KR20090035960A - Lactococcus lactis zen64 having antibacterial activity and probiotics comprising lactococcus lactis zen64 - Google Patents

Abstract

Provided is microbe formulation containing Lactococcus lactis zen64, which has antibacterial activity and shows high lactic acid generation and high rearing in seawater or fresh water. A microbe formulation comprises Lactococcus lactis zen64(KKACC 91328P) separated from gastrointestinal tract of fishes. The Lactococcus lactis zen64(KKACC 91328P) shows excellent lactic acid generation and high rearing in 0-7wt% of salt concentration. The microbe formulation including the Lactococcus lactis zen64(KKACC 91328P) is added to fodder of seawater and fresh water fishes.

Description

Lactococcus lactis zen64 having antibacterial activity and probiotics comprising lactococcus lactis zen64}

The present invention relates to lactococcus lactis gen 64 and antimicrobial preparations having the same, and particularly, as a lactic acid bacterium derived from the fish intestine, which exhibits high lactic acid production and high growth in fresh and seawater. It relates to a lactococcus lactis gen64 and a microbial agent comprising the same.

Hospital microorganisms that occur regardless of the season and habitat environment cause huge damage to fish and economic loss, and many antibiotics are used to prevent disease caused by pathogenic microorganisms. Accumulation in the body for a long time will cause problems such as antibiotic resistance as well as allergies. Therefore, the development of a natural antimicrobial agent that exhibits antimicrobial activity against pathogenic bacteria causing bacterial diseases of fish is of great significance in terms of preventing fish disease and ensuring aquatic product stability.

Various intestinal bacteria live in the intestines of fish, which plays an important role in digesting and digesting foods that enter the intestine and generating energy for nutrient synthesis and bio-maintenance. However, if the balance of gut flora is destroyed by poor environmental conditions, malnutrition, climate, drugs, and stress, the gut ecosystem is destabilized and adversely affects living organisms. Therefore, the emphasis on maintaining the stability of the intestinal bacterial flora has emerged the concept of probiotics (probiotics) or probiotics or probiotics as a microbial agent that has a beneficial effect on the living body. Probiotics is a live microbial feed additive that has a beneficial effect on the host by improving the host's intestinal microbial balance, and in recent years lactic acid bacteria have settled in the intestine to maintain the normal flora in the intestine, inhibit the growth and harmful effects of harmful bacteria and increase the absorption of toxic substances. As it turns out that there is an effect, the development of probiotics using lactic acid bacteria is being actively made.

Lactobacillus is non-pathogenic and forms a normal bacterial flora in the human intestine, and has the function of suppressing harmful bacteria by producing antibacterial substances in Gram-positive, cocci or bacilli form, and catalase negative (proven for anaerobic fermentation). In the case of lactic acid bacteria, a high biological availability and the availability of the strain belonging to the like according to the convenience of merchantability Lactobacillus bacteria (Lactobacillus) genus Bifidobacterium (Bifidobacterium) and Enterococcus genus (Enterococcus) genus.

Although most lactic acid bacteria as probiotics are separated from the intestines of animals and used for livestock, only a few species of lactic acid bacteria due to fish are still separated from the world.

In the present invention, to develop probiotics suitable for aquaculture environment, the lactic acid bacteria having excellent antibacterial activity were isolated from the gastrointestinal tract of fish, and the characteristics thereof were investigated to complete the present invention.

The present invention provides lactococcus lactis gen 64 (KACC 91328P), which is isolated in the gastrointestinal tract of fish and has antibacterial activity.

In addition, the present invention exhibits excellent lactic acid production and high growth at a salt concentration of 0 to 7.0% by weight, microorganisms including lactococcus lactis gen 64 having an antimicrobial activity characterized in that it is mixed in the feed of seawater and freshwater fish Provide formulations.

Therefore, according to the present invention, there is an effect of providing lactococcus lactis gen64 64 having an antimicrobial activity as a lactic acid bacterium derived from the fish intestine and exhibiting high lactic acid production and high growth in fresh and seawater.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The present invention provides lactococcus lactis gen 64 (KACC 91328P), which is isolated in the gastrointestinal tract of fish and has antibacterial activity.

In another aspect, the present invention provides a microbial agent comprising lactococcus lactis gen 64 having an antimicrobial activity characterized in that it is mixed in the feed of seawater and freshwater fish.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited by the following Examples.

Experimental fish and Use strain

Among the most frequently farmed fish species purchased in the fish market and sashimi, Pohang, Korea, flounder and rockfish, freshwater eel and sea eel (Anago), which live mainly in the wild state, were used as experimental fish. Was used as a bacterium that causes fish disease and was distributed at the Korea Fisheries Research and Development Institute, National Fisheries Research and Development Institute, in 20% glycerol (-70 ℃).

Used badge

The culture of lactic acid bacteria was MRS broth (proteose peptone) 10g / l, beep extract (10g / l), yeast extract (5g / l, dextrose) 20g / l , 1 g / l polysorbate 80, 2 g / l ammonium citrate, 5 g / l sodium acetate, K ₂ HPO _{4 2} g / l, MgSO ₄ 7H ₂ O 0.1 g / l L, MnSO ₄ 7H ₂ O 0.05 g / L, pH 6.5 0.2 0.2) were used, and separation was performed using MRS agar medium with 0.01% bromocresol green added. In addition, in order to measure the antimicrobial activity, the culture of the bacteria causing fish disease was pre-cultured Mueller Hinton Broth with 1% NaCl, followed by MRS broth with 1% NaCl.

Example 1

Isolation and Antimicrobial Activity of Lactic Acid Bacteria

In order to isolate the lactic acid bacteria having strong lactic acid-producing activity in the gastrointestinal tract, the stomach and intestines of each fish species were removed, and then chopped as much as possible, followed by shaking culture with 0.8% physiological saline to obtain a suspension. 0.1 ml of each suspension was plated in a pre-prepared MRS agar medium and cultured at 30 ° C. for 48 hours to obtain colonies of bacteria inhabiting the fish gut. Colonies formed were punctured in MRS agar medium containing 0.01% bromocresol green and cultured at 30 ° C. for 48 hours to select colonies showing clear zones. Selected strains were used by pure separation of single colonies obtained by repeating twice in MRS agar medium.

In order to measure the antimicrobial activity against the bacteria causing fish diseases of the selected lactic acid bacteria, Vibrio anguillarum , Edward C. Inoculate 200 μl into MRS soft agar (0.7% agar) maintained at 45 ° C with Edwardsiella tarta , Streptococcus sp., And Staphylicoccus sp. MRS hard agar (1.5% agar) was poured into the plate and stratified. Lactic acid bacteria selected on the surface of the soft agar were punctured and cultured at 30 ° C., and strains forming the clear zone in all four pathogenic microorganisms were finally selected.

345 colonies were isolated from the most commonly used flatfish, rockfish, freshwater eel and wild eel (Anago). Among them, as shown in Table 1 below, a total of 70 colonies of lactic acid bacteria showing clear zones were separated by puncturing the culture of P. a. Named Gen 70 and identified through Morphology and sequencing of 16S rRNA, Weissella spp. 1, Lactococcus spp. 25, Leukonostok species ( Leuconostoc spp.) 3, Carnobacterium spp. 14, Enterococcus spp. 4, Streptococcus spp. 3, Kocuria spp. ) 3 types of distribution.

Fish species Sample number Colony count Dominant group (number of species) Flatfish ( Paralichthys olivaceus ; flatfish) Camouflage 1 - Built 1 3 Weissella Bell (1) Camouflage 2 4 Lactococcus species (1) Enterococcus species (2) Built 2 10 Lactococcus species (5) Enterococcus species (2) Streptococcus species (2) Uruk ( Sebastes schlegeli ; jacopever) Camouflage 4 Lactococcus species (3) Enterococcus species (1) guts 9 Leukonostock species (3) Enterococcus species (1) Carnobacterium species (1) Cocoria species (1) Eel ( Conger verreauxi ; conger eel) Camouflage - guts One none Freshwater eel ( Anguilla japonica ; Japanese eel) Camouflage 5 Lactococcus species (2) Carnobacterium species (2) guts 34 Lactococcus species (14) Carnobacterium species (11) Streptococcus species (1) Cocoria species (2)

Among seven strains of 70 strains, strains with excellent lactic ability, zen2 (Weissella species), zen10 (Ryukonostock species), zen14 (Carnobacterium species), zen61 (Enterococcus species), zen64 (lactococcus species) Antimicrobial activity against fish disease bacteria was tested in five species, and the results are shown in Table 2 below.

Strain Sympathy microbe Vibrio anguillarum Edwardsiella tarta Streptococcus spp. Staphylococcus species zen 2 Weissella Bell +++ ++ + ++ zen10 Leukonostock species +++ ++ - ++ zen14 Carnobacterium species + + - - zen61 Enterococcus species + + - - zen64 Lactococcus species +++ +++ + ++  : Inhibitor zone; -: Negative, +: weak positive, ++: positive, +++: strong positive

zen14 and zen61 showed weak antimicrobial activity in Gram-negative bacteria, but did not show antimicrobial activity in Gram-positive bacteria. On the other hand, zen2, zen10 and zen64 showed strong antimicrobial activity against Gram-negative bacteria, while zen2, zen10 and zen64 showed strong activity against Gram-positive bacteria Staphylicoccus sp., But Streptococcus sp. Zen2 and zen64 showed weak antimicrobial activity, but zen10 did not show antimicrobial activity. As a result, Gram-negative bacteria Vibrio anguillarum , Edwardsiella tarta and As the final isolate, zen64, which exhibits antimicrobial activity against both Gram-positive bacteria Streptococcus sp. And Staphylicoccus sp., Has better antimicrobial activity than Gram-positive bacteria.

Example 2

Identification of isolated bacteria

Gram staining was performed to identify the selected lactic acid bacteria, and their morphology was observed under a microscope. Biochemical properties were investigated using API 50 CHL kit (Biomereux, France). In addition, 16S rRNA of the selected strain was PCR (Solgent Co., Ltd.) using primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3 ′) and 1492R (5′-GGTTACCTTGTTACGACTT-3 ′). After amplification of the polymerase chain reaction, the amplified sequence was compared with the nucleotide sequence registered in the National Center for Biotechnology Information (NCBI) to examine homology, and finally identified based on the experimental results.

As a result of observing the isolated bacterium zen64 under a microscope, it was identified as Gram-positive cocci, and the characteristics of the bacterium were examined to identify them. Biochemical properties of the isolates were investigated using the API 50 CHL kit. To this end, the isolates were shaken overnight in an MRS liquid medium, and the cells were recovered, suspended in suspension medium, and diluted with AIP 50 CHL medium to McFarland 2 standard turbidity. After inoculating the bacterial suspension into the tube and incubating for 24 to 48 hours to determine whether the carbohydrates were fermented, the isolated bacteria were L-arabinose, L-arabinose, ribose, xylose, Galaxtose, glucose, frucose, mannose, maltose, cellobiose, lactose, sucrose, trehalose, etc. D-arabinose, melibiose, D-tagatose and the like could not be fermented (Table 3). Comparing the results of the fermentation of these carbohydrates with the database, it was confirmed that the isolate was 99.8% similar to L. lactis subsp. Lactis .

Features reaction Features reaction Morphological shape coccus Amigdalin + Gram Dyeing + Arbutin + Spores - Esculin + motility - Salinity + Catalase - Cellobiose + Glycerol - Maltose + Erythritol - Lactose + Di-arabinos - Melibiose - L-Arabinos + Sucrose + Ribose + Trehalose + D-Xylose + Inulin - L-Xylose - Melezitos - Adonitol - Raffinos - Methyl-βdi-xylpyranoside - starch + D-galactose + Glycogen - Di-glucose + Xylitol - D-fructose + Genthiobiose + D-Mannos + D-Turannos - L-Sorboth - D-Rixos - Rhamnos - Di-tagatose - Dulcitol - D-fucose - Inositol - L-fucos - Mannitol + Di-Arabitol - Sorbitol - L-Arabitol - α-methyl-di-mannoside - Gluconate + α-methyl-di-glucoside - 2-keto-gluconate - N-acetyl-glucosamine + 5-keto-gluconate -  +: Positive reaction,-: negative reaction

The 16S rRNA gene fragment was amplified by polymerase chain reaction (PCR) to analyze the nucleotide sequence of 16S rRNA of the isolated bacterium. Polymerase chain reaction was carried out using primers 5′-AGAGTTTGATCCTGGCTCAG-3 ′ (27F) and 5′-GGTTACCTTGTTACGACTT-3 ′ (1492R) of the conserved region of the bacterial 16S rRNA gene, isolated from Zen64. One total chromosomal DNA was used as template. PCR reaction solution composition and reaction conditions were the same as those performed by Kweun et al. (Felsenstein, J. (2002) PHYLIP (phylogenyinference package), version 3.6a, seattle: Department of Genetics, University of Washington, Seattle , WA, USA.). The amplified 16S rRNA gene fragment was purified and sequenced using a die terminator cycle sequencing kit and a 373 automated DNA sequencer (Perkin Elmer Co, USA). It was. The determined nucleotide sequences were primarily selected from a group of bacteria that showed high homology compared to the corresponding nucleotide sequences of various previously registered bacteria using the BLAST search method of the US NCBI. For accurate phylogenetic analysis of the isolates, the CLUSTAL W software was used to compare the 16S rRNA sequences of the selected strains and to remove or appropriately modify 5 ′ and 3 ′ gaps, and then generate molecular phylogenetic tree. Was performed using the PHYLIP program package (Jukes, TH and cantor, CR (1969) Evolution of protein molecules.pp. 21-132. In HN Munro. (Ed.), Mammalian protein Metabolism , Academic Press, New York. See). Molecular evolutionary distances were based on the neighbor-joining method in the DNADIST program (Sautou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees.Mol. Biol. Evol . 4, 406-425.). As a result, as shown in Figure 1, 16S rRNA of the isolate was the highest similarity to L. lactis subsp. Lactis , and these results were consistent with the results of biochemical properties, so the isolated bacteria (Chung, HM and Kim, JM (2001) Isolation and identification of Probiotic candidate Isolated from the Gastrointestinal (GI) Track of Flounder . Bull.Fish.Sci.Inst., Yosu Nat'l) See Univ. 10, pp . 14-17, 2001).

Experimental Example 1

Acid and Bile Resistance of Lactobacillus Lactis Zen64 Isolates

Acid and bile resistance were examined to determine whether the isolated Lactococcus lactis gen64 strain possessed the basic properties required as a probiotic microorganism. Experiments for measuring acid resistance include a method of directly checking the survival rate in vivo and an indirect measurement using artificial gastric juice. The killing of live bacteria is mainly caused by a low pH. It has been reported that the results of the experiments in and in viro are almost identical. In order to investigate the acid resistance of the Lactococcus lactis gen64 strain selected in the present invention, after washing the cells cultured in MRS twice with 0.85% sodium chloride solution, 50mM phosphoric acid adjusted to pH 3, 4 and 5 It was left to the sodium (sodioum phosphate) solution. After standing, the bacterial solution was taken at regular intervals between 10 to 120 minutes and smeared on an MRS plate medium to check the survival rate as shown in FIG. 1, and as shown in FIG. 2, at pH 4 or higher, the survival rate was 100% after 120 minutes. At pH 3, the mortality by acid was not significant because it showed a survival rate of 90% or more from 10 minutes to 120 minutes. On the other hand, Gilliland et al. Reported that the resistance of bile to probiotics should be able to grow in medium containing oxgall (0.3% by weight) (Gilliland, SE, Staley, TE and Bush). , LJ (1984) Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J. dairy Sc i. 67, 3045-3051 and Lee, MS and Yoon, KH ( 2006) Characterization of Pediococcus pentosaceus isolated from Porcine intestine. Kor J. Microbiol. Biotechnol . 34 , 74-77.). Therefore, in order to investigate the resistance to the bile of the gen 64 strains, inoculated into MRS broth containing 0.3% (W / V) oxgal and MRS broth containing no oxal, and left at 37 ° C for 2 hours, The number of viable cells was measured. As a result, normal growth was shown regardless of the addition of oxal, and it was confirmed that the isolate Bacteria gen 64 was resistant to oxal. In Fig. 2,? Is curves representing the survival rate at pH 3, Δ is pH 4 and ■ is pH 5.

Experimental Example 2

Lactobacillus Lactis Zen 64 ( Lactococcus lactis zen64) Culture Characteristics of Isolated Bacteria

In order to investigate the growth and lactate productivity of Lactococcus Lactose xen64, 1% of MRS broth was adjusted to 7.0 and inoculated in sterile 500ml and incubated in a 30 ° C incubator for 3 hours to 36 hours. optical density (OD), pH and total acidity values were measured. OD was measured at 600 nm with a spectrophotometer (UV-1601 UV-Vis Spectrophotometer, Shimadzu Co., Ltd., Japan), and the pH was measured at pH (PHM210). The radioactivity was measured by a radiometer analytical in France, and the total acidity was measured by the AACC (02-31) method (Approved methods of the American Association of Cereal chemists (AACC) (1985) Minnesota, pp.2-31). 10 ml of the culture solution was added to an Erlenmeyer flask, 90 ml of distilled water was mixed, and 0.1 N sodium hydroxide (F = 1.0) was added to 1.0% phenolphthalein 50% ethanol indicator (DukSan pure Chemical Co., Ltd., Korea). The effect of sodium lactate on lactococcus lactis zen64 was investigated by preliminizing the selected lactococcus lactis zen64 in MRS medium. Applied to farmed fish In order to measure the concentration of 0, 1, 3.5, 5, 7, 10% by weight based on the salt concentration of 3.5% by weight of the sea.

In order to use the lactococcus lactis gen64 64 isolated through the present invention as a probiotic, various studies are required. The lactic acid produced by Zen 64 lactic acid bacteria, which had the highest antibacterial activity and growth, was related to the intestinal effect. 3 shows the results of measuring the growth of the bacteria and the resulting pH and total acidity change in units of 3 hours while culturing the isolated bacteria for 36 hours. The change in OD value at 600 nm with the culture time of strain Gen64 was small at 0.018 initially and 0.075 at 3 hours of cultivation. Could be confirmed. The growth slowed down after 21 hours of incubation, showing no pattern of bacterial growth. The logarithmic phase of the gen 64 strains thus appeared to range from 6 to 18 hours. On the other hand, the change in pH of the culture medium was inversely proportional to the growth of the bacteria from 6.28 to 6.11 at 3 hours of cultivation. It was found to be lowered. In Figure 3, ● represents OD, ○ represents Total Titratible acidity (TTA) and ▲ represents pH.

In addition, the total acidity value of the broth was proportional to the growth of the fungi, which increased rapidly from 2.84 to 4.97 at 6 hours of cultivation and to 12.89 at 21 hours of cultivation. After that, there was little change, and after 27 hours, the pH decreased.

4 shows the results of investigating whether the LTC is excellent in the antibacterial activity against the fish disease bacteria of Lactose gen 64 but affects the salt concentration. Gen64 showed high growth and acidity at sea salt concentrations below 3.5% by weight sodium chloride, while growth and acidity of bacteria were lowered at 3.5% sodium chloride concentration, but relatively high growth and acidity. As a result, lactococcus lactis zen64 derived from sea fish, which can grow at 3.5% sodium chloride concentration, is considered to be a probiotic (microbial agent). In Fig. 4,? Indicates OD and? Indicates total acidity, respectively.

The present invention is a lactic acid bacteria derived from the fish gut, Lactococcus lactis zen 64 having antibacterial activity and showing a high lactic acid production and high growth in fresh and sea water, and can be used in fisheries and the like by providing a microbial agent comprising the same .

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and such modifications and modifications are within the scope of the appended claims.

1 is a diagram showing the results of identification of Lactococcus lactis gen64 according to the present invention.

Figure 2 is a graph showing the acid resistance of the lactococcus lactis gen 64 strain according to the present invention.

Figure 3 is a graph showing the results of measuring the growth of bacteria and the resulting pH and total acidity change in units of 3 hours while culturing Lactococcus lactis gen64 according to the present invention for 36 hours.

4 is a graph showing the effect on the salt concentration of lactococcus lactis zen64 according to the present invention.

Claims (2)

Lactococcus lactis gen 64 (KKACC 91328P), which is isolated in the gastrointestinal tract of fish and has antimicrobial activity. A microbial agent comprising Lactococcus lactis zen64 having antimicrobial activity, characterized in that it is mixed in feeds of seawater and freshwater fish.

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