KR20090060510A - Pediococcus pentosaceus zen1 having antibacterial activity and probiotics comprising pediococcus pentosaceus zen1 - Google Patents

Abstract

A lactic acid bacteria which is derived from a fish intestine is provided to ensure high productivity of lactic acid, viability, and antibiotics activity as probiotics. A Pediococcus pentosaceus zen 1(KACC 91336P) is isolated from an intestine of fish and has antibiotic activity. A method for isolating the Pediococcus pentosaceus zen 1 comprises: a step of extracting stomach and internal organs and performing shaking culture with 0.8% saline solution to obtain suspension; a step of spreading on MRS agar medium and culturing at 30°C for 48 hours to obtain colonies; a step of culturing the colonies on MRS agar medium containing 0.01% of bromocresol green and screening colonies having clear zone; a step of isolating single colony; a step of inoculating pathogenic microorganisms including vibrio anguillarum, edwardsiella tarta, streptococcus sp. and Staphylicoccus sp. onto MRS medium containing 1% NaCl; and a step of culturing the selected lactic acid bacteria at 30°C and selecting a colony having clear zone to the 4 pathogenic microorganisms.

Description

Pediococcus pentosaceus zen1 having an antimicrobial activity and a microbial preparation including the same {Pediococcus pentosaceus zen1 having antibacterial activity and probiotics comprising Pediococcus pentosaceus zen1}

The present invention relates to a pediococcus pentosaceus gen 1 and a microbial agent comprising 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 Pediococcus pentosaceus gen 1 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 pediococcus pentosaceus gen 1 (KACC 91336P), which is isolated in the gastrointestinal tract of fish and has antibacterial activity.

In addition, the present invention is pediococcus pentosaceus gen 1 having an antimicrobial activity characterized in that it is mixed in the feed of seawater and freshwater fish (representing excellent lactic acid production and high growth at salt concentration 0 to 7.0% by weight) It provides a microbial agent comprising a.

Therefore, according to the present invention, as a lactic acid bacterium derived from the fish intestine, pediococcus pentosaceus gen 1 having an antimicrobial activity showing high lactic acid production and high growth in fresh water and seawater has an effect of providing a microbial agent comprising the same. have.

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

The present invention provides pediococcus pentosaceus gen 1 (KACC 91336P), which is isolated in the gastrointestinal tract of fish and has antibacterial activity.

The present invention also provides a microbial preparation comprising Pediococcus pentosaceus gen 1 having 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 2g / ℓ, MgSO 4? 7H} 2 O 0.1g / ℓ, MnSO 4? Was used _{7H 2 O 0.05g / ℓ, pH} 6.5 ± 0.2), the separation of 0.01% Bromocresol Green (bromocresol green) was added MRS agar medium was used. In addition, in order to measure the antimicrobial activity, the culture of the bacteria causing fish disease was pre-cultured Mueller Hinton Broth added with 1% NaCl, and then MRS broth added with 1% NaCl was used.

Example  One

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 of the bacteria causing fish disease of the selected lactic acid bacteria, Vibrio Anguillalum ( Vibrio) anguillarum ), Edwardsiella tarta ), Streptococcus sp., and Staphylicoccus sp., which were isolated from themselves, were inoculated into 200 μl of MRS soft agar (0.7% agar) maintained at 45 ° C. 1.5% agar) was poured into a 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 that clearly show a clear zone were separated by puncture in an MRS agar medium to which 0.01% bromocresol green was added and cultured at 30 ° C. for 48 hours. Named Gen 70, the results of morphology and sequencing analysis of 16S rRNA revealed 1 Pediococcus spp., 1 Weissella spp., 1 Lactococcus sp. Lactococcus spp.) 25 species, Leuconostoc spp. 3 species, Carnobacterium spp. 14 species, Enterococcus spp. 4 species, Streptococcus spp. .) Three species, Kocuria spp. And three species were distributed. Among eight 70 strains, genotype 1 (Pediococcus spp.) With excellent lactic acid production ability was selected as the final isolated strain, and the antimicrobial activity against fish disease bacteria was tested for genotype 1 strains. 1 is shown.

Pathogenic microorganisms Degree of antimicrobial activity Vibrio anguillarum ++ Edwardsiella tarta +++ Streptococcus spp. ± Staphylococcus spp. +++  : Inhibitor zone; -: Negative, ±: weak, +: weak positive, ++: positive, +++: strong positive

Zen 1 is a Gram-negative bacillus Vibrio anguillarum ), Edwardsiella tarta ) and Gram-positive bacteria Staphylicoccus spp. sp.), but the Gram-positive bacteria Staphylicoccus species In sp.), Staphylococcus spp. showed weak antimicrobial activity. In particular, Gen 2 showed Staphylicoccus spp. sp.) and Edwardsiella tarta showed strong antimicrobial activity.

Example  2

Isolated  Sympathy

Gram staining was performed to identify the selected lactic acid bacteria, and their morphology was observed under a microscope. Biochemical properties were investigated using an 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 at the National Center for Biotechnology Information (NCBI), and the homology was investigated.

As a result of observing the isolated bacterium Gen 1 under a microscope, it was identified as Gram-positive bacillus, and the characteristics of the bacterium were examined to identify it. Biochemical properties of the isolates were investigated using the API 50 CHL kit. To this end, the isolates were cultured overnight in an MRS liquid medium, 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 inoculating the carbohydrate while incubating for 24 to 48 hours, the isolated bacteria were L-arabinose, ribose, xylose, Galactose, glucose, frucose, mannose, acetylglycosamine (N-acetylglucosamine), cellobiose, maltose, melibiose, trehalose (trehalose) trehalose) and the like, D-arabinose (D-arabinose), lactose (lactose), sucrose (sucrose) and other sugar alcohols were not fermented (Table 2). Comparing the results of the fermentation of these carbohydrates with the database, it was confirmed that the isolate was 99.8% similar to P. pentosaceus .

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 the 16S rRNA of the isolate. The polymerase chain reaction was performed using primers 5′-AGAGTTTGATCCTGGCTCAG-3 ′ (27F) and 5′-GGTTACCTTGTTACGACTT-3 ′ (1492R) of the conserved regions of the bacterial 16S rRNA gene. Total chromosomal DNA isolated 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 most similar to P. pentosaceus ( P. pentosaceus ), and these results were consistent with the results of biochemical properties, so the isolate was (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 Univ. 10, pp. 14-17, 2001).

Experimental Example  One

Pediocaucus Pentosaceus Zen 1 Isolated Acid resistance Bile resistance

Acid and bile resistance were examined to determine whether the isolated Pediococcus pentosaceus gen 1 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 Pediococcus pentosaceus gen 1 strain selected in the present invention after washing the cells cultured in MRS twice with 0.85% sodium chloride solution, the pH was adjusted to 3, 4 and 5 It was left to 50 mM sodium phosphate solution. After standing, the bacterial solution was taken at regular intervals between 10 and 120 minutes and smeared on an MRS plate medium to check the survival rate as shown in FIG. 2, and as shown in FIG. At pH 3, the survival rate was more than 95% 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. See). Therefore, by aligning the concentration of the Ox go to MRS broth in order to investigate the tolerance to bile, 0, 0.15, 0.3, 0.5, 1% (W / V) of Zen 1 strain, the entire cultured Zen 1 strain 10 ⁵ After inoculation with CFU / ml, the cells were incubated at 37 ° C. for 24 hours, and the viable cell count was measured. As a result, even in 1% of Oxgal, the bacteria showed normal growth, and the isolate Gen 1 was confirmed to be resistant to Oxgal. Even if bile acid is secreted in the body, the administered lactic acid bacteria can grow, so it is considered to be an excellent lactic acid bacterium that can serve as a probiotic. In Fig. 2,? Is curves representing the survival rate at pH 3, Δ is pH 4 and ■ is pH 5.

Oxgall Pediococcus pentosaceus (Gen. 1) 0% 3.4 × 10 ⁸ 0.15% 3.7 × 10 ⁸ 0.3% 3.6 × 10 ⁸ 0.5% 3.4 × 10 ⁸ One% 3.2 × 10 ⁸

Experimental Example  2

Pediocaucus Pentosaceus Zen 1 ( Pediococcus pentosaceus zen1 ) Isolated  Culture characteristics

In order to investigate the growth and lactic acid productivity of Pediococcus pentosaceus gen 1, the pH of MRS broth was adjusted to 7.0 and inoculated 1% in sterile 500ml and incubated in a 30 ° C incubator for 30 hours in 3 hour increments. Changes in optical density, 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). ) Was consumed to give a total acidity.

Pediococcus pentosaceus gen 1 isolated through the present invention requires various studies to use as a probiotic. The lactic acid produced by Gen 1 lactic acid bacteria, which had the best 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 according to the incubation time of strain Gen 1 was small at 0.072 initially and 0.377 at 3 hours of cultivation, but the increase was sharply increased at 6 to 15 hours of cultivation. Could be confirmed. After 18 hours of incubation, the growth slowed down and showed no pattern of growth. The logarithmic phase of the gen 1 strain was therefore found to range from 3 to 15 hours. On the other hand, the pH change of the culture medium was inversely proportional to the growth of the bacteria, and the pH was rapidly decreased from the initial 6.45 to 6.24 at 3 hours of cultivation and from 3 to 12 hours of cultivation, which was lowered to the final pH 4.10 after 15 hours. In FIG. 3,? Denotes OD, ○ denotes TTA (Total Titratible acidity), and ▲ denotes pH.

In addition, the total acidity value of the culture medium was proportional to the growth of the fungi, and it increased rapidly from the initial 3.0 to 5.60 at 6 hours, and 18 hours at nearly 15.17. After that, the change decreased and the final acidity reached 18.00.

Experimental Example  3

Pediocaucus Pentosaceus Zen 1 ( Pediococcus pentosaceus zen1 ) Isolated  Growth temperature and salt effect

In order to investigate the effect of Pediococcus pentosaceus gen 1 on the growth temperature, the selected Pediococcus pentosaceus gen 1 was precultured in MRS medium, and then the growth temperature was increased to 25, 30 and 37 ° C. After adjusting for 24 hours, the number of viable cells was measured. Gen 1 showed high growth and acidity not only at 30 ° C. but also at lower temperatures, and slightly lower growth and acidity at temperatures above 30 ° C. (Table 4).

In addition, to investigate the effect of Pediococcus pentosaceus gen 1 on sodium chloride concentration, the preselected Pediococcus pentosaceus gen 1 was precultured in MRS medium and then used for marine fish. The salt concentration was adjusted to 0, 1, 3.5, 5, 7, 10 wt% based on 3.5 wt%. Pediacoccus pentosaceus gen 1 is a good antimicrobial activity against the fish disease bacteria, but the results of the investigation whether it affects the salt concentration is shown in FIG. Gen 1 showed high growth and acidity under the salt concentration of 3.5 wt% sodium chloride in the sea, and relatively high growth and acidity even at the concentration of 3.5% sodium chloride. As a result, pediococcus pentosaceus gen 1 derived from sea fish capable of growing at 3.5% sodium chloride concentration is considered to be a probiotic (microbial product) for application to aquaculture fish. In Fig. 4,? Indicates OD and? Indicates total acidity, respectively.

Growth temperature (℃) Absorbance (OD _600nm ) Total acidity (ml) 25 3.620 15.71 30 3.784 17.35 37 2.383 11.51

The present invention is a lactic acid bacterium derived from the fish gut, pediococcus pentosaceus gen 1 having an antimicrobial activity, showing a high lactic acid production and high growth in fresh and sea water, and a microbial agent comprising the same to be used in fisheries and the like Can be.

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 view showing the identification results of Pediococcus pentosaceus gen 1 according to the present invention.

Figure 2 is a graph showing the acid resistance of the Pediococcus pentosaceus gen 1 strain according to the present invention.

Figure 3 is a graph showing the results of measuring the bacterial growth and pH and total acidity change according to the three-hour unit while incubating for 36 hours Pediococcus pentosaceus gen 1 according to the present invention.

Figure 4 is a graph showing the effect on the salt concentration of Pediococcus pentosaceus gen1 in accordance with the present invention.

Claims (2)

Pediococcus pentosaceus gen 1 (KACC 91336P), which is isolated in the gastrointestinal tract of fish and has antimicrobial activity. A microbial agent comprising Pediococcus pentosaceus gen 1 having antimicrobial activity, characterized in that the mixture is made in feed of seawater and freshwater fish.

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