KR101999531B1 - Lactobacillus perolens SRCM102283 strain having immunity activity, antiviral activity and probiotics properties and uses thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus perolens SRCM102283 strain having immunological activity, antiviral activity and probiotics properties, and to uses thereof. The Lactobacillus perolens SRCM102283 strain of the present invention, considered to be a useful strain, has all the characteristics of the probiotics, and thus can be usefully used as an intestinal drug; can be used as a starter strain for the production of high quality fermented food; and can be used as an antimicrobial agent against harmful microorganisms including Bacillus cereus, Bacillus sphaericus, Enterococcus faecium, Listeria ivanovii, Listeria monocytogenes, Streptococcus mutans, Enterococcus aerogenes, Escherichia coli, Shigella flexneri or Shigella sonnei.

Description

Lactobacillus ferorensis SRCM102283 strain having immunoactivity, antiviral activity and probiotics properties and use thereof (Lactobacillus perolens SRCM102283 strain having immunity activity, antiviral activity and probiotics properties and uses thereof)

The present invention relates to Lactobacillus ferocensis SRCM102283 strain having immunological activity, antiviral activity and probiotic characteristics and uses thereof.

Probiotics is derived from the Greek word for life. Probiotics was defined in 1965 as "a substance secreted by a microorganism that stimulates the growth of other microorganisms." In 1974, it was defined as "a creature or substance that has a beneficial effect on the host animal." Then in 1989, Fuller redefined probiotics as "a probiotic agent that beneficially influenced the host by improving the intestinal microflora" and introduced the concept of living microorganisms. Aerobic bacteria, anaerobic bacteria, lactic acid bacteria, and yeast bacteria are used in these probiotics, but lactic acid bacteria are mainly used as a major strain.

Lactic acid bacteria are widely found in nature and produce carbohydrates by anaerobic use of carbohydrates. The natural environment in which lactic acid bacteria are found is not only present in human or animal intestines but also in various vegetables and fruits and plays an important role in the fermentation process in fermented food such as kimchi, yogurt and German sauerkraut in Korea . These lactic acid bacteria enter the intestines and multiply in the intestinal epithelium, thereby preventing the colonization of harmful microorganisms and secreting antimicrobial substances, thereby suppressing the growth of harmful microorganisms, improving diarrhea and constipation, , Serum cholesterol lowering and the like. In addition, it is generally known that these lactic acid bacteria are directly or indirectly added to foods to improve the storage stability of foods by lactic acid, which is a metabolite thereof, and to improve the flavor and texture of foods.

Korean Patent Laid-Open Publication No. 2017-0128133 discloses 'a composition for enhancing immunity comprising lactic acid bacteria', Korean Patent No. 0354315 discloses 'a lactobacillus microorganism producing a novel antimicrobial substance' , As in the present invention, there is no information on the Lactobacillus ferroence SRCM102283 strain having immunological activity, antiviral activity and probiotic properties and its use.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide an antimicrobial composition which is free of antiviral activity, immunological activity, antioxidant activity, ), Lactobacillus perolens SRCM102283 strain (KCCM12239P), which has an antimicrobial activity against secretory activity, γ-hemolytic activity and harmful microorganisms and does not produce biogenic amines, harmful substances and harmful enzymes, was isolated.

Since the lactobacillus ferroence SRCM102283 strain of the present invention has all the characteristics of the above-mentioned provitics, it can be used effectively as a formulating agent and can be used as a starter strain for producing a fermented product of good quality and a strain of Bacillus sp. , Enterococcus faecium, Listeria monocytogenes, Listeria monocytogenes, Streptococcus mutans, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sinnai, as antibacterial agents against harmful microorganisms The present invention has been completed.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition comprising an antiviral activity, an immunological activity, an antioxidative activity, a milk protein coagulant, an acid tolerance, a bile acid tolerance, an antibiotic resistance, a β-glucosidase secretion, Lactobacillus perolens strain SRCM102283 (KCCM12239P), which has antibacterial activity against harmful microorganisms and does not produce biogenic amines, harmful substances and harmful enzymes, is provided.

The present invention also provides a probiotic preparation comprising the strain, a culture thereof, a concentrate of the culture broth, or a dried product thereof as an active ingredient.

The present invention also relates to a method for producing a microorganism, which comprises culturing the above strain, a culture thereof, a concentrate of the culture broth or a dried product thereof as an active ingredient, such as Bacillus cereus, Bacillus sparkicus, Enterococcus lasserium, Listeria ivanovi, Listeria monocytogenes, There is provided an antimicrobial composition for mutans, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sinnai.

The present invention also provides a starter composition for the production of a fermented food, which contains the strain, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.

The Lactobacillus perolens strain SRCM102283 strain (KCCM12239P) of the present invention is useful as an antiviral activity, an immunological activity, an antioxidative activity, a milk protein solidifying property, an acid resistance, an anti-biliary property, an antibiotic resistance,? -Glucosidase, Secretion activity, γ-hemolytic activity, antimicrobial activity against harmful microorganisms, and did not produce biogenic amines, harmful substances and harmful enzymes.

Therefore, the Lactobacillus ferorensis SRCM102283 strain of the present invention is considered to be a useful strain that can be used in various industries such as a dressing agent, a starter strain for fermented food preparation, and an antibacterial microorganism preparation.

1 shows the effect of inhibiting Influenza A virus infection with FACS of Lactobacillus perolens strain SRCM102283 isolated in the present invention.
FIG. 2 shows the measurement result of the secretion ability of IFN-? Of Lactobacillus perolens strain SRCM102283 isolated in the present invention.
Figure 3 shows the immunological activity of Lactobacillus perolens strain SRCM102283 isolated in the present invention.
FIG. 4 shows the results of comparing antioxidative activities of Lactobacillus perolens SRCM102283 isolated from the present invention with ascorbic acid.
FIG. 5 shows the flow diagram of Lactobacillus perolens strain SRCM102283 isolated in the present invention.

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition comprising an antiviral activity, an immunological activity, an antioxidative activity, a milk protein coagulant, an acid tolerance, a bile resistance, an antibiotic resistance, a β- Lactobacillus perolens strain SRCM102283 (KCCM12239P), which has antibacterial activity against active and harmful microorganisms and does not produce biogenic amines, harmful substances and harmful enzymes, is provided.

In the present invention, strains were isolated from vinegar. Among them, antiviral activity, immunological activity, antioxidant activity, lipid-lowering activity, acid-fast bacilli, antibiotic resistance, β-glucosidase secretion, Lactobacillus perolens strain SRCM102283, which has antimicrobial activity against active and harmful microorganisms and does not produce biogenic amines, harmful substances and harmful enzymes, was identified, and this strain was identified as Lactobacillus perolens strain SRCM102283 It was identified and deposited with the Korean Center for Microorganism Conservation (KCCM) on March 28, 2018, and received the deposit number KCCM12239P.

In a strain according to an embodiment of the present invention, the acid tolerance may be acidic at pH 3-5, and the bile bile may be bile bile in 0.1-0.5% bile.

In a strain according to an embodiment of the present invention, the virus is Influenza A viruses and the antibiotic is selected from the group consisting of fusidic acid, netilmicin, methicillin, nalidixic acid Nalidixic acid, Pipemidic acid, Trimethoprim and Trimethoprim-Sulfamethoxazole. The harmful microorganisms are Bacillus cereus , Bacillus sp. Lee kusu (Bacillus sphaericus), Enterococcus passive help (Enterococcus faecium), L. Ivanova ratio (Listeria ivanovii), L. monocytogenes cytokines to Ness (Listeria monocytogenes), Streptococcus mutans (Streptococcus mutans), Enterobacter nose kusu Eroge Ness (Enterococcus aerogenes), Escherichia coli (Escherichia coli), a Shh Gela flex Neri (Shigella flexneri) and Shh Gela hand Nei (Shigella sonnei), the The biogenic amines histamine and tyramine, the harmful substances are indole and phenylpyruvic acid, the harmful enzymes are as β- glucuronidase dehydratase and urea can be best, but is not limited thereto.

The Lactobacillus page Lawrence (Lactobacillus perolens) SRCM102283 strain and secrete β- glucosidase, further leucine aryl amidase (Leucine arylamidase), valine aryl amidase (valine arylamidase), α- chymotrypsin (α-chymotrypsin), But are not limited to, acid phosphatase, alpha-glucosidase, and N-acetyl-beta-glucosaminidase.

The present invention also provides a probiotic preparation comprising the strain, a culture thereof, a concentrate of the culture broth, or a dried product thereof as an active ingredient.

The probiotic agent can be prepared and administered in various formulations and methods according to methods known in the art. For example, the Lactobacillus perolens strain SRCM102283 strain of the present invention, a culture thereof, a concentrate of the culture broth or a dried product thereof may be mixed with a carrier commonly used in the pharmaceutical field to prepare powders, liquids and may be formulated and administered in the form of solutions, tablets, capsules, syrups, suspensions or granules, and the like. Examples of the carrier include, but are not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring matters and fragrances. The administration dose can be appropriately selected depending on the degree of absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, sex, strain, condition and severity of the disease.

The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, and is not limited to a specific method.

When the strain obtained in the step of culturing the strain of the present invention or the culture of the strain or the concentrated liquid of the culture medium of the strain is used as an additive, the culture of the strain or the strain or the concentrated liquid of the culture of the strain is directly added Other additives can be used together and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the purpose of use thereof.

The present invention also provides a food comprising the probiotic agent.

The probiotic agent of the present invention can be used as an antibiotic agent for the prevention and treatment of various diseases such as antiviral activity, immunological activity, antioxidant activity, milk protein solidity, acid resistance, biliary cholesterol, antibiotic resistance,? -Glucosidase secretion ability, And Lactobacillus perolens strain SRCM102283 which does not produce harmful substances and harmful enzymes, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.

When the probiotics of the present invention are used as a food additive, the probiotics can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the probiotics of the present invention are added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of foods or beverages. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the mixing amount may be less than the above range, and since there is no problem in terms of safety, the mixing amount may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the probiotics can be added include breads, candies, snacks, confectionery, gums, dairy products including ice cream, various soups, drinks, tea, drinks, and vitamin complexes. .

The present invention also relates to a method for producing a microorganism, which comprises culturing the above strain, a culture thereof, a concentrate of the culture broth or a dried product thereof as an active ingredient, such as Bacillus cereus, Bacillus sparkicus, Enterococcus lasserium, Listeria ivanovi, Listeria monocytogenes, There is provided an antimicrobial composition for mutans, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sinnai.

The antimicrobial composition of the present invention may have the same meaning as antibiotics, which is generically referred to as an antimicrobial agent, and may have the same meaning as an antifungal agent, a bactericide, an antiseptic, a preservative or a bactericidal agent, and preferably Bacillus cereus, Bacillus sp. Development and life of pathogenic microorganisms including Enterococcus lasserium, Listeria monocytogenes, Streptococcus mutans, Enterococcus erogenes, Escherichia coli, Shigella flexneri, and Shigella sinnai. But is not limited to, a substance capable of inhibiting or inhibiting the function of the compound.

The antimicrobial composition of the present invention may contain carbohydrates, proteins, lipids, vitamins and minerals in addition to Lactobacillus perolens strain SRCM102283, a culture thereof, a concentrate of the culture or a dried product thereof. The saccharide may be selected from the group consisting of honey, dextrin, sucrose, palatinose, glucose, fructose, starch syrup, sugar alcohol and the like. The saccharide, protein, lipid, vitamins or minerals, , Sorbitol, xylitol and maltitol. The protein may be milk-derived proteins such as casein, whey protein, soybean protein, animal-derived enzymes such as trypsin and pepsin of these proteins, and neutrase The lipid may be a first saturated fatty acid, a sunflower oil containing a polyunsaturated fatty acid, a rapeseed oil, an olive oil, a safflower oil, a corn oil, Derived fat such as soybean oil, palm oil and palm oil, middle-chain fatty acid, EPA, DHA, soybean-derived phospholipid, milk-derived phospholipid, The RAL fluids may be potassium phosphate, potassium carbonate, potassium chloride, sodium chloride, calcium lactate, calcium gluconate, calcium pantothenate, casein calcium, magnesium chloride, ferrous sulfate, sodium bicarbonate, but are not particularly limited by each example .

The present invention also provides a starter composition for the production of a fermented food, which contains the strain, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.

In the present invention, a starter for fermented food preparation means a preparation or a composition containing microorganisms involved in fermentation for producing fermented food. And is used to provide a microorganism capable of growing in a fermented food or a microorganism capable of growing as a dominant species by adding at the time of fermented food production. When the foodstuff is produced using the foodstuff fermenting starter, the microorganism contained in the foodstuff fermentation starter may be used to control the quality of the foodstuffs constantly or to control the quality of the foodstuffs for a specific purpose, Purpose can be achieved. In the present invention, by using Lactobacillus perolens strain SRCM102283 or a culture thereof as a starter strain, antiviral activity, immunological activity, antioxidative activity, milk protein solidity, acid resistance, bile resistance, antibiotic resistance, A fermented food having β-glucosidase secretion ability, γ-hemolytic activity, antimicrobial activity against harmful microorganisms, and which does not produce biogenic amines, harmful substances and harmful enzymes can be produced.

In the starter composition for manufacturing fermented food according to an embodiment of the present invention, the fermented food may be, but not limited to, kimchi, soybean paste or fermented milk.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Isolation of lactic acid bacteria from vinegar

For the isolation of lactic acid bacteria, 1 ml of refrigerated vinegar sample was diluted with each step in 9 ml of sterilized 0.85% NaCl solution. 100 μl of the diluted solution was plated on a plate containing Lactobacillus MRS agar (Difco ™, MI, USA) medium and cultured at 37 ° C for 48 hours, and strains having morphological characteristics similar to those of lactic acid bacteria were firstly screened. The purely isolated lactic acid bacteria were cultured on MRS solid medium, suspended in 10% skim milk (BD Difco, Sparks, MD, USA) and stored at -80 ° C.

Preparation of Lactic Acid Bacteria Culture and Cell Culture for Measurement of Immune and Antiviral Activity

In order to prepare a culture medium for measuring immunity and antiviral activity of the lactic acid bacteria, 50 ml of MRS broth was inoculated and cultured at 30 DEG C and 150 rpm for 24 hours. The culture broth of the lactic acid bacteria was centrifuged at 3000 rpm for 30 minutes to remove the cells, and the recovered supernatant was titrated with neutral pH and filtered with a 0.22 μm filter. RAW264.7 (TIB-71), a macrophage cell line, was used to confirm the antiviral activity and immunostimulating effect of the selective lactobacillus. RAW264.7 (TIB-71) was cultured in DMEM containing 10% FBS (Fetal Bovine Serum, Gibco, # 16000-044 500 mililiter) and 1% antibacterial and antifungal substances (Anti-Anti 100X, Gibco, # 15240-062) (Dulbecco's Modified Eagles Medium, High Glucose, HyClone # SH30243.01) medium at 37 ° C in a 5% CO ₂ incubator once a day.

Antiviral activity

In order to measure the antiviral activity of the isolated strain, a mouse macrophage inhibited the A / PR (Puerto Rico) / 8/34-GFP infection and a cytokine, a vital defense agent against viral infection (IFN-?), Which is known to be an inhibitor of interferon-beta.

RAW264.7 (TIB-71) cells were cultured in DMEM medium containing 10% FBS and 1% anti-bacterial and antifungal substances to confirm the inhibitory effect of A / PR (Puerto Rico) / 8 / Well plate (CORNING) at a concentration of 2 × 10 ⁵ cells / well, and cultured in a 5% CO ₂ incubator at 37 ° C. for about 16 hours. Then, 500 μl of PBS buffer was added to remove FBS Were added to each well to wash the cells. The isolated culture supernatant was diluted to a final concentration of 4% using DMEM medium containing 1% FBS and 1% antibiotic, and cultured for 8 hours at 37 ° C in a 5% CO ₂ incubator. The supernatant was removed and washed with PBS buffer. (-) DMEM (serum-free) to be 0.1 MOI for influenza A virus (Green Fluorescent Protein (GFP) -tagged A / PR / 8/34, A / PR / 8/34-GFP, H1N1- After dilution with the medium, the cells were added to the cells at 500 μl / well, followed by infection for 1 hour. After washing with PBS buffer, DMEM medium containing 1% FBS and 1% antibiotic was added at a rate of 500 μl / well and cultured at 37 ° C in a 5% CO ₂ incubator for 12 hours. Raw cell 264.7 was tested for inhibition of influenza A virus infection using fluorescence-activated cell sorting (FACS, Guava ^® easyCyte Flow Cytometers). LPS (lipopolysaccharide) and Leuconostoc mesenteroides BLS35, a patented antiviral activity strain, were used as positive control substances to confirm infection inhibition effect of influenza A virus.

RAW264.7 (TIB-71) cells were cultured in a 24-well culture plate (CORNING) using DMEM medium containing 10% FBS and 1% anti-bacterial and antifungal substances at a concentration of 2 x 10 < ⁵ cells / well, and cultured at 37 ° C in a 5% CO ₂ incubator for about 16 hours. To remove FBS, 500 μl PBS buffer was added to each well to wash the cells. The isolated culture supernatant was diluted to a final concentration of 4% using DMEM medium containing 1% FBS and 1% antibiotic, and cultured for 24 hours at 37 ° C in a 5% CO ₂ incubator. Then, Was measured using a Verkine Interferon beta ELISA kit (PBL Assay Science, NJ, USA). The recovered supernatant samples were diluted 10-fold with dilution buffer and added to the 96-well plate coated with antibody in an amount of 10 μl, and then left at room temperature for 1 hour. The washing solution was washed three times with 300 μl / well of the washing buffer, and the antibody solution was diluted with the concentrated solution. Then, 100 μl of the antibody solution was added to the plate, followed by standing at room temperature for 1 hour. Washing buffer was added 300 μl / well and washed three times. HRP conjugate concentrate was diluted with concentrated solution, and 100 μl / well was added thereto. Then, the mixture was left at room temperature for 1 hour. Wash buffer was added at a rate of 300 μl / well. After washing three times, the TMB substrate was added at a rate of 100 μl / well and left in a dark place for 15 minutes. The reaction was stopped by adding 100 μl of stop solution per well and the absorbance was measured at 450 nm using a SpectraMax 190 microplate reader (Molecular Devices, US). The OD value obtained by measuring the absorbance at 450 nm was compared with a standard curve prepared using a standard substance to measure the amount of IFN- ?. Mouse IFN-β standard was used as a standard, and the concentration was calculated by comparing with the standard curve obtained by diluting 2 times with dilution buffer from 15.6 pg / ㎖ to 1000 pg / ㎖. Lipopolysaccharide (LPS) and Leuconostoc mesenteroides BLS35, a patented antiviral activity strain, were used as positive control substances for IFN-β secretion.

Evaluation of immunologically active microorganisms

In order to confirm the immunostimulating effect of the isolated strain, the secretion ability of the cytokine TNF-α (tumor necrosis factor-α) was measured. RAW264.7 (TIB-71) cells were cultured in a 24-well culture plate (CORNING) using DMEM medium containing 10% FBS, 1% anti-bacterial and antifungal substances at a concentration of 2 x 10 < ⁵ cells / well, and cultured at 37 ° C in a 5% CO ₂ incubator for about 16 hours. To remove FBS, 500 μl PBS buffer solution was added to each well to wash the cells. The isolated culture supernatant was diluted to a final concentration of 4% using DMEM medium containing 1% FBS and 1% antibiotic, and cultured in a 37 ° C, 5% CO ₂ incubator for 24 hours. The amount of secreted TNF-α was measured using OptEIATM ELISA kit (BD Biosciences, San Diego, Calif., USA). anti-cytokine capture antibody was diluted in a coating buffer (0.2 M sodium phosphate, pH 6.5) and added to a 96-well plate (NUNC Co., Roskilde, Denmark) at a rate of 100 μl / well. Respectively. Tween 20 (Sigma) is washed three times with PBS / Tween solution added to PBS (phosphate buffered saline) to 0.05% (v / v). Assay diluent (PBS with 10% FBS) was added to the plate at 200 μl / well and left at room temperature for 1 hour. After washing three times with PBS / Tween, 100 μl of the culture supernatant was added to each well, followed by standing at room temperature for 2 hours. After washing 5 times with PBS / Tween, a biotinylated antibody as a detection antibody and a streptavidin-horseradish peroxidase conjugate (an enzyme solution, streptavidin-horseradish peroxidase conjugate) were diluted (PBS containing 10% FBS) And 100 쨉 l / well was added to the plate, followed by allowing to stand at room temperature for 1 hour. After washing 7 times with PBS / Tween, 100 μl / well of a substrate solution (TMB substrate reagent set, BD Biosciences) was added and left in a dark place for 30 minutes. The reaction was stopped by adding 50 μl of 2 N sulfuric acid solution as a stop solution, and the absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, US). The OD value obtained by measuring the absorbance at 450 nm was compared with a standard curve prepared using a standard substance to measure the amount of TNF- ?. Recombinant mouse TNF was used as the reference material and the concentration was calculated by comparing the standard curve obtained by diluting 2 times with the assay diluent (PBS with 10% FBS) from 15.6 pg / ㎖ to 1000 pg / ㎖. Positive control substances of TNF-a secretion ability include LPS (lipopolysaccharide) and the patented antiviral activity strain Leuconostoc mesenteroides BLS35 was used.

Identification of biogenic amine production

It was confirmed by the qualitative test method for confirming the generation of biogenic amine. Identification of biosynthetic amine production of isolated strains The solid medium was prepared by using MRS agar (pH 5.3) medium containing 0.25% glycerol, 0.006% bromocresol purple, 0.1% histidine or tyrosine. The isolated strains were streaked on each medium and cultured at 37 ° C for 24 hours. Then, the generation of the biogenic amines was confirmed by comparing the degree of color development with the control MRS medium.

Antioxidant activity measurement

DPPH (2,2-diphenyl-1-picryl-hydrazyl, Sigma-aldrich, MO, USA) was used to measure the antioxidant activity of the strain. The isolates were streaked on MRS agar medium and cultured in a 37 ° C incubator for 48 hours. Single colonies were inoculated into 5 ml MRS broth and cultured at 37 ° C and 150 rpm for 24 hours. 1 ml of the culture was taken in a 1.7 ml tube and centrifuged at 13,000 rpm for 10 minutes to recover the supernatant. 20 μl of supernatant was added to 180 μl of 100 μM DPPH methanol solution. The reaction product was reacted at room temperature for 30 minutes, and the absorbance at 517 nm was measured with a microplate reader. The DPPH radical scavenging activity was compared using ascorbic acid, a natural antioxidant, as a standard.

Antioxidant activity (%) = [1- (Abs _517nm of sample) / (Abs _{517nm of} sample)] X100

Abs: Absorbance of the DPPH solution of the sample at 517 nm

Abc: Absorbance of sample-free DPPH solution at 517 nm

Measurement of antimicrobial activity against food harmful microorganisms

To investigate the antibacterial activity, isolated lactic acid bacteria were inoculated into MRS liquid culture medium, cultured at 37 ° C for 24 hours, and centrifuged at 13,000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was sterilized with a 0.45 μm syringe filter (Sartorius, Frankfurt, Germany), and 100 μl of the supernatant of the isolated strain was dispensed into wells of 6 mm in accordance with the well diffusion method using a 0.8% soft agar plate. And the size of the inhibitory ring formed was measured. Twelve indicative strains were purchased from the Korean Collection for Type Cultures (KCTC) and the Korean Culture Center of Microorganisms (KCCM) (Table 1), stored at -80 ° C in 20% glycerol stock Respectively. The harmful bacteria were cultured to the appropriate medium, adjusted to a uniform concentration (OD ₆₀₀ , 0.4), and then inoculated on a 0.8% soft agar plate to prepare a medium for antimicrobial activity measurement.

The food-harmful microorganisms used for testing the antimicrobial activity of the strain of the present invention Strain Gram stain Oxygen requirement badge Temperature (℃) Bacillus cereus KCTC1013 positivity Aerobic LB Bros 30 Bacillus spericus KCCM1184 positivity Aerobic LB Bros 30 Enterococcus Passium KCTC3095 positivity Aerobic Brain-Heart Infusion (BHI) Bros. 30 Listeria Ivanovi Subspecies Ivanovi KCTC3444 positivity Aerobic BHI Bros 37 Listeria monocytogenes KCCM3710 positivity Aerobic BHI Bros 37 Staphylococcus aureus KCCM11593 positivity Aerobic Trypic soy broth 37 Streptococcus mutans KCCM40105 positivity Aerobic BHI Bros 37 Enterococcus erogenes KCCM12117 voice Aerobic Nutrient Bros 30 Escherichia coli ATCC10798 voice Aerobic Nutrient Bros 37 Escherichia coli KCCM11234 voice Aerobic Nutrient Bros 37 Shergella flexneri KCTC2517 voice Aerobic Trypic soy broth 37 Shigella Sonnay KCTC2518 voice Aerobic Trypic soy broth 37

Analysis of hemolytic and noxious metabolites and harmful enzyme production

Blood agar medium was prepared by adding 5% sheep blood (MBcell, Korea) to a blood agar base (BD, Difco) to identify the hemolytic activity of the selected strains. The selected strains were cultured for 24 hours at 37 ℃ after streaking, and α, β and γ hemolytic activity were confirmed in the form of rings around the cells. In order to confirm the production of indole and phenylpyruvic acid, which are harmful metabolites related to cancer, MRS (BD, Difco) liquid culture medium was inoculated with the selected strains, followed by shake culture at 37 ° C for 24 hours, and the indole solution dropper (BBL, BD Difco) was added to determine whether indole formation was caused by color change of the medium surface. The production of phenylpyruvic acid was determined by inoculating a strain in a phenylalanine agar (fluka, Buchs, switzerland) medium, culturing at 37 ° C for 24 hours and 48 hours, adding 3-4 drops of 10% ferric chloride (MBcell, Korea) And the presence of phenylpyruvic acid was confirmed by color change. And the production of a colon cancer-inducing enzyme such as urease and? -Glucuronidase was confirmed. Urease production was assessed by color change after 4-24 h incubation at 37 ° C in anaerobic condition with Vaseline oil (MB cells, Korea) added to the urea rapid test kit (MB cells). β-Glucuronidase was inoculated into the tryptone bile X-glucuronide (TBX, Oxoid) agar medium and incubated at 37 ° C. for 24 hours. The color change of the strain was observed to determine β-glucuronide And the activity thereof was confirmed.

Coagulability of milk protein in selected lactic acid bacteria

The coagulability of milk proteins was determined by incubating selected lactic acid bacteria in MRS broth at 37 ° C for 24 hours and then inoculating 1% in sterilized 10% skim milk (difco, USA). The skim milk inoculated with the strains was incubated for 5 days at 37 ° C and the degree of curd formation was observed. The degree of curd formation was determined according to the degree of coagulation as a starter for fermented milk and fermented food.

Selection of lactic acid bacteria having? -glucosidase activity

MRS-EI medium was prepared by mixing Esculin Iron agar with MRS broth to measure β-glucosidase activity. The strains were inoculated on MRS-EI medium and incubated at 37 ° C for 48 hours. The strains forming the brown or black zone were selected by examining the color change around the colonies.

Determination of acid resistance and bile resistance

In order to select strains resistant to gastric juice and bile, acid tolerance and bile test were performed. For acid resistance analysis, the strain was inoculated into the MRS liquid medium at a concentration of 10 ⁸ CFU / ml. Then, 1% of each strain was inoculated into the MRS liquid medium adjusted to pH 2.0, pH 3.0, pH 4.0 and pH 5.0, and the number of viable cells was measured after incubation at 37 ° C for 3 hours. The number of viable cells before the pH control was used as a control. In order to analyze the tolerance to bile acids, the selected strains were inoculated into the MRS broth at an initial bacterial count of 10 ⁸ CFU / ml, and the concentration of OX-Vyl (Neogen, USA) was 0.1%, 0.3%, 0.5% and 1.0 %, Respectively. After incubation at 37 ° C for 6 hours, the number of viable cells was measured. The number of viable cells before addition of Oxgall was used as a control, and the survival rate was calculated to confirm the degree of biliary bite.

Confirm antibiotic susceptibility

The antibiotic susceptibility of selected strains was examined by diffusion method. Amikacillin (25 μg, AML), Amikacin (30 μg, AK), amoxicillin + clavulanic acid (3 μg, AFP), Ampicillin (10 μg, AMP), Azithromycin (15 μg, AZM), Cefaclor (30 μg, CEC), Cefotaxime Cefuroxime (30 μg, CZM), cephalexin (30 μg, CL), ciprofloxacin (5 μg, CIP), clindamycin CD, Erythromycin, 15 μg, E, Fusidic acid, 30 μg, FC, Gentamicin, 30 μg, CN, Imipenem, 10 μg, IMI, Kanamycin , 30 μg of K), lincomycin (15 μg of MY), netilmicin (30 μg of NET), methicillin (5 μg of MET), nalidixic acid NA), neomycin (30 mu g, N), nitrofuran (Nifroxacin, 10 μg, NOR), Ofloxacin (5 μg, OFX), Penicillin G (1 IU, P), Penicillin G (10 IU, Pipemidic acid (20 μg, PI), Piperacillin (100 μg, PRL), Rifampicin (5 μg, RD), Roxithromycin Rexithromycin, 15 μg, RXT), streptomycin (10 μg, S), Teicoplanin, 30 μg, TEC, Tetracycline, 30 μg TE, Trimethoprim (Vectomycin, 30 μg, VA) were used in total of 38 species. For the susceptibility test, MRS solid medium was prepared using the final strain, and antibiotics were inserted into disk dispenser 8 (Liofilchem, Italy), and the disks were placed in order and cultured at 37 ° C for 24 hours. The inhibitory ring size for each antibiotic was measured and the sensitivity and tolerance were confirmed by CLSI (Clinical Laboratory Standards Institute).

Confirmation of availability

API 50 CHL kit (BioMerieux, France), based on the total 49 carbohydrate availability, was used to confirm the glycosyltransferase activity. The selective strains were subcultured in 5 ml of MRS liquid medium and cultured at 30 ° C for 24 hours. 1 ml of the culture was centrifuged at 13,000 rpm for 20 minutes to remove the supernatant. Cell pellets were recovered and washed with PBS (phosphate buffered saline, 0.1 M, pH 7.0) and then used in the experiment. Using a PBS and a McFarland standard, a suspension was prepared at a turbidity of 2, and 1 ml of the culture suspension suspended in API 50 CHL medium was added, mixed, and dispensed in an amount of 120 μl into an API 50 CH strip. Mineral oil 2 ~ 3 drops per tube were added to each tube and cultured at 37 ℃ for 24 hours and 48 hours. The sugar fermentation pattern was confirmed by positive and negative color change of the tube.

Confirm enzyme production ability

To investigate the enzyme production of Lactobacillus ferocensis SRCM102283, 19 enzymatic activities including alkaline phosphatase were examined using API ZYM kit (bioMeriux Co., France). Lactobacillus ferulens SRCM102283 was cultured in MRS solid medium, and the cells were collected, suspended in 0.85% NaCl solution, and adjusted to 5 ~ 6 Macfarland standard. The suspension was added to each cupule of the API ZYM kit and incubated at 37 ° C for 4 hours. After that, the ZYM-A and ZYM-B reagents were dropped one by one to observe the change of color, and the degree of enzyme activity was examined.

Identification of strain and selection of strain

The strain was inoculated on TSB broth and incubated at 37 ° C for 24 hours. The cells were recovered by centrifugation and the ZR Fungal / Bacterial DNA Miniprep kit (Zymo Research Corp., CA., USA). The extracted DNA was synthesized with primers 785F (5'-GGATTAGATACCCTGGTA-3 ': SEQ ID NO: 2) and 907R (5'-CCGTCAATTCMTTTRAGTTT-3': SEQ ID NO: 3) to amplify 16S rRNA gene fragments. The product was purified using a QIAquick PCR purification kit (QIAGEN), and then submitted to Macrogen Co., Ltd. for analysis of the base sequence. The nucleotide BLAST of the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) was used to compare the homology with the nucleotide sequence registered in GeneBank. The nucleotide sequence of the standard strain was obtained through the ExTaxone-e server (http://www.eztaxon.org) and the MEGA 7.0 program was used. Neighbor-joining algorithm was used for the system diagram, and the robustness of the system was confirmed by bootstrapping over 1,000 iterations.

Example 1. Identification of a selection strain

Based on the results of 16S rRNA gene sequencing analysis of SRCM102283, BLAST was found to be Lactobacillus perolens and the homology with standard strains was compared and analyzed on Eztaxon server. The parameters and option values used for the sequence alignment analysis were the Gonnet series as the protein weight matrix, the gap opening 15, the gap extension 6.66 and the delay transition weight 0.5, and the gap penalty was 5 , A K-tuple size of 2, top diagonals of 4, and a window size of 4. As a result, SRCM102283 (1,593 bp, SEQ ID NO: 1) showed 99% homology with Lactobacillus ferorens (L532). In order to construct the phylogenetic tree using the nucleotide sequence, the taxonomic distance inference was modeled by the maximum composite Likehood method. The finally selected strain was designated as Lactobacillus perolens SRCM102283 and deposited on March 28, 2018 with the Korean Society for Microbiological Conservation (KCCM) and assigned the deposit number KCCM12239P.

Example 2. Antiviral activity

To confirm the antiviral activity of SRCM102283, Raw cell 264.7, a mouse macrophage cell line, was treated with culture filtrate of lactic acid bacteria, and GFP was infected with the tagged virus (A / PR (Puerto Rico) / 8/34-GFP) (FACS) in order to confirm the degree of inhibition of virus infection (FIG. 1). Influenza A virus infection inhibitory effect was examined. As a result, 71% of Lactobacillus ferocensis SRCM102283, Leuconostoc mesenteroides as a control, BLS35 was found to be 65%. The Lactobacillus ferocensis SRCM102283 strain was found to be superior to the control in inhibiting influenza A virus infection.

The secretion potency of IFN-? Of SRCM102283 was measured using Reucomostomyces teneroides BLS35, which is an antiviral patented strain, as a control, and the measurement results are shown in Fig. IFN-β is a cytokine secreted by reacting with external substances such as viruses and cancer cells, and induces an immune response by causing an anti-cancer and anti-viral action. The secretion of IFN-β from the BLS35 strain was measured to be 0.15 ± 0 ng / ml, and the Lactobacillus ferorensis SRCM102283 strain was measured to be 0.38 ± 0.03 ng / ml. Control group Ryukono Stokeshen terroidis It was confirmed that the lactobacillus ferroence SRCM102283 strain was superior to the BLS35 strain by 253%.

Example 3. Immunological activity

The immunological activity of SRCM102283 is shown in FIG. 3, using Ryukono Stokeshenceteroids BLS35, which is an antiviral patented strain, as a control. TNF-α of the BLS35 strain was 65.7 ± 3.23 ng / ml and that of Lactobacillus ferulensis SRCM102283 was 420 ± 21.2 ng / ml. Control group Ryukono Stokeshen terroidis It was confirmed that the Lactobacillus ferroence SRCM102283 strain was more excellent in 639% immunity than the BLS35 strain.

Example 4. Confirmation of production of biogenic amine

Biogenic amines are mainly detected in fermented foods containing protein, and are produced by amino acids or decarboxylase-producing microorganisms during the fermentation, aging and storage of foods. Among biogenic amines, histamine can cause food poisoning and allergies when consumed, and tyramine can secrete vasoconstrictors to cause hypertension. Therefore, as a result of biosgenic amine qualitative experiment, it was confirmed that SRCM102283 strain did not use the precursor amino acid histidine and tyrosine in the medium, so that it did not produce biogenic amine.

Example 5. Antioxidant activity measurement

DPPH is one of the indicators of antioxidant activity against phenolic substances as a representative substrate for measuring antioxidant activity. It inhibits lipid oxidation in foods by donating electrons to free radicals, inhibits aging by reactive oxygen species in the body, And to prevent aging. Therefore, the antioxidative activity of SRCM102283 strain was measured using DPPH. As a result, the antioxidative activity of SRCM102283 strain was as high as 33.59% when 30 ㎍ / ㎖ of ascorbic acid was 22.93%.

Example 6. Measurement of antimicrobial activity against food harmful microorganisms

The antimicrobial activity of the 12 pathogenic microorganisms was measured and the antimicrobial activity against various indicator strains was found to be excellent. In particular, SRCM102283 discloses that Bacillus sp. KCTC1184 and Escherichia coli KCCM11234 showed high antimicrobial activity. The use of microorganisms with a variety of antimicrobial spectra in traditional soybean fermentation and food manufacturing is expected to be useful in addressing the problem of contamination by pathogenic microorganisms in food manufacturers, as well as natural preservatives and feed preservatives, as well as new antibiotic substitutes Application.

Comparison results of antibacterial activity of selected strains No Antioxidant activity SRCM102283 One Bacillus cereus KCTC1013 +++ 2 Bacillus spearicus KCTC1184 +++++ 3 Enterococcus Passium KCTC3095 ++ 4 Listeria Ivanovi Subspecies Ivanovi KCTC3444 + 5 Listeria monocytogenes KCTC3710 ++ 6 Staphylococcus aureus KCCM11593 - 7 Streptococcus mutans KCCM40105 ++ 8 Enterococcus erogenes KCCM12117 +++ 9 Escherichia coli ATCC10798 ++++ 10 Escherichia coli KCCM11234 +++++ 11 Shergella flexneri KCTC2517 ++ 12 Shigella Sonnay KCTC2518 ++ +: <1 cm, ++:> 1 cm, +++:> 1.5 cm, ++++:> 2 cm, +++++:> 2.5 cm.

Example 7. Analysis of hemolytic and noxious metabolites and harmful enzyme production

The SRCM102283 strain was tested for hemolysin by dissolving red blood cells or hemolysis which discolored the blood. As a result, the strain was found to exhibit the gamma-hemolysis phenomenon. The selective strains did not produce enzymes that could be involved in cancer formation such as β-glucuronidase and urease, and the indole and phenylpyruvic acid, known as the noxious metabolites, ). Therefore, it has been confirmed that it is safe as a probiotic strain because it does not have hemolysis, harmful enzymes and harmful metabolites.

Physiological characteristics of SRCM102283 strain Lactobacillus ferroence SRCM102283 Hemolysis γ-hemolysis Glucuronidase &lt; RTI ID = 0.0 &gt; ND Urease ND Indole ND Phenyl pyruvic acid ND ND: not-detected.

Example 8. Protein coagulation of selected lactic acid bacteria

The curd formation characteristics in reduced skim milk were investigated to confirm the coagulability of SRCM102283 strain. The SRCM102283 strain was found to have an excellent fermentation activity of milk protein by forming a curd at 48 hours of incubation. It is believed that excellent curd forming ability can be suitably used as a fermentation starter.

Protein coagulation of SRCM102283 Strain SRCM102283 Coagulation of milk protein + +: Coagulation of milk protein after 120 hours at 37 ° C

Example 9. Selection of lactic acid bacteria having? -Glucosidase activity

The qualitative evaluation results of β-glucosidase of strain SRCM102283 using Esculin iron agar are shown in Table 6. Soybean fermented foods contain soybean protein, peptides, oligosaccharides, phospholipids, isoflavones, saponins, minerals and vitamins, which are beneficial to the human body. Examples of the aglycone-type glycosides in which such sugar components are mixed include isoflavones. Isoflavones present as glycosides are converted into non-glycosylated forms of daisyin and genistein by β-glucosidase produced by lactic acid bacteria and then absorbed into the body. Genistein is known to have anticancer activity, and SRCM102283 showed the possibility of grafting of probiotic food because it could be used as a fermentation seed when the enzyme producing microorganism was used for fermentation.

Results of β-glucosidase activity assay No. Strain Sympathy ? -glucosidase activity One SRCM102283 Lacto Bacillus Per Lawrence +++ * ++++, >2.0; +++, 1.5-1.9; ++, 1.0 to 1.4; ND, < 1.0.

Example 10. Identification of acid resistance and bile resistance

Among the probiotics characteristics, the pH of the lower gastric juice and the tolerance to the bile acid causing the microbial cell membrane to be affected were confirmed. The survival rate of acid tolerance was determined at pH 5.0, pH 4.0, pH 3.0 and pH 2.0. As a result, the survival rate was over 90% at pH 3.0, pH 4.0 and pH 5.0 except pH 2.0. In the case of biliary excretion, the survival rate was more than 90% at all concentrations except 1.0%, indicating that the strain was resistant to bile acid. Therefore, the selective strains are excellent in acid resistance and bile resistance, and it is considered that they can tolerate low gastric juice pH and bile acid microbial cell membrane decomposition and move to the intestines when using this strain.

Analysis of acid resistance and brittleness of SRCM102283 Strain No. Survival rate (%) pH Solution (%) 5.0 4.0 3.0 2.0 0.1 0.3 0.5 1.0 SRCM
102283 99.56 99.13 98.08 40.22 97.37 96.13 90.24 79.61

Confirm antibiotic susceptibility

The antimicrobial susceptibility of SRCM102283 was investigated and the antibiotic susceptibility of the strains was investigated. As a result, the antimicrobial susceptibility of Amoxicillin, Ampicillin, Azithromycin, Ceftazidime, Ceftazidime, Clindamycin, Erythromycin, (1IU), Penicillin G (10 IU), Penicillin G (2 IU), Piperacillin G (Penicillin G) (10 IU), Imipeneme, Lincomycin, Nitrofurantoin, (Piperacillin, Rifampicin, Roxithromycin, and Tetracycline), and the growth was inhibited by Fusidic acid, Netilmicin, Methicillin, It has been confirmed that it is resistant to antibiotics such as Nalidixic acid, Pipemidic acid, Trimethoprim, and Trimethoprim-Sulfamethoxazole.

Antibiotic susceptibility analysis of SRCM102283 No. Name of antibiotic density SRCM102283 One Amoxicillin (AML) 25 [mu] g S 2 Amikacin (AK) 30 [mu] g M 3 Amoxicillin + Clavulanic acid (AUG) 3 [mu] g M 4 Ampicillin (AMP) 10 [mu] g S 5 Azithromycin (AZM) 15 [mu] g S 6 Cefaclor (CEC) 30 [mu] g M 7 Cefotaxime (CTX) 30 [mu] g S 8 Ceftazidime (CAZ) 30 [mu] g S 9 Cefuroxime (CXM) 30 [mu] g M 10 Cephalexin (CL) 30 [mu] g M 11 Ciprofloxacin (CIP) 5 [mu] g M 12 Clindamycin (CD) 10 [mu] g S 13 Erythromycin (E) 15 [mu] g S 14 Fusidic acid (FC) 30 [mu] g ND 15 Gentamicin (CN) 30 [mu] g M 16 Imipenem (IMI) 10 [mu] g S 17 Kanamycin (K) 30 [mu] g M 18 Lincomycin (MY) 15 [mu] g S 19 Netilmicin (NET) 30 [mu] g ND 20 Methicillin (MET) 5 [mu] g ND 21 Nalidixic acid (NA) 30 [mu] g ND 22 Neomycin (N) 30 [mu] g W 23 Nitrofurantoin (F) 300 [mu] g S 24 Norfloxacin (NOR) 10 [mu] g M 25 Ofloxacin (OFX) 5 [mu] g M 26 Penicillin G (P) 1 IU S 27 Penicillin G (P) 10 IU S 28 Penicillin G (P) 2 IU S 29 Pipemidic acid (PI) 20 [mu] g ND 30 Piperacillin (PRL) 100 [mu] g S 31 Rifampicin (RD) 5 [mu] g S 32 Roxithromycin (RXT) 15 [mu] g S 33 Streptomycin (S) 10 [mu] g M 34 Teicoplanin (TEC) 30 [mu] g M 35 Tetracycline (TE) 30 [mu] g S 36 Trimethoprim (TM) 5 [mu] g ND 37 Trimethoprim-Sulfamethoxazole (SXT) 25 [mu] g ND 38 Vancomycin (VA) 30 [mu] g W ND: no inhibition, W: ≤ 1 cm, M: ≤ 2 cm, S:> 2 cm

Confirmation of availability

Lactobacillus ferocensis SRCM102283 strain was identified using an API 50 CHL Kit (BioMerio) to confirm the sugar availability. As a result, the sugar availability was evaluated by using D-galactose, D-glucose, D-fructose, D-mannose, methyl- alpha -D-glucopyranose, methyl- alpha -D- glucoside, N- D-lactose, sucrose, D-meligitose, starch, genothiobiose, D-tagatose, glycogen, Carbon source.

Analysis of sugar availability of Lactobacillus ferroensis SRCM102283 strain by API 50 CH kit Test Reaction Test Reaction Contorl + Esculin + Glycerol - Salicin + Erythritol - D-cellobiose + D-arabinose - D-maltose + L-arabinose - D-lactose + D-ribose - D-melibiose - D-xylose - Sucrose + L-Xylose - Trehalose - D-adonitol - Inulin - Methyl-D-xylopyranoside - D-melezitose + D-galactose + D-raffinose - D-glucose + Amidon (Starch) + D-fructose + Glycogen - D-mannose + Xylitol - L-sorbose - Gentiobiose + Rhamnose - D-turanose - Dulcitol - D-lyxose - Inositol - D-tagatose + Mannitol - D-fucose - Sorbitol - L-fucose - Methyl-alpha-D-glucopyranose + D-arabitol - Methyl-α-D-glucoside + L-arabitol - N-Acetyl-D-glucosamine + Glycogen + Amygdalin + 2-Ketogluconate - Arbutin + 5-Ketogluconate - -: negative effect, +: positive effect.

enzyme Generation  Confirm

The productivity of 19 enzymes was confirmed by using API ZYM Kit (BioMerio) to confirm the enzyme production of Lactobacillus ferocensis SRCM102283 strain. The results are shown in Table 10. The selective strains were selected from the group consisting of leucine arylamidase, valine arylamidase,? -Chymotrypsin, acid phosphatase,? -Glucoside Glucosidase, N-acetyl-β-glucosaminidase, and the like, as well as the activity of α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase.

Lactobacillus ferroence by API ZYM kit Analysis of enzyme activity of SRCM102283 strain Lactobacillus ferroence SRCM102283 Control - Alkaline phosphatase - Esterase (C4) - Esterase lipase (C8) - Lipase (C14) - Leucine arylamidase + Valine arylamidase + Cystine arylamidase - Trypsin - alpha-chymotrypsin + Acid phosphatase + Naphthol-AS-BI-phosphohydrolase - alpha -galactosidase - β-galactosidase - β-glucuronidase - α-glucosidase + β-glucosidase + N-acetyl-β-glucosaminidase + alpha-mannosidase - alpha-fucosidase - -: negative effect, +: positive effect.

Korea Microorganism Conservation Center (overseas) KCCM12239P 20180328

<110> Microbial Institute for Fermentation Industry <120> Lactobacillus perolens SRCM102283 strain having immunity          activity, antiviral activity and probiotics properties and uses          the <130> PN18204 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1593 <212> DNA <213> Lactobacillus perolens SRCM102283 <400> 1 ttctccccgg gtcccgtcac ttcgttccta ggggttggtt ttaagttttt ttttttctgc 60 tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gaacgaggtt tgatcagttt 120 gcggtggtgc ttgcatcacc aattaccgat taaaccgagt ggcggacggg tgagtaacac 180 gtgggtaacc tgcccttcag caggggataa catttggaaa cagatgctaa taccgtataa 240 ccacggagac cgcatggtct ccgggtaaaa gatggcgcaa gctatcactg agggatggac 300 ccgcggcgta ttagccagtt ggtggggtaa tggcctacca aagcgatgat acgtagccga 360 cctgagaggg taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca 420 gcagtaggga atcttccaca atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag 480 aaggctttcg ggtcgtaaaa ctctgttatt gaagaagaac gtgtgtgaga gtaactgctc 540 atgcagtgac ggtattcaac cagaaagtca cggctaacta cgtgccagca gccgcggtaa 600 tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa agcgagtgca ggcggttttt 660 taagtctgat gtgaaagcct tcggcttaac cgaagaagtg catcggaaac tgggaaactt 720 gagtgcagaa gaggagagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa 780 gaacaccagt ggcgaaggcg gctctctggt ctgtaactga cgctgaggct cgaaagcgtg 840 ggtagcaaac aggattagat accctggtag tccacgccgt aaacgatgaa tactaagtgt 900 tggggggttt ccgcccctca gtgctgcagc taacgcatta agtattccgc ctggggagta 960 cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 1020 ggtttaattc gaagacacgc gaagaacctt accaggtctt gacatcttct gccaagctga 1080 gagatcagcc gttcccttcg gggacagaat gacaggtggt gcatggttgt cgtcagctcg 1140 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttatgatt agttgccagc 1200 attcagttgg gcactctagt cagactgccg gtgataaacc ggaggaaggc ggggatgacg 1260 tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatggg tggtacaacg 1320 agcagcgaga ccgcgaggtc aagcgaatct ctaaaaacca tcctcagttc ggattgtagg 1380 ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca cgccgcggtg 1440 aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa 1500 agccggtagg acaaccgcaa ggagtcagcc gtctaaggtg ggacaaatga ttagggtgaa 1560 gtcgtacaaa gggtaagcca aaccgcccca ccc 1593 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggattagata ccctggta 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccgtcaattc mtttragttt 20

Claims (5)

Antibiotic activity, β-glucosidase secretion activity, γ-hemolytic activity, antimicrobial activity against harmful microorganisms, and bioavailability, bioavailability, antioxidant activity, antioxidant activity, Lactobacillus perolens SRCM102283 strain (KCCM12239P) which does not produce genic amines, harmful substances and harmful enzymes,
The virus is Influenza A viruses,
The microorganisms are Bacillus cereus (Bacillus cereus), Bacillus sphaericus (Bacillus sphaericus), Enterococcus passive help (Enterococcus faecium), L. Ivanova ratio (Listeria ivanovii), L. monocytogenes cytokines to Ness (Listeria monocytogenes), Streptococcus Streptococcus mutans , Enterococcus aerogenes , Escherichia coli , Shigella flexneri , and Shigella sonnei ,
The biogenic amine is histamine and tyramine,
Wherein said harmful substance is indole and phenylpyruvic acid,
Wherein said harmful enzymes are? -Glucuronidase and urease. The method of claim 1, wherein the antibiotic is selected from the group consisting of fusidic acid, netilmicin, methicillin, nalidixic acid, pipemidic acid, trimethoprim, And Trimethoprim-Sulfamethoxazole. A probiotic formulation comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture broth, or a dried product thereof as an active ingredient. A method for the production of Bacillus cereus, Bacillus spericus, Enterococcus lasserium, Listeria monocytogenes, Listeria monocytogenes, Streptococcus sp. A composition for antimicrobial use against cocus mutans, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sinnai. A starter composition for producing a fermented food comprising the strain of claim 1 or 2, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.

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