KR100865075B1 - Novel probiotic strain Lactobacillus sp. SM1 showes high cell adherence - Google Patents

Abstract

The present invention relates to a novel Lactobacillus SM1 strain and a probiotic produced therein, and more particularly, to a novel Lactobacillus SM1 strain having antibacterial activity against Vibrio pathogenic strains and various pathogenic microorganisms. The novel Lactobacillus SM1 strain of the present invention is resistant to gastric acid and bile acids, and is excellent in adsorption ability of intestinal cells and is caused by pathogenic microorganisms because of its growth inhibitory activity against pathogenic microorganisms including Vibrio microorganisms. It can be usefully used as a composition for the treatment and prevention of diseases.

Lactobacillus, Vibrio, Acid Resistance, Bile Resistance, Antibacterial Activity, Intestinal Fixation, Probiotics

Description

Novel probiotic strain Lactobacillus sp. SM1 showes high cell adherence}

1 is a molecular diagram showing the analysis of the 16S rRNA sequence of the new strain of the present invention.

2 is an antibacterial investigation conducted by the V. cholera (Vibrio cholerae KCTC 2715), using the culture supernatant of the novel strain of the present invention.

Figure 3 is a comparison of intestinal fixation ability between the new strain and the comparative strain Lactobacillus rhamnosus GG of the present invention.

The present invention relates to a novel Lactobacillus SM1 strain and the probiotic they produce, and more particularly to a novel Lactobacillus SM1 strain and its probiotics having antibacterial activity against Vibrio strains and various hospital strains.

Vibrio are classified bibeuriohgwa (family Vibrionaceae) (Vibrio) Twelve species have been reported in the genus. In particular, as a human pathogen, Vibrio bulnipicus ( Vibrio) vulnificus ) was first reported in 1979 and is known as Vibrio cholera ( Vibrio). cholerae ) and Vibrio parahaemolyticus are well known as important causative agents of acute diarrhea. Vibrio bacteria, including Vibrio bulnipicus, are halophilic motile Gram-negative rods that live in seawater, tidal flats, and marine life (Hollis DG, et. al . J Clin Microbiol 3: 425-431, 1976). Vibrio bacteria are infected when people who drink alcohol every day, people with chronic illnesses such as diabetes or cirrhosis eat raw seafood, or when skin wounds are exposed to seawater, fishing, or by fishing or shelling (Blake PA, et. al . N Engl J Med 300: 1-5, 1979). There is no effective vaccine for the infection of the Vibrio genus pathogen, and prevention is important because it is classified as a fatal disease at the onset. Therefore, the development of vaccines against the pathogenic bacteria is urgent and for this purpose, active researches on probiotics, probiotics, enzymes, oligosaccharides, etc. are being actively conducted.

Probiotics are generally defined as live microbial food ingredients that are beneficial to health (Liilly and Stillwell, Science 147: 747-748, 1965), but recently Salminen et al . ( Int . J. Food) . Microbiol . 44: 93-106, 1998), defined as living microbial products in food and feed or dietary additives designed to promote human or animal health. Bacteria that are most commonly used as a probiotic is a Lactobacillus bacteria (Lactobacillus) and streptococcus (Streptococcus) as a lactic acid bacteria. Lactobacillus Among the Lactobacillus genus know Fig filler's (Lactobacillus acidophilus), Lactobacillus Bulgaria kusu (Lactobacillus bulgaricus ), Lactobacillus plantarum ), Lactobacillus casei ) , Lactobacillus lactis ) , among the Streptococcus genus Streptococcus faecium), Streptococcus Thermo filler's (Streptococcus thermophilus ), Streptococcus diacetilactis ) is the main species. Probiotic Lactobacillus is most effective when it is able to settle in the intestine to proliferate and inhabit. Settling to the intestinal cell wall is an important prerequisite for inhabiting the human intestine. It is known that probiotic lactic acid bacteria can have various beneficial effects in the intestine by fixing on the intestinal cell surface (Donohue et. al , Asia pacific J. Clin . Nutr . 5: 25-28, 1996). Settlement demonstrates the potential for probiotic lactic acid bacteria to live in the human intestine and to grow in intestinal conditions. In addition, lactic acid bacteria must be resistant to oral and stomach enzymes, low pH of gastric juice, bile and mucus in the small intestine in order to function well in human intestines (Havenaar et al ., Probiotics : the Scientific Basis (Fuller, R., ed.), Pp. 209-224, 1992).

Patents on probiotic lactobacillus include Lactobacillus paracasei CLW-011 (Korean Patent No. 446148) isolated from the intestine of pigs, and Lactobacillus permantum YL-3 (Korean Patent No. 351177) isolated from the caecum of chickens. ) And Lactobacillus ashidophilus 30SC (Korean Patent No. 2001-0070689) isolated from human feces, but there are no lactic acid bacteria microorganisms isolated from fish intestines.

Accordingly, the present inventors have completed the present invention by confirming the novel Lactobacillus sp. SM1 strain isolated from the shrimp intestine has the effect of inhibiting the growth of vibrio strains and various pathogenic microorganisms, and excellent in acid resistance, bile resistance and intestinal fixation. .

It is an object of the present invention to provide novel Lactobacillus strains and the probiotics they produce.

In order to achieve the above object, the present invention provides a novel Lactobacillus strain.

The present invention also provides a probiotic comprising the Lactobacillus strain or its culture supernatant.

The present invention also provides a feed additive comprising the Lactobacillus strain or its culture supernatant.

The present invention also provides a composition for the treatment and prevention of diseases caused by pathogenic microorganisms containing the Lactobacillus strains or their culture supernatants or the above probiotics as an active ingredient.

The present invention also provides a health food for preventing diseases caused by pathogenic microorganisms containing the Lactobacillus strains or their culture supernatants or the probiotics as an active ingredient.

In addition, the present invention provides a lactobacillus strain or a culture supernatant thereof or a lactic acid bacterium preparation containing the probiotics as an active ingredient.

In another aspect, the present invention provides a fermented milk containing the Lactobacillus strain or the culture supernatant thereof or the probiotics as an active ingredient.

The present invention also provides a method for producing probiotics from the novel Lactobacillus strain of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Lactobacillus strain.

The inventors of the present invention were collected from the sea near Anmyeondo located on the west coast of Chungcheongnam-do ( Penaus). Lactobacillus strains were isolated from the viscera of orientalis KISHINOUYE ), and new Lactobacillus SM1 strains with excellent antibacterial activity were isolated from Vibrio strains. This strain was Gram positive and bacilli. In addition, the strain of the present invention through 16S rRNA gene sequencing and analysis method is Lactobacillus casei ( Lactobacillus) casei ) was identified as a new strain with 97% homology (see Figure 1). Since the sugar fermentation pattern of the new strain of the present invention was analyzed, as a result, glycerol (glycero), D-ribose (D-Ribose), D-galactose (D-Galactose), D-Fractose (D-Fractose), D-Mannose, D-Mannitol, N-Acetylglucosamine, Amygdalin, Arbutin, Esculin Ferric Citrate, Salicycin Salicin, D-Delbiose, D-Maltose, Gentiobiose, Potassium Gluconate, Potassium 2-Ketogluconate Was shown (see Table 1). The new strain was written on December 4, 2006 to the Lactobacillus Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology. sp . SM1 "(KCTC 11043BP). The new strain of the present invention was then examined for acid resistance by investigating the survival rate in artificial gastric juice. As a result, the strain L1 of the genus Lactobacillus sp. The survival rate was 67% after the last minute (see Table 2) .Bile resistance test also showed growth on MRS plate added with 3% bile acid, indicating strong resistance to bile acid (see Table 3). In addition, the new strain of the present invention was confirmed to have a growth inhibitory ability against the pathogenic Vibrio strain and several pathogenic strains (see Table 4 and Figure 2).

The present invention also provides a probiotic comprising the Lactobacillus strain or its culture supernatant.

Probiotics can function when they can settle and live in the gut. The present inventors investigated the intestinal fixation ability of the Lactobacillus rhamnosus GG strain known to be excellent in intestinal fixability and the Lactobacillus genus SM1 strain of the present invention. As a result, it was confirmed that the Lactobacillus SM1 strain of the present invention showed higher intestinal fixation ability than the comparative strain (see FIG. 3). Through this, it can be seen that the Lactobacillus genus SM1 strain of the present invention and its culture supernatant can be usefully used as probiotics. The probiotics of the present invention can be used in animals, including livestock, and can be added to food or animal feed. It can also be used in veterinary medicines and medicines.

The present invention also provides a feed additive comprising the Lactobacillus strain or its culture supernatant.

The feed additive of the present invention preferably joins the novel Lactobacillus SM1 strain or its culture supernatant at 1 to 20 parts by weight, and more preferably at 10 to 20 parts by weight, based on 100 parts by weight of the feed.

The present invention also provides a composition for the treatment and prevention of diseases caused by pathogenic microorganisms containing the Lactobacillus strains or their culture supernatants or the probiotics as an active ingredient.

The pathogenic microorganism is a vibrio genus microorganism or a pathogenic microorganism is preferred, the vibrio microorganism is Vibrio parahemoriticus ( Vibrio parahaemolyticus ), Vibrio fluvialis ), Vibrio anguilarum), Vibrio know tease tee Syracuse (Vibrio alginolyticus), Vibrio cholera (Vibrio cholarae ) and Vibrio vulnificus are preferably selected from the group consisting of, but are not limited thereto. The pathogenic microorganism is Yersinia garvieae), the Monastir to live as a child Monica Monica is a subspecies live (Aeromonas salmonicida subsp . salmonicida ), Edwardsiella tarda ) and Leucothrix mucor are preferably selected from the group consisting of, but are not limited thereto.

The disease is preferably selected from the group consisting of acute diarrhea, enteritis, gastroenteritis, constipation, abdominal pain, bloating, cholera and food poisoning, but is not limited thereto.

The composition for the treatment and prevention of diseases caused by pathogenic microorganisms comprising the Lactobacillus strains or their culture supernatants or probiotics as an active ingredient of the present invention can be administered orally or parenterally during clinical administration, the main component One or two or more pharmaceutically acceptable conventional carriers or one or two or more excipients may be selected for the effective amount of the Lactobacillus strains or their culture supernatants or probiotics into the compositions of conventional pharmaceutical formulations. have.

The carrier may be used by selecting one or two or more from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of the fragrances, vitamins, and antioxidants. Can be used.

In the present invention, the carrier and the additive may be used in any pharmaceutically acceptable, specifically, as a diluent lactose (lactose monohydrate), corn starch (cornstarch), soybean oil (soybean oil), microcrystalline cellulose (microcrystalline cellulose) ) Or mannitol (D-mannitorl) is preferable, magnesium stearate or talc is preferred as a lubricant, polyvinyipyrolidone (PVP: polyvinyipyrolidone) or hydroxypropyl cellulose (HPC :) hydroxypropylcellulose). In addition, the disintegrant may be selected from among carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polyacrylin potassium or cross-linked polyvinylpyrrolidone. Preferably, the sweetening agent is selected from sucrose, fructose, sorbitol or aspartame, and as a stabilizer, carboxymethylcellulose sodium (Ma-CMC), beta-cyclodextrin, It is selected from white bee's wax or xanthan gum, and preservatives include methyl p-hydroxy benzoate (methhlparaben), propyl p-hydroxy benzoate (propylparaben), or sorbic acid. It is preferable to select from potassium sorbate.

The composition for the treatment and prevention of diseases of the present invention is used for the purpose of efficiently treating or preventing diseases caused by pathogenic microorganisms is not particularly limited in the formulation, beverages, powders, capsules, soft capsules, tablets It can be used as a gum, adhesive type liquid composition, and can be used as a transdermal dosage form such as lotion, ointment, gel, cream, patch or spray, and can be used as a preparation for milk, pills, tablets, powders, granules and the like. have.

In addition to the above main components, the composition for the treatment and prevention of diseases of the present invention includes minerals such as vitamins B, C, E, beta-carotene, Ca, Mg, Zn, phospholipids such as lecithin or maltose, oligosaccharides, amino acids Compounds of the above can be used, and among them, the use of 2 to 3 components in vitamin C, E or beta-carotene, maltol and the like is more preferable because it can enhance the bioactive effect.

In addition to the above components, in order to enhance the taste as a known additive, natural flavors such as plum, lemon, pineapple or herb, or natural fruit juice, natural pigments such as chlorophyllin and flavonoids, and fructose, sweet sugar Can be mixed with honey, sugar alcohol, sugar and acidulants such as citric acid and sodium citrate.

The present invention also provides a health food for preventing diseases caused by pathogenic microorganisms containing the Lactobacillus strains or their culture supernatants or the probiotics as an active ingredient.

The pathogenic microorganism is a vibrio genus microorganism or a pathogenic microorganism is preferred, the vibrio microorganism is Vibrio parahemoriticus ( Vibrio parahaemolyticus ), Vibrio fluvialis), not Anguilla Vibrio Room (Vibrio anguilarum), Vibrio know tease tee Syracuse (Vibrio alginolyticus), Vibrio cholera (Vibrio cholarae ) and Vibrio vulnificus are preferably selected from the group consisting of, but are not limited thereto. The pathogenic microorganism is Yersinia garvieae), the Monastir to live as a child Monica Monica is a subspecies live (Aeromonas salmonicida subsp . salmonicida ), Edwardsiella tarda ) and Leucothrix mucor are preferably selected from the group consisting of, but are not limited thereto.

The disease is preferably selected from the group consisting of acute diarrhea, enteritis, gastroenteritis, constipation, abdominal pain, bloating, cholera and food poisoning, but is not limited thereto.

Examples of health foods containing the probiotics of the present invention include folk remedies for the purpose of improving liver function, such as tea, jelly, juice, extract, and beverages, which contain the probiotics as an active ingredient. As described above, the disease preventing health food of the present invention processed in various forms is easy to take and can be stored for a long time without any side effects on the human body.

When the Lactobacillus strain of the present invention or the culture supernatant or probiotics thereof are used as food additives, each of the extracts may be used as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. have. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food or beverages, it is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the Lactobacillus strain of the present invention or their culture supernatant or probiotic raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the kind of food. Examples of the food to which the Lactobacillus strain of the present invention or the culture supernatant or probiotics thereof may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gum, and ice cream. Includes dairy products, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional components, as in a conventional beverage. The natural carbohydrates described above are glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin, aspartame, and the like can be used. The ratio of the natural carbohydrate is generally 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 ml of the Lactobacillus strains or their culture supernatants or probiotics of the present invention.

The above-mentioned Lactobacillus strains of the present invention or culture supernatants or probiotics thereof are various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH It may contain a regulator, stabilizer, preservative, glycerin, alcohol, carbonation agent used in the carbonated beverage. In addition, the probiotics of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but the probiotics of the present invention are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

In addition, the present invention provides a lactobacillus strain or a culture supernatant thereof or a lactic acid bacterium preparation containing the probiotics as an active ingredient.

Lactobacillus strains or their culture supernatants or probiotics of the present invention is preferably 0.01 to 50 parts by weight per 100 parts by weight of lactic acid bacteria preparation, but is not limited thereto.

In another aspect, the present invention provides a fermented milk containing the Lactobacillus strain or the culture supernatant thereof or the probiotics as an active ingredient.

Lactobacillus strains or their culture supernatants or probiotics of the present invention is preferably 0,01 to 50 parts by weight per 100 parts by weight of fermented milk, but is not limited thereto.

The present invention also provides a method for producing probiotics from the novel Lactobacillus strain of the present invention.

The present invention provides the following process: 1) culturing the Lactobacillus SM1 strain of the present invention; And 2) obtaining probiotics produced by the cultured Lactobacillus SM1 strain of step 1).

Hereinafter, the present invention will be described in detail by examples and formulation examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples.

< Example  1> Neomycology  Separation and Identification

For the development of lactic acid strains, the inventors diluted the shrimp gut collected from the sea and incubated for 48 hours at 30 ℃ condition in MRS (Difco, USA) medium. After cultivation, dozens of colonies with gloss milky white in the form of a typical lactic acid bacterium were screened and separated. Selected colonies were purely separated by culturing in fresh medium three times. Pure cultured microorganisms were stored in a culture medium added with 20% glycerol and stored at minus 80 ℃.

Vibrio of Isolated Strains The SM1 strain having the best antibacterial activity against the strain was selected and analyzed. The strains were identified by physiological biochemical and molecular lineage methods. Morphological characteristics were Gram-positive and bacillus, and it was identified as a novel microorganism with 97% homology with Lactobacillus casei through 16S rRNA gene sequencing and analysis. The nucleotide sequence of the analyzed 16S rRNA of the isolated strain is shown in SEQ ID NO: 1, the phylogenetic tree showing the molecular phylogenetic location is shown in FIG. The sequence analysis method is as follows. DNA was isolated and cultured cells were suspended in 100 µl of STES buffer [0.4 M NaCl, 0.2 M Tris-HCl (pH 7.6), 0.01 M EDTA, 1% SDS]. Glass beads were added to the samples and disrupted with TOMY (micro tube mixer MT-360) for 5 minutes to elute the cytoplasm. The cell eluate was suspended in 200 μl of TE buffer (pH 8.0) and 200 μl of phenol / chloroform and centrifuged at 12,000 rpm for 5 minutes, and then the supernatant was treated with 5 μl of RNase A, followed by 37 ° C. The reaction was carried out for 1 hour under the conditions, and chloroform was added thereto, followed by centrifugation for 5 minutes at 12,000 rpm. Then, the supernatant of the centrifuged sample was washed twice with ethanol and dried (vacuum dry: SpeedVac). Finally, the recovered DNA was dissolved and stored in sterile distilled water. For PCR amplification of 16S rDNA, PCR premix (Bionia, Korea) and universal primer were used.Forward primer (5 '-GA GTT TGA TCC TGG CTC AG -3': SEQ ID NO: 2) and reverse primer (5) Amplification using '-GGT TAC CTT GTT ACG ACT T-3' (SEQ ID NO: 3). PCR amplification was performed by adjusting 20 μl of the total reaction solution. 1 minute at 94 ° C for DNA denaturation, 1 minute at 50 ° C for binding of primers, and 1 minute 50 seconds at 72 ° C for the synthesis of DNA strands was repeated 30 times. Sequencing and phylogenetic analysis of the amplified DNA is described by Chang et al. al ., Int . J. Syst . Evol . Microbiol . 52: 377-381, 2002). The novel microorganism of the present invention identified by the above method was identified as “ Lactobacillus sp.” On December 4, 2006 at the Korea Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, located at 52, Eeun-dong, Yuseong-gu, Daejeon, Korea. Deposited as SM1 ”(KCTC 11043BP).

< Example  2> Lactobacillus ( Lactobacillus sp .) SM1  Physiological and Biochemical Characterization of Strains

For biochemical characterization of new strains, sugar fermentation patterns of the strains were analyzed after incubation at 37 ° C. for 24 and 48 hours using API 50CHL system (BioMrieux, Marcy I'Etolite, France) (Table 1).

Carbohydrates Lactobacillus SM1 strain Incubation time (hr) 24 48 Glycerol + + _{D -} Ribose + + _D- galactose _(D- Galactose) + + _{D -} glucose + + _{D -} Fractose + + _D- mannose _(D- Mannose) + + _{D -} Mannitol + +  N-acetylglucosamine + + Amygdalin + + Arbutin w + Esculin ferric citrate + + Salincin w + _D- Delhi BIOS _(D- Delibiose) + + Maltose _{D- (D-} Maltose) + + Gentiobiose + + Potassium Gluconate - w Potassium 2-Ketogluconate - w

+, Positive; -,voice; w, weak positive

< Example  3> Lactobacillus SM1  Strain Acid resistance  Research

Synthetic gastric juice: 3.5 g D-glucose 3.5 g, NaCl 2.05 g, KH ₂ PO ₄ 0.6 g titrated to pH 2.5 using 1 N HCl buffer to determine the acid resistance of Lactobacillus SM1 strains , 0.1 g of CaCl ₂ , 0.37 g of KCl, 0.05 g of Bile extract porcine (B8631, Sigma, USA), 0.1 g of lysozyme and 13.3 mg of pepsin in 1 L of distilled water] , And treatment for 30 and 120 minutes, and then compared to the survival rate (%) by measuring the number of viable cells by plating on MRS agar (Difco, USA).

As a result, Lactobacillus SM1 strain showed 67% survival rate when stress was applied to artificial gastric juice for 30 minutes, and 10% survival rate after 2 hours treatment.

New strain CFU ml ^-1 (% survival rate) 0 min 30 minutes 120 minutes Lactobacillus SM1 1.2 × 10 ⁸ (100) 8.0 × 10 ⁵ (0.67) 1.2 × 10 ⁵ (0.1)

< Example  4> Lactobacillus SM1  Strain Bile resistance  Research

To test the resistance of bile acids of Lactobacillus SM1 strains, the strains were plated on MRS agar plates containing 0, 0.3, 1, 3 and 5% (w / v) of bile acid (porcine bile extract, Sigma). After 48 hours incubation at 37 ℃ conditions the growth of SM1 strain was measured

As a result, the Lactobacillus SM1 strain showed no growth in the experimental group added with 5% bile acid, but showed a strong resistance to bile acid by growing up to the experimental group added with 3% bile acid.

New strain Bile Acid Concentration (%) 0 0.3 One 3 5 Lactobacillus SM1 T T T W NG

T, bile tolerance ; W, weak growth ; NG , no growth

< Example  5> Investigation of growth inhibitory properties of pathogenic bacteria

To test pathogenic bacterial antagonism, Lactobacillus SM1 strains were inoculated in 10 ml MRS broth and incubated at 37 ° C for 24 hours, using a 0.22 μl particle size filter (Sartorius, Palaisequ, France). The cells were removed to obtain only the supernatant. Pathogenic vibrio strains were cultured in Marine Agar (Difco, USA), Edwardsiella tarda , Aeromonas salmonicada, and Aeromonas salmonicida. subsp . salmonicida ), Leucothrix mucor), Streptococcus para Ube lease (Streptococcus parauberis), Streptococcus am a child (Streptococcus iniae), Lactobacillus nose kusu the garbage (Lactococcus garvieae) is TSA (Tryptic Soy Agar: by adjusting the concentration of each culture from Difco, USA) medium with 10 ^6-7 were plated in the medium. 60 μl of the Lactobacillus SM1 strain supernatant was added to a paper disc (paper disc: 6 mm, Yoyo Roshi Kaisha, Japan) and cultured for 24 hours, and then the diameter of the inhibitory ring (mm) was measured. As a control, a medium without cultured bacteria was used.

As a result, it was confirmed that the Vibrio genus pathogen strains and various pathogenic microorganisms have growth inhibition ability (Table 2 and FIG. 2).

Pathogens Suppression (diameter, mm, mean ± SD) Vibrio parahemoticus ( Vibrio parahaemolyticus KCTC 2729) 10.0 ± 1.0 Vibrio fluvilis ( V. fluvialis KCTC 2473) 9.5 ± 1.5 Vibrio Anguilarum ( V. anguilarum ) 14.0 ± 0.0 Vibrio alginolitis ( V. alginolyticus KCTC 2472) 9.8 ± 1.2 Vibrio cholera ( V. cholarae KCTC 2715) 15.0 ± 0.3 Vibrio vulnyificus ( V. vulnificus KCTC 2959) 11.0 ± 0.0 Streptococcus parauberis KCTC 3651) 0 Streptococcus am a child (S. iniae KCTC 3657) 0 Lactococcus garvieae KCTC 3772) 0 Yersinia ruckeri KCTC 12265) 9.5 ± 1.5 Aeromonas Salmonicada Subspecies Salmonicada salmonicida subsp . salmonicida KCTC 12266) 8.0 ± 1.0 Edwardsiella tarda KCTC 12267) 11.6 ± 0.4 Leucothrix Leucothrix mucor KCTC 12450) 15.0 ± 0.2

< Example  6> Lactobacillus SM1  Intestinal Settleability of Strains

In this example, HT-29 cell line, which is a human colon adenocarcinoma cell, was used for experiments on the degree of fixation ability to the intestinal cells, and the existing intestinal fixation ability was excellent as a comparative strain with the Lactobacillus genus SM1 strain. L. rhamnosus , known as Lactobacillus GG strain was used. HT-29 cell line was grown in culture in RPMI 1640 medium (Gibco BRL, USA) containing 10% (v / v) Fetal Bovine Serum (FBS). Lactobacillus SM1 strains grown in MRS broth were prepared by washing with PBS (phosphate-buffered saline) and then adding RPMI 1640 medium without serum. The cultured HT-29 cell line was divided into 16-well cell culture plates (BD science, USA), cultured to form a single layer, and then, the prepared Lactobacillus strain SM1 (10 ⁹ CFU / mL) was added thereto. The cells were cultured in a 37 ° C. cell incubator containing 5% CO ₂ . After 2 hours of incubation, Lactobacillus SM1 strain that failed to attach to HT-29 cell line was washed with PBS, and the strain attached with 0.1% triton X-100 was detached and then used with MRS agar (Difco, USA). After incubating at 37 ° C. for 72 hours, the number of Lactobacillus genus SM1 strains was grown. In the same manner as described above, Lactobacillus rhamnosus, a comparative strain, also examined the intestinal fixability of HT-29 cell line.

As a result, it was confirmed that the novel lactobacillus SM1 strain of the present invention has superior fixation ability than the Lactobacillus rhamnosus GG comparative strain known to be excellent in intestinal fixation (FIG. 3).

< Formulation example  1> Manufacture of food

Food containing the Lactobacillus SM1 strain of the present invention and its culture supernatant were prepared as follows.

1. Preparation of Cooking Seasonings

Cooking seasoning was prepared by mixing 8 to 12 parts by weight of the culture supernatant of the Lactobacillus SM1 strain of the present invention to 100 parts by weight of cooking seasoning for improving liver function.

2. soup  And juicy ( gravies Manufacturing

Culture supernatant of the Lactobacillus SM1 strain of the present invention 8 to 12 parts by weight, Injinho extract 0.1 to 4 parts by weight, brown root extract 0.1 to 3 parts by weight, dermis extract 0.1 to 3 parts by weight, creation extract 0.1 to 3 parts by weight, licorice 0.1-3 parts by weight of the extract and 0.01-3.24 parts by weight of the red ginseng extract were added to 100 parts by weight of the soup and the juice to prepare meat products for improving liver function, soups and noodles of noodles.

3. Dairy products ( dairy products Manufacturing

Culture supernatant of the Lactobacillus SM1 strain of the present invention 8 to 12 parts by weight, Injinho extract 0.1 to 4 parts by weight, brown root extract 0.1 to 3 parts by weight, dermis extract 0.1 to 3 parts by weight, creation extract 0.1 to 3 parts by weight, licorice 0.1-3 parts by weight of the extract, 0.1-3.24 parts by weight of the red ginseng extract were added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

4. Linear  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh.

The culture supernatant of the genus Lactobacillus SM1 strain of the present invention was decompressed and concentrated in a vacuum concentrator, dried by spraying and a hot air dryer, and ground to a particle size of 60 mesh by a grinder to obtain a dry powder.

The grains, seeds, and extracts of the present invention prepared above were formulated in the following proportions.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Dry powder (10 parts by weight) of the culture supernatant of the genus Lactobacillus SM1 strain,

Ganoderma lucidum (0.5 parts by weight), and

Sulfur (0.5 part by weight).

< Formulation example  2> Manufacture of beverage

1. Preparation of carbonated drinks

5 to 10 parts by weight of sugar, 0.05 to 0.3 parts by weight of citric acid, 0.005 to 0.02 parts by weight of caramel, 0.1 to 1 parts by weight of vitamin C, and 10 parts by weight of an additive of the culture supernatant of the genus Lactobacillus SM1 of the present invention, ~ 94 parts by weight of purified water is mixed to make a syrup. The syrup is sterilized at 95 to 98 ° C. for 20 to 180 seconds, mixed with cooling water at a ratio of 1: 4, and then carbonated gas is injected into 0.5 to 0.82 parts by weight to prepare a carbonated beverage. It was.

2. Preparation of Functional Beverages

Culture of Lactobacillus SM1 strain of the present invention in 0.1 parts by weight of vitamin C, 5.8 parts by weight of fructose, 3.8 parts by weight of sugar, 0.12 parts by weight of citric acid, 0.03 parts by weight of malic acid, 0.04 parts by weight of sodium citrate and 0.02 parts by weight of gardenia blue pigment 10 parts by weight of the supernatant was combined and completely dissolved in metered water. The dissolved beverage was adjusted with metered purified water such that the total amount was 1 part by weight.

3. Manufacture of health drinks

Instant sterilization was carried out by homogeneously mixing 10 parts by weight of the supernatant of the Lactobacillus genus SM1 strain of the present invention with subsidiary materials such as 0.5 parts by weight of liquid fructose, 2 parts by weight of oligosaccharide, 2 parts by weight of sugar, 0.5 parts by weight of salt and 75 parts by weight of water. Then it was packaged in a small packaging container such as glass bottles, plastic bottles to prepare a healthy drink.

4. Preparation of Vegetable Juice

10 g of the vacuum dried liquid of the Lactobacillus SM1 strain culture supernatant of the present invention was added to 1 L of tomato or carrot juice to prepare a vegetable juice for health promotion.

5. Preparation of Fruit Juice

10 g of the vacuum dried of the Lactobacillus SM1 strain culture supernatant of the present invention was added to 1 L of apple or grape juice to prepare a fruit juice for health promotion.

< Formulation example  3> Preparation of Fermented Milk

The skim milk powder was sterilized for 15 seconds at 72-75 ° C. of the raw material oil, in which the non-fat solid content was adjusted to 8-20 parts by weight. The sterilized raw milk was cooled to a certain temperature and then inoculated with Lactobacillus SM1 strain at a concentration of 10 ⁶ cfu / mL and incubated until pH 4-5. After the culture material, the culture solution was cooled. Syrup was prepared by dissolving 0.1-50 parts by weight of fruit juice, 0.1-20 parts by weight of dietary fiber, 0.5-30 parts by weight of glucose, 0.1-15 parts by weight of oligosaccharide, 0.001-10 parts by weight of calcium, 0.0001-5 parts by weight of vitamins, and the like. . The syrup prepared in this way was sterilized, cooled, mixed with a cultured fructose at a predetermined ratio, stirred and packaged in a homogenized container to prepare fermented milk.

< Formulation example  4> Lactobacillus Uniform  Produce

Lactobacillus SM1 strains were inoculated in MRS broth at a concentration of 10 ⁶ cfu / ml and pH-control fermentation (pH-control fermentation) was performed at 37 ° C for 18-24 hours. After completion of the culture, the cells were recovered by centrifugation at 10,000 × g at 4 ° C. The recovered cells were mixed in the same amount with a protective agent containing 2.5% whey and 5% sucrose in 5% skim milk and then powdered through a lyophilizer. The dry powders of the Lactobacillus genus SM1 strain thus prepared showed a viable cell count of 1 × 10 ¹¹ cfu / g or more.

< Formulation example  5> Preparation of Lactic Acid Bacteria Formulations

Lactic acid bacteria formulations, such as lactic acid bacteria foods, formal preparations were prepared using the lactic acid bacteria powder prepared in Preparation Example 4. 20 parts by weight of dry powder of Lactobacillus SM1 strain, 10 parts by weight of oligosaccharide, 20 parts by weight of anhydrous glucose, 5 parts by weight of fructose, 2 parts by weight of vitamin C, 5 parts by weight of fruit powder, 5 parts by weight of aloe, dietary fiber 15 18 parts by weight and 18 parts by weight of chaff were mixed, and a predetermined amount was dispensed into a stick or a bottle to spore. The lactic acid bacterium preparation thus prepared maintained a viable cell count of 5 × 10 ⁸ cfu / g or more.

The novel Lactobacillus SM1 strain of the present invention is resistant to gastric acid and bile acids, and is excellent in adsorption ability of intestinal cells and is caused by pathogenic microorganisms because of its growth inhibitory activity against pathogenic microorganisms including Vibrio microorganisms. It can be usefully used as a composition for the treatment and prevention of diseases.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel probiotic strain Lactobacillus sp. SM1 showes high cell          adherence <130> 6p-12-45 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1348 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA of Lactobacillus sp. SM1 <400> 1 tggcggacgg gtgagtaaca cgtgggtaac ctgccctaaa gtgggggata acatttggaa 60 acagatgcta ataccgcata aatcttggaa cctcatggtt ccaagctgaa agatggcgta 120 agctatcgct tttggatgga cccgcggcgt attagctagt tggtgaggta aaggctcacc 180 aaggcgatga tacgtagccg acctgagagg gtgatcggcc acattgggac tgagacacgg 240 cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg 300 agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt tggagaagaa 360 cgtacgtgag agtaactgtt cacgtagtga cggtatccaa ccagaaagcc acggctaact 420 acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggattt attgggcgta 480 aagcgagcgc aggcggtttc ttaagtctga tgtgaaagcc ctcggctcaa ccgaggaagt 540 gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 600 ggaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg 660 acgctgaggc tcgaaagcat gggtagcgaa caggattaga taccctggta gtccatgccg 720 taaacgatga atactaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 780 aagtattccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt gacgggggcc 840 cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct 900 tgacatcttc tgctattcta agagattaga agttcccttc ggggacagaa tgacaggtgg 960 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1020 cccttatgac tagttgccag cattaagttg ggcactctaa tcagactgcc ggtgacaaac 1080 cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1140 gctacaatgg atggtacaac gagtcgcaaa accgcgaggt taagctaatc tcttaaagcc 1200 attctcagtc cggactgtag gctgcaactc gcctacacga agtcggaatc gctagtaatc 1260 gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1320 atgagagttt gtaacacccg aagccggt 1348 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 gagtttgatc ctggctcag 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 ggttaccttg ttacgact 18

Claims (14)

Vibrio bacteria and Yersinia garvieae , Aeromonas salmonicida subsp. Salmonicida , Edwardsiella tarda and Leukotrikirix radish Lactobacillus sp . Strain characterized by inhibiting a pathogen selected from the group consisting of Leucothrix mucor and having the following mycological characteristics and having the 16S rRNA sequence shown in SEQ ID NO: 1: 1) bacilli that show positive upon gram staining; 2) Glycerol (D-Ribose), D-Glactose (D-glactose), D-Glucose (D-glucose), D-Fractose, D-Mannose (D- Mannose, D-Mannitol, N-Acetylglucosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-deli Biomass (D-Delibiose), D-Maltose, Gentiobiose, Potassium Gluconate, Potassium 2-Ketogluconate (Potassium 2-Ketogluconate) as a carbon source . The Lactobacillus strain according to claim 1, which is described in Accession No. KCTC 11043BP. delete Probiotics comprising the Lactobacillus strain of claim 1 or the culture supernatant thereof. A feed additive comprising the Lactobacillus strain of claim 1 or a culture supernatant thereof. Claim 1 Lactobacillus strain or a culture supernatant or a composition for the treatment and prevention of diseases caused by pathogenic microorganisms containing the probiotics of claim 4. The method of claim 6, wherein the pathogenic microorganism is Vibrio parahemoriticus ( Vibrio) parahaemolyticus ), Vibrio fluvialis ), Vibrio anguilarum), Vibrio know tease tee Syracuse (Vibrio alginolyticus ), Vibrio cholera ( Vibrio cholarae ), Vibrio vulnificus , Yersinia garvieae ), Salmonica subtype Salmonica ( Aeromonas) salmonicida subsp . salmonicida ), Edwardsiella tarda ) and Leucothrix mucor ( Leucothrix mucor ) composition for treatment and prevention, characterized in that selected from the group consisting of. The composition for treating and preventing diseases of claim 6, wherein the disease is any one selected from the group consisting of acute diarrhea, enteritis, gastroenteritis, constipation, abdominal pain, bloating, cholera and food poisoning. Health food for preventing diseases caused by pathogenic microorganisms comprising the Lactobacillus strain of claim 1 or the culture supernatant thereof or the probiotics of claim 4. 10. The pathogenic microorganism of claim 9, wherein the pathogenic microorganisms are Vibrio parahemoriticus, Vibrio fluvialis, Vibrio anguillalum, Vibrio alginolitis, Vibrio cholera, Vibrio bulnipicus, Yersinia Luceri, Iromonas salmonica A subspecies Salmonicada, Edwad Cielar Tarda and Leukotrikirix Mukor are health foods characterized in that it is selected from the group consisting of. 10. The health food for preventing diseases of claim 9, wherein the disease is any one selected from the group consisting of acute diarrhea, enteritis, gastroenteritis, constipation, abdominal pain, bloating, cholera and food poisoning. Lactobacillus strains of claim 1 or lactic acid bacteria preparation comprising the culture supernatant thereof or the probiotics of claim 4 as an active ingredient. Lactobacillus strain of claim 1 or fermented milk comprising the culture supernatant thereof or the probiotic of claim 4 as an active ingredient. 1) culturing the Lactobacillus strain of claim 1; And 2) A method for producing probiotics from the Lactobacillus strain comprising the step of obtaining the probiotics produced by the cultured Lactobacillus strain of step 1).

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