens. In Korea, various kinds of antibiotics have been used for the prevention and treatment of the diseases of fish cultured in Korea since the 1980s, and the use of antibiotics for a long period of time has been used in marine bacteria causing fish bacterial diseases. There is a concern about generation of resistant bacteria. Factors that determine the antibiotic resistance of bacteria are easily transferred to other bacteria by plasmid transfer. These factors can be transferred to bacteria that cause human diseases. In this case, It has been reported that it can play a role. Therefore, in order to solve the antibiotic resistance problem, many attempts have been made to utilize natural materials which can replace them. Herbal medicinal plant extracts and plant essential oils have been used for edible or medicinal purposes for a long time. Recently, researches on antimicrobial, antifungal and anticancer substances have been actively conducted using these resources. Although there have been many reports on the antimicrobial effect of bacteria on food, which is harmful to humans, research on the antimicrobial effect of natural products against bacteria causing diseases of aquaculture fish is in the beginning stage. For the specific efficacy against the bacterial diseases occurring in the farms, that is, the antimicrobial effect, the natural resource extracts were used to identify the effects on bacteria causing fish diseases and the antimicrobial composition to replace the antibiotics used in the streptococci It is urgent to do.

Streptococcus sp. , A causative organism of Streptococcus spp. , Causes enormous economic losses in freshwater and seawater farming in the United States, Israel, Australia and Japan, including Korea. Streptococcus is a genus of bacteria, and various species of this genus cause disease in many animals, including humans. Bacteria that cause disease in most fish are gram-negative bacteria with pink color in Gram stain, while Streptococcus species are Gram-positive bacteria with purple color in Gram stain. Fish diseases caused by Streptococcus are not common, but once they develop, they carry many losses. Aquatic Streptococcus is not common, but it can cause disease in people who are weak. In addition to Streptococcus, strains such as Lactococcus, Enterococcus and Vagococcus also cause diseases similar to Streptococcus. Staphylococcal disease in fish was first discovered in 1957 in Japanese aquaculture trout. It has been reported that salmon, mullet, golden shiner, pinfish, eel, sea trout, tilapia, sturgeon and perch are easily infected with staphylococcal disease. Examples of Streptococcus species cause disease in fish is present in the S. difficile lease (S. difficilis), the Mille S. Lee (S. milleri) found in the kidney of carp found in Israel, turbot in northern Spain ( turbot) am found in both the S. S. Farah Ube lease (S. parauberis), found in fish and mammals include Ke (S. iniae). In addition, as the species that causes disease in fish, and very much like the staphylococcal as the gram-positive bacterium is a Lactobacillus nose kusu taught sorrow found in Japanese eels and defense (L. garvieae) and the L. pisyum found in rainbow trout (L. piscium ) And Vagococcus salmoninarum ) and the like.

In general, fish infected with Streptococcus tend to darken, become cloudy and prominent in the eyeballs, and have a reddened eye margin and fins. When infected fish are dissected, inflammation and hemorrhage of the liver, hypertrophy and constriction of the digestive tract, cerebral hemorrhage and in situ formation of the gill lid are observed. In addition, infectious fish have been shown to be effective in drug susceptibility testing because of the rapid decrease in feed intake due to early appetite decline, but they are often ineffective when used in actual farms. Therefore, it is required to introduce a more fundamental and new concept for prevention of fish diseases in order to prevent the damage caused by the chain of strains of the aquaculture in the treatment-oriented countermeasures against the existing drugs.

To date, known drugs for streptococci include Florfenicol, Ampicillin, Doxycyclin, and Spiramycin. These are all healable when given for symptomatic use in the aquatic environment, but most pathogens have antibiotic resistance to them and often do not have a clear effect.

Probiotics are known to have antimicrobial activity against bacterial pathogens in a variety of normal intestinal flora, and they play an important role in maintaining normal intestinal flora balance and inhibiting the growth of bacterial pathogens (Fuller , RJ, Appl. Bacteriol., 1989, 66, 365-378; Shahani, KM, Am. J. Clin. Nutr., 1980, 33, 2448-2457). Probiotics used in humans and livestock are not suitable for cultivation at an optimum development temperature of 37 ° C and at 15 to 25 ° C, and probiotics capable of withstanding high salt concentrations are essential, especially for seawater cultivation. As the water quality of seafood is increasing rapidly, the quality of the water environment gradually worsens and the frequency of fish diseases increases. Therefore, it is essential to find a probiotic that can replace antibiotics. Most of the probiotic products for fish use in Korea are imported from foreign countries and they are not well adapted to the domestic fish growth environment. In addition, the colony forming unit is low due to lack of stability due to time difference from import to domestic distribution.

In general, lactic acid bacteria used for probiotics are gram-positive bacilli, Non-emulsifying and catalase negative bacteria which are widely distributed in nature and also found in animal fields and fermented foods (Ringo, E., Aquaculture, 1998, 160: 177-203). It is known that these lactic acid bacteria are important microorganisms for improving intestinal microflora by antibacterial activity and the like. Antimicrobial activity factors of such lactic acid bacteria are organic acid, hydrogen peroxide and bacteriocin (Piard, JC, Lait., 1992, 72: 113-142) . In addition, lactic acid bacteria adhere to and colonize intestinal epithelial cells to inhibit infection and growth of pathogens. A study of the antibacterial activity against pathogenic bacteria of Lactobacillus are L. monocytogenes Jeans (Listeria monocytogenes ), Escherichia coli ), Salmonella typhimurium ( Salmonella typhimurium , Vibrio anguillarum ) have been reported to have antibacterial activity against microorganisms. However, most fish microbes and pathogenic bacteria are Gram-negative, while lactic acid bacteria as probiotics have a limitation in that they exhibit antimicrobial activity only against Gram-positive bacteria which are systematically similar to their antimicrobial activity (Suma, K., Int. J. Food Microbiol , ≪ / RTI > 1998, 40: 17-25). Therefore, for industrial application as a probiotic strain, isolated strains must have a broad antimicrobial spectrum that can inhibit the growth of gram-negative bacteria as well as gram-positive bacteria. In addition, in order to exhibit the continuous antibacterial effect and beneficial effect of probiotics, it is necessary to survive intestinal environmental conditions such as low-pH stomach, bile and various digestive enzymes. In other words, another important condition that a probiotic strain should have is resistance to acid and biliary cholesterol and adequate resistance to antibiotics (Verschuere, L., Microbiol. Mol. Biol. Rev., 2000, 64: 655-656).

Accordingly, the present inventors have made intensive studies to discover new strains which can exhibit antimicrobial activity against bacterial pathogens of fish among natural resources and have excellent acid resistance and bile resistance and can be used as biotics for fish. As a result, Lactococcus lactis KR-WW-2 strain and its culture, which exhibit excellent acid resistance and bile resistance, among the novel strains of Lactococcus lactis and their cultures, have been shown to be effective against Streptococcus strains ( Streptococcus iniae , Vibrio anguillarum and Vibrio vulnificus , and exhibited acid resistance, bile resistance and the like, and completed the present invention.

One object of the present invention is to provide a lactococcus lactis strain KR-W.W-2 of Accession No. KCTC 12237BP.

Another object of the present invention is to provide an antimicrobial composition comprising the strain or a culture thereof as an active ingredient.

It is still another object of the present invention to provide a probiotic composition comprising the strain as an active ingredient.

It is still another object of the present invention to provide a feed additive for fish culture comprising the above-described antimicrobial composition or probiotic composition as an active ingredient.

It is another object of the present invention to provide a feed for fish culture comprising the feed additive.

It is still another object of the present invention to provide a method for preventing diseases caused by pathogenic bacteria in aquacultured fish, comprising feeding the fish that are being cultured.

It is still another object of the present invention to provide a method for preventing infection by pathogenic bacteria in aquaculture, comprising the step of spraying the antimicrobial composition on a fish farm.

In one aspect for achieving the above object, the present invention provides a lactococcus lactis strain KR-W.W-2 of Accession No. KCTC 12237BP.

The term "Lactococcus lactis KR-WW-2 strain" of the present invention refers to a novel Lactococcus lactis KR-WW-2 strain newly isolated and identified by the present inventors from a fermented food such as various kimchi, 2 strain, it can be used in combination with "KR-WW-2 strain" or "KR-WW-2". The strains are characterized by their antibacterial activity against bacterial pathogens of fish . Single cells are of the cocci type (Fig. 1) with a minor axis of 0.7-1.0 [mu] m and a major axis of 0.7-1.6 [mu] m. The fungus forms a circular colony in the MRS agar medium and has a light cream color.

The 16S rRNA gene sequence of the novel strain is shown in SEQ ID NO: 1, and the gene sequence was analyzed and compared to show high homology with Lactococcus lactis ( L. lactis ), and a novel Lactococcus lactis strain Identified as Lactococcus lactis KR-WW-2.

Lactococcus genus The strains have various kinds, and the present inventors have selected strains showing excellent antibacterial activity against fish bacterial pathogens and exhibiting high acid resistance and biliary cholesterol. The KR-WW-2 strain identified by the present inventors It was confirmed that the strain was more resistant to antimicrobial activity and gastric acid and bile than other strains of Lactococcus.

Specifically, the inventors of the present invention found that strains isolated from sardine samples collected in Sokcho, Gangwon-do were the best isolates of six strains selected by using MRS agar medium (LB-agar) and exhibited the best thermostability and resistance to many antibiotics The KR-WW-2 strain of the present invention was found to be able to survive under acidic conditions and high proportion of bile conditions because of its excellent acid resistance and biliary excretion. These results indicate that the strain newly identified in the present invention is more effective than the conventional strain of Lactococcus Since it exhibits excellent antimicrobial activity, it can be used as a composition for preventing fish microbial diseases against aquaculture fish. Further, since it is excellent in acid resistance and bile resistance, it is possible to prevent fish microbial diseases by mixing with feeds with feed additives. It suggests.

The present inventors have deposited a strain of the present invention, designated as Lactococcus lactis KR-WW-2, in the Korean Collection for Type Culture (KCTC), an international depository under the Budapest Treaty, And received grant number KCTC 12237BP.

In another aspect, the present invention provides an antimicrobial composition comprising lactococcus lactis KR-W.W-2 strain of deposit number KCTC 12237BP or a culture thereof as an active ingredient.

In another aspect, the present invention provides a probiotic composition comprising the strain.

The term "culture product" of the present invention refers to a cultured strain obtained by culturing the strain for a certain period in a medium capable of supplying nutrients so that the KR-WW-2 strain of the present invention can grow and survive in vitro, , Extra nutrients, and the like, and also includes the culture medium from which the strain has been removed after culturing the strain. The KR-W. W-2 strain or the culture thereof may be used as an antimicrobial composition, since the KR-W. W-2 strain has an antibacterial activity, particularly an antibacterial activity against a bacterial pathogenic bacterium of a fish. Preferably as an antimicrobial composition against fish bacterial pathogens.

The term " probiotics " of the present invention refers to live microorganisms, i.e., live fungi, which provide beneficial health benefits to intestinal flora, i.e., the health of the host. Generally, probiotics are consumed as part of fermented foods such as yogurt or as dietary supplements. Microorganisms known as probiotics include lactic acid bacteria (LAB), bifidobacteria, some yeast, and bacillus.

Therefore, the KR-WW-2 strain of the present invention has been identified as a strain of Lactococcus lactis, which is a kind of lactic acid bacteria, which is a well-known probiotic along with Bifidobacterium, and exhibits its antibacterial activity. Therefore, probiotics for fish, .

Preferably, the fish bacterial pathogen is Streptococcus (Streptococcus) or may be a Vibrio (Vibrio), but may be more preferably Streptococcus am cliff, Vibrio central nine days rarum or Vibrio vulnificus, but is not limited thereto.

According to a specific embodiment of the present invention, KR-WW-2 not only exhibits an excellent antibacterial activity against Streptococcus species (Table 6 and Fig. 3 to 5), but also has excellent acid resistance and biliary properties, (Table 4 and 5), it can be used not only as an antimicrobial agent for bacterial bacterial pathogens but also as a suitable microbial strain for fish.

Therefore, the novel lactokoxlactis The KR-WW-2 strain and its culture provide compositions and fish probiotic compositions exhibiting antimicrobial activity against fish bacterial pathogens. The composition of the present invention may contain, in addition to the above-mentioned effective ingredient, a known carrier or additive which is acceptable for pharmaceutical, food or feed use.

In another aspect, the present invention provides a feed additive for fish culture comprising the antibacterial composition or probiotic composition as an active ingredient. The feed additive of the present invention may further contain additives such as known carriers or stabilizers that are acceptable for pharmaceutical, food or feed use in addition to the above-mentioned effective ingredients. If necessary, various additives such as vitamins, amino acids, minerals, Antioxidants, antibiotics, antibacterial agents, and other additives. At this time, the form may be in a suitable state such as powder, granule, pellet, suspension and the like.

When the antimicrobial composition of the present invention is contained in feed as a feed additive, the composition can be added as it is or mixed with other feed ingredients, and can be suitably used according to a conventional method. . Since the composition of the present invention is derived from a strain and is environmentally friendly, there is no particular problem in terms of stability, so there is no great limitation on the amount of the mixture.

The fishes to be subjected to the present invention are preferably fishes such as sea breams, halibut, oysters, red sea breams, sea cucumbers, mullet and linguard and sea fishes such as eel, But it is not particularly limited thereto. However, the feed additive of the present invention is not limited to the above-mentioned fishes, and can be used for aquaculture fish. Preferably, the feed additive of the present invention can be used for aquaculture fishes which are susceptible to Streptococcus species, Vibrio angulilla or Vibrio vulnificus Lt; / RTI >

In another aspect, the present invention provides a feed for fish culture comprising the feed additive. The form of the feed of the present invention is not particularly limited, and any feed such as powdery feed, solid feed, wet pellet feed, dry pellet feed, EP (extruder pellet) feed,

In another aspect, the present invention provides a method for preventing disease caused by pathogenic bacteria in aquaculture, comprising feeding the fish that are being cultured. At this time, it is preferable that the feed is supplied at the same amount and feed interval as the ordinary feed. The pathogenic bacteria that can be prevented by the feed of the present invention are preferably, but not limited to, Streptococcus species, Vibrio angulorum or Vibrio vulnificus.

In another aspect, the present invention provides a method for preventing infection by a pathogenic bacterium of aquaculture comprising the step of spraying the antimicrobial composition on a fish farm. Since the antimicrobial composition comprising the KR-WW-2 strain of the present invention or a culture thereof as an active ingredient exhibits antimicrobial activity against bacterial bacterial pathogens, it is possible to prevent the bacterial pathogenic bacteria Infection by pathogens can be prevented. The pathogen that can prevent the infection by spraying the antimicrobial composition of the present invention on a farm is preferably, but not limited to, Streptococcus species, Vibrio angulorum or Vibrio vulnificus.

The novel Lactococcus lactis KR-WW-2 strain having the antimicrobial activity of the present invention exhibits excellent antibacterial activity against strains of bacterial pathogens such as Streptococcus species, Vibrio angularum and Vibrio vulnificus, Because it has biliary properties, it is a suitable strain as a feed additive. Therefore, it can be added to feed to prevent fish aquatic diseases caused by bacterial pathogens. In addition, since the strain and the culture thereof exhibit an antimicrobial activity against a bacterial pathogenic microorganism of a fish, the microorganism can be sprayed on a farm to prevent infection by fish microbial pathogens of aquaculture.

1 is an electron microscope image showing the morphology of a novel lactococcus lactis KR-WW-2 strain having antibacterial activity according to the present invention.
2 is a diagram showing a 16S rRNA gene sequence of a novel Lactococcus lactis KR-WW-2 strain according to the present invention.
FIG. 3 is a graph showing antimicrobial activity against Staphylococcus aureus, a bacterial pathogenic bacterium of fish, in the culture medium of a strain of the genus Bacillus KR-OF1 according to the present invention.
FIG. 4 is a graph showing the antimicrobial activity of a culture broth of a novel Lactococcus lactis KR-WW-2 strain according to the present invention against a fish bacterial pathogen, Vibrio vulnificus.
FIG. 5 is a graph showing antimicrobial activity against Vibrio angulorum, a bacterial pathogenic bacterium of fish, in the culture medium of a novel Lactococcus lactis KR-WW-2 strain according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Isolation and selection of lactic acid bacteria

As a sample for isolating the lactic acid bacteria strain from the natural world, the sardine samples were collected in Sokcho, Gangwon Province. Samples were diluted with sterilized water, shaken at 30 ° C for 30 minutes, diluted to 10 ⁴ times with an agar plate dilution method, and 0.1 ml of the diluted solution was added to MRS-Agar medium (Table 1) And spread evenly over the entire surface of the culture medium. After 3 to 5 days of incubation at 30 ° C in a thermostat, morphological analysis of the single colony showed that only the lactic acid bacteria strain was purely cultured, and then cultured on MRS-Agar slope medium, suspended in 20% glycerol, And stored in a 70 ° C freezer. Each colony was finally screened for resistance to various antibiotics with excellent survival rate at high temperatures through a thermostability test and various antibiotic resistance tests (Table 2).

The single cells observed on the electron microscope of the selected strains were cocci type (Fig. 1) having a minor axis of 0.7 to 1.0 탆 and a major axis of 0.7 to 1.6 탆, and the fungus was circular in the MRS agar medium colony) and it was confirmed to have a light cream color.

Medium component Concentration (g / l) Proteose Peptone 10.0 Beef Extract 10.0 Yeast Extract 5.0 Glucose 20.0 Polysorbate 80 (polysorbate 80) 1.0 Ammonium Citrate 2.0 Sodium Acetate 5.0 Magnesium Sulfate 0.1 Manganese Sulfate 0.05 Dipotassium Phosphate 2.0 Agar 15.0

number Selection strain Thermal Stability (60 ℃) Antibiotic resistant material Remarks One W.W-2 0.29% Survival Gentamicin, Cephalexin, Colistin, Zincbacitracin ◎ 2 Lac-6 0.3 x 10 ^-7 % Survival Cephalexin, Oxytetracycline, Zincbacitracin △ 3 Lac-7 - Zincbacitracin × 4 Lac-8 0.5 × 10 ^-6 % Survival Colistin, Zincbacitracin △ 5 Lac-9 0.9 x 10 ^-6 % Survival Erythromycin, Zincbacitracin △ 6 Lac-10 0.4 x 10 ^-6 % Survival Gentamicin, Erythromycin, Zincbacitracin △

Example  2: Identification of selected strains

In order to identify the phylogenetic location of the selected strains, an internal transcribed spacer of the 16S ribosomal RNA gene (57S bp) containing 1, 5.8S ribosomal RNA gene and an internal transcriptional spacer 2 Were analyzed. The 16S rDNA fragment 5'-TCC GTA GGT GAA CCT GCG G-3 '(SEQ ID NO: 2) was amplified from the selected strains by PCR using primers set, and the amplified product was purified and purified using an automated sequencer (Applied Biosystems sequence analysis kit (BigDye Terminator Cycle Sequencing Kit, Perkin-Elmer Applied Biosystems). The 16S rDNA of the finally selected strain was analyzed for homology using the blast program of the gene bank, and it was confirmed that the 16S rDNA showed high homology with lactococcus lactis, the standard strain (SEQ ID NO: 1; FIG. 2). In addition, the lactic acid isolated using various biochemical methods was analyzed and found to have a high similarity with the standard strain Lactococcus lactis (Table 3). Therefore, the strain of the present invention was confirmed to have high homology with lactococcus lactis. The lactic acid bacteria strain of the present invention was identified and named as Lactococcus lactis KR-WW-2, (KCTC) on July 6, 2012, and has been granted accession number KCTC 12237BP.

Example  3: Acid resistance  exam

Lactococcus lactis KR-WW-2 strain isolated and isolated in Example 1 was inoculated into the sterilized MRS liquid medium. After culturing at 30 ° C for 24 hours, the strain was recovered by centrifugation at 9000 rpm for 25 minutes. Then, the isolated strain recovered in the MRS liquid medium adjusted to pH 3.0 with HCl was inoculated at a concentration of 1.4 × 10 ⁹ CFU / ml and cultured at 30 ° C. for 90 minutes. After incubation for 90 minutes, KR-WW-2 was inoculated. Each sample was collected every 30 minutes, diluted in sterile physiological saline, plated on MRS plate medium, and cultured at 30 ° C for 48 hours. And the viable cell count was measured (Table 4).

Example  4: My bile  exam

To test the bile resistance of lactococcus lactis KR-WW-2 isolated and isolated in Example 1, MRS medium containing 1, 2, 3 and 4% oxgall, respectively, was prepared And used as an artificial bile solution. Bile is secreted from the upper part of the small intestine to promote the digestion and absorption of lipid foods, microorganisms and detergents similar to the action of the microorganisms composed of fatty acids and fatty acids that affect microbial bactericidal action . Therefore, tolerance to bile acids is one of the basic characteristics that a probiotic microorganism must possess in addition to resistance to acidic conditions above.

The isolated lactococcus lactis KR-W.W-2 strain of the present invention was cultured at 30 DEG C for 24 hours and then centrifuged at 9000 rpm for 25 minutes to recover the cells. The recovered cells were washed twice with 0.01 M phosphate buffered saline (PBS), and an MRS liquid medium containing 1 to 4% of bovine bile was added in the same amount as the supernatant. The cells were incubated at 30 ° C for 3 hours , Followed by stepwise dilution with 0.85% salt buffer, and the number of colonies of Lactococcus lactis KR-WW-2 strain on the MRS agar plate medium was measured and the survival rate was calculated. As a result, the number of viable cells tended to decrease compared with the initial number of inoculated bacilli after treatment of bacillus juice, but it was judged that the bacillus was possessed (Table 5).

Example  5: Lactococcus lactis KR -W.W-2 culture

The Lactococcus lactococcus lactis KR-WW-2 strain used in the present invention was firstly cultured using the MRS liquid medium, and the second main culture was inoculated in a medium containing 6 to 8% of skimmed milk at a concentration of 1% And then cultured at 30 DEG C for 48 hours. The cultures used in the experiments were obtained by centrifuging the culture of the strain at 10,000 rpm, recovering the supernatant, and filtering with a 0.45 μm filter.

Experimental Example  1: Antibacterial activity against bacterial bacterial pathogens in fish

In order to confirm the antimicrobial activity of the lactococcus lactis KR-WW-2 strain isolated, sorted and identified by the above examples against the bacterial pathogenic microorganism of fish, bacterial strains of Streptococcus species, Vibrio vulnificus, The antimicrobial activity against Vibrio angularum was measured. The isolated strain was shake cultured in MRS liquid medium for 48 hours and centrifuged at 10,000 rpm to recover the supernatant. The supernatant was recovered by adding BHI + 1% NaCl agar, NB + 0.5% NaCl agar and 1.4 × 10 ⁹ ml / The fish pathogenic fungus of CFU was inoculated 0.5%, mixed with the medium uniformly, and then solidified in a Petri dish. Then, 100 μl of the supernatant of the isolated strain of the present invention which had been cultivated was dispensed on a paper disk, and the water was dried and placed on a medium in which the pathogenic bacteria had been preliminarily distributed. The mixture was allowed to stand at 4 ° C. for 1 hour to absorb the component in the medium, , And the size of the ring formed by the inhibition of the pathogen activity around the paper disk was measured. As a result, it was confirmed that the culture solution of the strain Lactococcus lactis KR-WW-2 showed excellent antibacterial activity against Streptococcus species, Vibrio vulnificus and Vibrio angulillaum (Table 6, Figs. 3 to 5) .

Therefore, the KR-WW-2 strain of the present invention and the culture thereof exhibit antimicrobial activity against bacterial pathogens of fish, and thus can be used as an antimicrobial composition therefor. In particular, strains having excellent acid resistance and bile resistance are selected, And it was confirmed that it could be fed to aquaculture by mixing with feed.

Korea Research Institute of Bioscience and Biotechnology KCTC12237BP 20120706

<110> Korea Research Institute of Bioscience and Biotechnology <120> Lactococcus lactis KR-W.W-2 as a novel strain with antibacterial          activity and use thereof <130> PA121116 / KR <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 494 <212> DNA <213> Artificial Sequence <220> <223> Lactococcus sp. KR-W.W-2 <400> 1 gctggcggcg tgcctaatac atgcaagttg agcgctgaag gttggtactt gtaccgactg 60 gatgagcagc gaacgggtga gtaacgcgtg gggaatctgc ctttgagcgg gggacaacat 120 ttggaaacga atgctaatac cgcataaaaa ctttaaacac aagttttaag tttgaaagat 180 gcaattgcat cactcaaaga tgatcccgcg ttgtattagc tagttggtga ggtaaaggct 240 caccaaggcg atgatacata gccgacctga gagggtgatc ggccacattg ggactgagac 300 accggcccaa actcctacgg gaggcagcag tagggaatct tcggcaatgg acgaaagtct 360 gaccgagcaa cgccgcgtga gtgaagaagg ttttcggatc gtaaaactct gttggtagag 420 aagaacgttg gtgagagtgg aaagctcatc aagtgacggt aactacccag aaagggacgg 480 ctaactacgt gcca 494 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16s rRNA fragment <400> 2 tccgtaggtg aacctgcgg 19

Claims (8)

&Lt; RTI ID = 0.0 &gt; Lactococcus &lt; / RTI &gt; lactis ) KR-WW-2 strain.
Streptococcus iniae , Vibrio anguillarum and Vibrio vulnificus (hereinafter, referred to as &quot; Lactococcus lactis KR-WW-2 &quot; 0.0 &gt; Vibrio &lt; / RTI &gt; vulnificus ).
A probiotic composition comprising the lactococcus lactis KR-WW-2 strain of accession number KCTC 12237BP as an active ingredient.
The method of claim 3,
Wherein the composition has an antimicrobial activity against at least one strain selected from the group consisting of Streptococcus iniae , Vibrio anguillarum and Vibrio vulnificus .
A feed additive for fish culture comprising the antimicrobial composition of claim 2 or the probiotic composition of claim 3 as an active ingredient.
A feed for fish cultivation comprising the feed additive of paragraph 5.
A method for preventing disease caused by pathogenic bacteria in aquaculture, comprising feeding the fish of the formula of claim 6.
A method for preventing infection by a pathogenic bacterium of aquaculture comprising the step of spraying the antimicrobial composition of claim 2 on a fish farm.

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