KR101961069B1 - NOVEL STRAIN OF Weissella cibaria AND USE OF THE SAME - Google Patents

Abstract

The present invention relates to a Weissella cibaria D30 strain (KCCM 12211P) having antimicrobial, antioxidant, anti-inflammatory and immunoregulatory activities; an antimicrobial composition comprising the same as an active ingredient; and a composition, comprising the same as an active ingredient, for preventing, ameliorating or treating inflammatory diseases or immune diseases.

Description

[0001] The present invention relates to novel strains of Weissella cibaria and their uses,

The present invention relates to a novel strain of Wyse Lactobacillus; An antimicrobial composition comprising the same as an active ingredient; And a composition for preventing, ameliorating or treating an inflammatory disease or immune disease comprising the same as an active ingredient.

Since most microbial communities in the intestines account for the largest proportion of microbial communities throughout the body, most of the beneficial effects of microbes are derived from intestines. Probiotics is a living microorganism that is used in the prevention and treatment of diseases by controlling the immune response when it is settled in the intestinal environment when the proper amount is ingested. Especially, it inhibits the colonization and proliferation of intestinal pathogenic bacteria such as food poisoning causative bacteria It is widely known that it is effective. However, in order to act as a probiotic, it must resist resistance to the digestive environment and be delivered to the intestine, so it must be resistant to acid and bile and have the ability to settle in the intestines. Also, from the viewpoint of safety, it is necessary to confirm the presence of hemolytic agents, the production of harmful enzymes such as carcinogenic enzymes, and resistance to antibiotics.

Y Cellar strains as one of relatively new lactic acid bacteria identified in, and involved in the initial fermentation of kimchi Leuconostoc mesenteroides and Lactobacillus plantarum are known as one of the dominant species of kimchi. It has been reported that the Wysella strain produces relatively small amounts of organic acids and produces functional extracellular polysaccharides. In addition, weissella koreensis , Weissella Functional properties of kimchii have been reported, and functional probiotic products using these strains have been produced.

On the other hand, detox means that materials showing toxicity in vivo are discharged to the outside of the body through intestines, kidneys, lungs and the like. The detoxification action comprehensively means detoxification of alcohol, drugs and toxic substances introduced into the body, removal of toxic metabolites produced by life activities, removal of harmful pathogenic bacteria, prevention of fat accumulation, and the like, Studies on this possible Wyse strains are required.

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Food and Fuction, Vol.9, pp.1254-1264 (Aug.

The present invention provides a strain of Weissella cibaria D30 (KCCM 12211P) having antibacterial, antioxidant, anti-inflammatory and immunomodulating activity.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a strain of Weissella cibaria D30 (KCCM 12211P) having antibacterial, antioxidant, anti-inflammatory and immunomodulating activity.

The antimicrobial activity may be one or more selected from the group consisting of Micrococyst, Staphylococcus, Listeria, Bacillus, Scleptococcus, E. coli, Salmonella, Campylobacter and Vibrio.

The strain may further have one or more of the following characteristics selected from the group consisting of i) to iv):

I) Excellent acid resistance, bile resistance, alcohol resistance and intestinal adherence;

Ii) inhibition of? -Glucuronidase activity;

Iii) inactivated hemolytic activity; And

Iv) Antibiotic resistance to Ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline. Antibiotic resistance to ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline.

The strain has a cohesive force with a self-cohesive force and a causative agent of food poisoning, or has an ability to inhibit intestinal adhesion of a causal agent of food poisoning, and the food poisoning causative agent may be at least one selected from the group consisting of Staphylococcus, Listeria, Salmonella and E. coli.

The strain may produce at least one selected from lactic acid, acetic acid and pyroglutamic acid, but not produce citric acid.

The strain may comprise the 16S rRNA base sequence of SEQ ID NO: 1.

In one embodiment of the present invention, weissella cibaria D30 strain (KCCM 12211P) as an active ingredient.

The strain may be a live cell, a dead cell, or a culture filtrate.

In another embodiment of the present invention, weissella cibaria) provides the inflammatory diseases or immune diseases prevention or treatment a pharmaceutical composition containing the D30 strain (KCCM 12211P), as an active ingredient.

In another embodiment of the present invention, weissella cibaria D30 strain (KCCM 12211P) as an active ingredient, or a health functional food for preventing or ameliorating an immune disease.

The present invention relates to a strain of Weissella cibaria D30 (KCCM 12211P) having antibacterial, antioxidant, antiinflammatory and immunomodulating activity and its use. The strain (KCCM 12211P) has an advantage of excellent stability and safety It is available as probiotics,

In addition, the above strain (KCCM 12211P) has antimicrobial activity against pathogenic bacteria, intestinal adhesion inhibition of food poisoning causative bacteria, weak organic acid production ability, excellent antioxidant, anti-inflammatory and immunomodulating activity, Or a composition for preventing, ameliorating or treating immune diseases. Therefore, it is expected that the strain (KCCM 12211P) will eventually be capable of detoxification.

1 is a Weissella It is a schematic illustration showing the cibaria D30 strain (KCCM 12211P).
Figure 2 shows the results of (A) Vibrio parahaemolyticus The strain (KCCM 11965) and Weissella This is a photograph showing the result of comparing the hemolytic activity of the cibaria D30 strain (KCCM 12211P).
FIG. 3 shows that when the inflammatory response was induced by LPS in the macrophage cell line RAW 264.7 cells, Lactobacillus rhamnosus GG strain (KCTC 12202BP) and Weissella the processing of cibaria strain D30 (KCCM 12211P) a graph comparing the activity of inhibiting nitric oxide generation.
FIG. 4 shows that Lactobacillus rhamnosus GG strain (KCTC 12202BP) (" LGG ") and Weissella (IL-1β, IL-6 and TNF-α) in the treatment of (a) inflammatory mediator enzyme (iNOS and COX-2) and (B) cibaria D30 strain (KCCM 12211P) mRNA < / RTI >

The inventors of the present invention have found that among the Bacillus bacteria, the fermented foods such as Weissella cibaria) strain (KCCM 12211P) and then separated and identified, verified by the probiotic properties thereof (reliability and safety), and the evaluation of antibacterial, antioxidant, anti-inflammatory and immunomodulatory activity, and completed the present invention.

The present invention provides a strain of Weissella cibaria D30 (KCCM 12211P) having antibacterial, antioxidant, anti-inflammatory and immunomodulating activity.

In accordance with the present invention, weissella cibaria ) D30 strain (KCCM 12211P) can be regarded as a subspecies of Wysselachia.

Conventional Wysella strains are involved in the early fermentation of kimchi and are known as one of the dominant species of kimchi. These Wyse strains are known to produce relatively small amounts of organic acids and produce functional extracellular polysaccharides.

The flow diagram of the strain (KCCM 12211P) is as shown in Fig. 1 and is isolated from kimchi. In order to identify the isolated strain, 16S rRNA sequence was analyzed. As a result, the strain (KCCM 12211P) was confirmed to have the 16S rRNA base sequence of SEQ ID NO: 1 and was deposited at the Korean Microorganism Conservation Center on Jan. 17, And received the accession number KCCM 12211P.

The strain (KCCM 12211P) is characterized in that it has excellent antibacterial, antioxidant, anti-inflammatory and immunomodulating activity among Wisceras strains.

First, in connection with the above-mentioned antimicrobial activity, the strain (KCCM 12211P) may have antimicrobial activity against pathogenic bacteria including Gram-positive bacteria and Gram-negative bacteria, and may have an antibacterial activity against microbes such as Micrococyst, Staphylococcus, Listeria, Bacillus, It is preferable to have an antimicrobial activity against at least one selected from the group consisting of Caucasians, Escherichia coli, Salmonella bacteria, Campylobacter bacteria and Vibrio bacteria, and preferably has an antimicrobial activity against at least one selected from the group consisting of Micrococcus, Staphylococcus, Listeria, Streptococcus, Escherichia coli, Salmonella, It is more preferable to have antimicrobial activity against at least one selected from Pichia pastoris, Pichia pastoris, Pichia pastoris, Pichia pastoris, Pichia pastoris, Pichia pastoris, Pichia pastoris, Pichia pastoris and Pichia pastoris

In connection with the antimicrobial activity, the strain (KCCM 12211P) may have cohesive power with cohesive force and cohesive force with food poisoning causative bacteria or may have intestinal adhesion inhibition ability of causative agent of food poisoning. At this time, the causative agent of food poisoning may be one or more selected from the group consisting of Staphylococcus, Listeria, Salmonella and E. coli. Specifically, the strain (KCCM 12211P) has a self-cohesive force of 60% or more when cultured at 37 ° C for 24 hours and a cohesive force of 40% or more when mixed with food-borne causative bacteria under the same conditions. Therefore, the strain (KCCM 12211P) can effectively prevent intestinal clustering through intestinal clustering, and has an advantage of inhibiting intestinal clustering of pathogens causing food poisoning.

Next, in relation to the antioxidant activity, the antioxidative activity of the strain (KCCM 12211P) was evaluated by radical scavenging ability evaluation using free radical models DPPH, ABTS ⁺ and hydroxyl radical model hydroxyl, or β-carotene Lt; RTI ID = 0.0 > inhibitory < / RTI > ability.

Next, in relation to the anti-inflammatory / immunomodulatory activity, the anti-inflammatory / immunomodulatory activity of the strain (KCCM 12211P) can be confirmed by evaluating Nitric oxide and an inflammatory mediator production inhibitory activity.

In addition, the strain (KCCM 12211P) may produce at least one selected from lactic acid, acetic acid, and pyroglutamic acid, but may not produce citric acid.

The strain (KCCM 12211P) can be utilized as a probiotic, and therefore, is characterized by excellent stability and safety. Thus, the strain (KCCM 12211P) may additionally have one or more of the following characteristics selected from the group consisting of i) to iv):

I) Excellent acid resistance, bile resistance, alcohol resistance and intestinal adherence;

Ii) inhibition of? -Glucuronidase activity;

Iii) inactivated hemolytic activity; And

Iv) Antibiotic resistance to Ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline. Antibiotic resistance to ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline.

In the above i), the survival rate of the strain (KCCM 12211P) may be 80% or more when reacted at pH 2.5 for 3 hours, and the resistance to inoculation is 0.3% (w / v) oxgall for 24 hours. (KCCM 12211P) may be more than 90%. The survival rate of the strain (KCCM 12211P) may be 70% or more when the alcohol resistance is reacted for 4 hours under the condition of 5 to 20% ethyl alcohol.

By suppressing the activity of? -Glucuronidase in the above-mentioned ii), the biosafety can be ensured. In addition, biotransformation of useful substances can be expected by producing beta -glucosidase.

The present invention also relates to a process for the preparation of cibaria D30 strain (KCCM 12211P) as an active ingredient.

The Weissella cibaria strain D30 (KCCM 12211P) is as described above. In addition, the strain (KCCM 12211P) may be a live cell, a dead cell culture, or a culture filtrate.

The capacity of the strain (KCCM 12211P) is preferably 1 μg / mL to 100 μg / mL, but is not limited thereto. At this time, when the capacity of the strain (KCCM 12211P) is less than the above range, there is a problem that the activity is not exhibited properly. If the capacity of the strain (KCCM 12211P) exceeds the above range, there may be a concern about toxicity have.

The term " antibacterial " in the present specification means antimicrobial activity against pathogenic bacteria including Gram-positive bacteria and Gram-negative bacteria, and includes micrococyst, staphylococcus, Listeria, Bacillus, Scleptococcus, Escherichia coli, Salmonella, Campylobacter and Vibrio spp., And more preferably at least one selected from the group consisting of Micrococyst, Staphylococcus, Listeria, Streptococcus, Escherichia coli, Salmonella, Campylobacter and Vibrio But it is not limited thereto.

In addition, the present invention can provide a pharmaceutical composition, a health functional food, a quasi-drug or a cosmetic composition containing the antibacterial composition.

The present invention also relates to a process for the preparation of cibaria) provides the inflammatory diseases or immune diseases prevention or treatment a pharmaceutical composition containing the D30 strain (KCCM 12211P), as an active ingredient.

The Weissella cibaria strain D30 (KCCM 12211P) is as described above. In addition, the strain (KCCM 12211P) may be a live cell, a dead cell culture, or a culture filtrate.

The term " inflammatory disease " as used herein refers to allergic diseases such as gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, Selected from the group consisting of infections, bacterial infections, fungal infections, burns, wounds due to surgical or dental surgery, PGE hyperaemia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis and eczema But it is not limited thereto.

The term " immune disease " as used herein refers to any disease or disorder that can be diagnosed and treated as a result of a disease or disorder, such as Behcet's disease, multiple myositis, dermatomyositis, autoimmune hematopoietic, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, eosinophilic epidermolysis, epididymitis, glomerulonephritis, Graves' Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, mucinous squamous cell carcinoma, ulcerative colitis, Diseases, and obesity, but is not limited thereto. Do not.

The pharmaceutical composition according to the present invention can be formulated in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

The solid preparation for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin or the like in the above strain (KCCM 12211P). In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the condition of the patient, the body weight, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also relates to a process for the preparation of cibaria D30 strain (KCCM 12211P) as an active ingredient, or a health functional food for preventing or ameliorating an immune disease.

The Weissella cibaria D30 strain (KCCM 12211P), and the above-mentioned inflammatory diseases or immune diseases are as described above. In addition, the strain (KCCM 12211P) may be a live cell, a dead cell culture, or a culture filtrate.

In the health functional food according to the present invention, when the above-mentioned strain (KCCM 12211P) is used as an additive for a health functional food, it can be used as it is or can be used together with other food or food ingredients, have. The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

Formulations of health functional foods may be in the form of powders, granules, pills, tablets, capsules, as well as in the form of ordinary foods or beverages.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

Generally, the above-mentioned strain (KCCM 12211P) may be 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 raw material. However, in the case of long-term ingestion intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The beverage in the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, and carbonating agents used in carbonated beverages. In addition, the health functional food according to the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food according to the present invention.

In addition, the present invention relates to the use of a compound of formula cibaria) D30 strain (KCCM 12211P) may provide a fermentation starter, seed composition, or fermented food comprising as an active ingredient.

As described above, the present invention relates to a method for producing an antimicrobial, antioxidant, anti-inflammatory and immunomodulating activity of Weissella cibaria) D30 strain (KCCM 12211P), and relates to their use, the strain (KCCM 12211P) is the stability and safety bar having excellent advantages, can be used as probiotics,

In addition, the above strain (KCCM 12211P) has antimicrobial activity against pathogenic bacteria, intestinal adhesion inhibition of food poisoning causative bacteria, weak organic acid production ability, excellent antioxidant, anti-inflammatory and immunomodulating activity, Or a composition for preventing, ameliorating or treating immune diseases. Therefore, it is expected that the strain (KCCM 12211P) will eventually be capable of detoxification.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

< Example >

Example  One: Weissella cibaria Strain KCCM  12211P) Isolation and identification

To separate the new strain, kimchi broth was sampled and used as a sample. Kimchi broth was heat - treated (80 ℃, 30 min), spread on TSA agar medium using 10 - fold dilution method, streaked several times, cultured and colonies were observed to isolate new strain. To identify the isolates, 16S rRNA sequence analysis was requested by Bionics (Seoul, Korea). Specifically, 16S rRNA sequencing was performed, and 16S rRNA gene was subjected to PCR using 27F and 1492R primers. Based on the results of analysis of nucleotide sequences, homology with other standard strains registered in Genbank database was compared using BLAST program. The new strains were Weissella cibaria , And the similarity was 99%. In addition, the homology was analyzed and a schematic diagram was obtained using the Mega 7 program (Fig. 1). At this time, Weissella cibaria On January 17, 2018, the strain was deposited with the Korean Society for Microbiological Conservation and received the accession number KCCM 12211P.

Experimental Example  1: Probiotic  Stability evaluation

(1) Acid resistance  And My answer is juicy  evaluation

Weissella according to Example 1 cibaria The acid resistance and brittleness of the strain (KCCM 12211P) were evaluated.

Specifically, for the evaluation of acid resistance, artificial gastric juice (pH 2.5, MRS medium supplemented with 0.3% (w / v) pepsin) was prepared, and the strains were reacted at 37 ° C for 3 hours to identify live bacteria. After addition of 0.3% (w / v) oxgall to the MRS medium, the strain was incubated at 37 ° C for 24 hours, and the number of live bacteria was measured.

process Number of bacteria (Log CFU / mL) Initial number of bacteria 8.30 ± 0.01 0.3% (w / v) pepsin, pH 2.5, 3 hours 7.35 ± 0.02 0.3% (w / v) oxgall, 24 hours 8.28 + 0.03

* Values are expressed as means ± SD (n = 3).

As shown in Table 1, weissella according to Example 1 cibaria As a result of the acid resistance evaluation of the strain (KCCM 12211P), the number of bacteria after cultivation decreased by 0.95 log CFU / mL compared with the initial number of bacteria, but it is considered to have sufficient resistance. In addition, as a result of the evaluation of biliary properties, the number of bacteria after culturing did not show any significant difference compared with the initial number of bacteria, and the bacteria were sufficiently transferred to the intestine with sufficient resistance to the digestive environment in the body, It is expected to be possible.

(2) Assessment of Alcohol Tolerance of Strain

Weissella according to Example 1 The alcohol tolerance of the cibaria strain (KCCM 12211P) was evaluated.

Specifically, for the evaluation of alcohol tolerance, strains were inoculated into MRS medium, activated at 37 ° C. for 18 hours, added with ethyl alcohol (5, 10, 20%) and then incubated at 37 ° C. for 2 hours and 4 After reacting for a period of time, living bacteria were measured.

process Number of bacteria (Log CFU / mL) Ethyl alcohol concentration (%) 5 10 20 Initial number of bacteria 8.90 + 0.03 8.97 ± 0.06 8.95 ± 0.05 2 hours 8.88 ± 0.06 8.53 + - 0.10 6.89 + - 0.10 4 hours 8.62 ± 0.09 7.61 + 0.04 6.44 ± 0.05

* Values are expressed as means ± SD (n = 3).

As shown in Table 2, weissella according to Example 1 The survival rate of the cibaria strain (KCCM 12211P) was 99.76 ± 0.67%, 95.14 ± 1.09%, and 79.96 ± 1.21%, respectively, for the ethanol concentration of 5, 10 and 20% The survival rates were 96.85 ± 1.06%, 84.90 ± 0.39% and 74.79 ± 0.59%, respectively. That is, regardless of the alcohol content and the incubation time, the survival rate is 70% or more, and it is expected that the intestinal probiotic activity will be sufficiently exhibited.

(3) Chapter Attachment  evaluation

Weissella according to Example 1 evaluate the ability of the sheet attachment cibaria strain (KCCM 12211P). At this time, Lactobacillus rhamnosus GG strain (KCTC 12202BP) was used as a control.

Specifically, for the evaluation of intestinal adhesiveness, the HT-29 (human colon adenocarcinoma cell line) cell line inoculated with 2 × 10 ⁵ cells / well was cultured until a single layer was formed, and the strain was inoculated. Lt; / RTI &gt; The cells were washed three times with phosphate-buffered saline to remove unattached microorganisms. After removing the cell line using 1% Triton X-100 solution, the number of cells attached to the cell line was measured using MRS medium.

process Number of bacteria (Log CFU / mL) Control group Example 1 Initial number of bacteria 8.26 + 0.03 8.09 ± 0.09 Number of attached bacteria 7.58 ± 0.08 7.65 ± 0.06 Adhesion (%) 17.83 ± 5.75 ^a 37.63 ± 4.96 ^b

* Different superscripts in the same row represent statistical differences between strains (p <0.05). Values are expressed as means ± SD (n = 3). * Attachment ability = number of bacteria attached / number of initial bacteria × 100.

As shown in Table 3, weissella according to Example 1 cibaria As a result of the evaluation of intestinal adhesiveness of strains (KCCM 12211P), 7.65 ± 0.06 log CFU / mL was adhered and the intestinal adherence was 37.63 ± 4.96% when 8.09 ± 0.09 log CFU / mL was inoculated, while Lactobacillus The binding affinity of rhamnosus GG strain (KCTC 12202BP) was only 17.83 ± 5.75%, indicating a significant difference.

Experimental Example  2: Probiotic  Safety evaluation

(1) Enzyme of strain Production capacity  evaluation

Weissella according to Example 1 cibaria The enzyme production ability of the strain (KCCM 12211P) was evaluated.

Specifically, API ZYM kit was used to evaluate enzyme production ability. More specifically, the strain was cultured to the logarithmic growth phase and centrifuged at 12,000 rpm for 10 minutes to remove the culture filtrate. After washing three times, only the cells were obtained, and the cells were inoculated on a cube containing each substrate and cultured for 4 hours. After that, solutions A and B were added, respectively, and enzyme production ability was confirmed according to the degree of color development.

enzyme Enzymatic productivity Control - Alkaline phosphatase + Esterase + Esterase lipase + Lipase - Leucine arylamidase + Valine arylamidase + Cystine arylamidase + Trypsin - α-Chymotrypsin - Acid phosphatase + Naphthol-AS-BI-phosphohydrolase + α-Galactosidase - β-Galactosidase + β-Glucuronidase - α-Glucosidase - β-Glucosidase + N-Acetyl-β-glucosaminidase - α-Mannosidase - α-Fucosidase +

* + Means positive, - means negative.

As shown in Table 4, weissella according to Example 1 As a result of evaluation of enzyme production ability of cibaria strain (KCCM 12211P), it was confirmed that β-glucuronidase, which is a cancer inducing enzyme, was not produced, and it can be considered that biosafety is secured. In addition, it has been confirmed to produce .beta.-glucosidase, one of useful enzymes, so that biotransformation of a useful substance can be expected.

(2) Evaluation of hemolysis of strains

Weissella according to Example 1 The hemolytic activity of the cibaria strain (KCCM 12211P) was evaluated. At this time, Vibrio parahaemolyticus The strain (KCCM 11965) was used as a control.

Specifically, in order to evaluate hemolytic activity, Bacillus bacteria were straiked on a Columbia agar medium containing 5% Sheep blood. When a clear zone is formed around the colony, an α-hemolytic phenomenon is shown, a β-hemolytic phenomenon occurs when a green-hued zone occurs, and a γ-hemolytic phenomenon occurs when there is no change, and the result is shown in FIG.

As shown in FIG. 2, the Vibrio parahaemolyticus strain (KCCM 11965) according to the control group exhibited β-hemolysis, whereas the Weissella cibaria strain (KCCM 12211P) according to Example 1 showed γ- It has been confirmed that it has hemolytic properties. Thus, it is confirmed that this is a safe strain.

(3) Antibiotic resistance of the strain

CLSI (Clinical and Laboratory Standards Institute) being adjusted to a reference, Weissella spp. To the non-standard sized relationship to a procedure similar to the reference Lactobacillus for the app., Weissella to Example No. 1 cibaria The antimicrobial susceptibility of the strain (KCCM 12211P) was evaluated.

In order to evaluate the antibiotic resistance, strains were inoculated into MRS medium at a concentration of 10 ⁷ CFU / mL using a flat plate method and 8 antibiotics (ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, ciprofloxacin, chloramphenicol, doxycycline ) Was loaded on a paper disk having a diameter of 8 mm, and then the resulting clear zone was measured after culturing at 37 ° C for 24 hours. The results are shown in Table 5 below.

Antibiotic Antibiotic susceptibility Ampicillin I Gentamicin R Kanamycin R Streptomycin R Tetracycline I Ciprofloxacin R Chloramphenicol I Doxycycline I

* S: susceptibility; I: medium; R: Resistance.

As shown in Table 5, weissella according to Example 1 cibaria The strain KCCM 12211P has been shown to be effective against Ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline. Have antibiotic resistance.

Experimental Example  3: Evaluation of the antimicrobial activity of the strain

(1) Pathogenicity of the strain Inhibition of growth on bacteria  Activity evaluation

Weissella according to Example 1 of cibaria strain (KCCM 12211P) was evaluated for growth inhibitory activity against pathogenic bacteria.

Specifically, 5 占 퐇 of the culture broth was loaded onto the MRS medium and cultured at 37 占 폚 for 24 hours. The pathogenic bacteria cultured for 24 hours were diluted in TSB medium supplemented with 0.7% agar at a concentration of 10 ⁷ CFU / mL, overlayed on the previously cultured MRS medium, and cultured at 37 ° C for 24 hours. By measuring the diameter of the clear zone formed by the deferred antagonism method, the growth inhibitory activity of pathogenic bacteria was verified. The results are shown in Table 6 below.

Pathogenic bacterial strain name Growth inhibitory activity (mm) Micrococcus flavus KCCM 10240 34.5 ± 0.82 Staphylococcus aureus KCCM 32395 24.5 ± 10.8 Staphylococcus aureus 1573 23.5 ± 2.45 Listeria monocytogenes ATCC 15313 25.0 + - 0.94 Bacillus cereus KCCM 29544 11.5 + -0.94 Streptococcus mutans KCTC 5124 18.5 ± 1.25 Escherichia coli ATCC 25922 22.5 + -0.94 Escherichia coli 20.5 ± 0.84 Salmonella Typhimurium 21.5 ± 0.45 Salmonella Enteritidis ATCC 13076 23.0 ± 0.54 Campylobacter jejuni 22.0 + - 0.84 Campylobacter coli 21.0 + - 0.94 Vibrio parahaemolyticus KCCM 11965 18.5 ± 0.95

* Values are expressed as means ± SD (n = 3).

As shown in Table 6, the growth inhibitory activity against Bacillus cereus was relatively weak compared to other pathogenic bacteria. However, except for Bacillus cereus , the growth inhibitory activity against Bacillus cereus was not distinguished between Gram positive bacteria and Gram negative bacteria, Inhibitory activity.

(2) Self-cohesion of the strain and evaluation of cohesion force with foodborne pathogens

Weissella according to Example 1 self cibaria strain (KCCM 12211P) was evaluated in the cohesive strength of the joint and the cohesive force and food poisoning organisms.

Specifically, the cultured strains were collected by centrifugation, washed three times with PBS, and suspended in PBS to adjust the absorbance at 600 nm to 0.30 ± 0.01. And incubated at 37 ° C for 4 hours and 24 hours. The absorbance was measured and auto-aggregation was calculated using the following equation.

Self-cohesion force (%) = (1 - A ₂ / A ₁ ) × 100

A ₁ , absorbance of the strain suspension before incubation; A ₂ , the absorbance of the strain suspension after incubation

Co-aggregation with food-borne pathogens was carried out at 37 ° C after mixing the same amount of suspension of each strain of food poisoning with the adjusted absorbance. 4 hours and 24 hours, and the cohesive force with foodborne pathogens was calculated using the following equation.

(%) = [1 - A ₁ / {(A ₂ + A ₃ ) / 2}] × 100

A ₁ , the absorbance of the suspension after incubation; A ₂ , Absorbance of pre-incubation causative bacteria suspension;

A ₃ , absorbance of the strain suspension before incubation

Self Cohesion (%) 4 hours 24 hours 22.95 + 1.54 66.25 + 1.10

* Values are expressed as means ± SD (n = 3).

A causative strain of food poisoning Culture time (h) Cohesive power with foodborne pathogens (%) Staphylococcus aureus 4 23.10 ± 1.15 24 58.58 ± 1.61 Listeria monocytogenes 4 18.81 + 1.49 24 58.93 + - 1.16 Salmonella Enteritidis 4 19.18 ± 1.08 24 40.69 1.51 Escherichia coli 4 18.31 ± 2.26 24 56.16 ± 1.76

* Values are expressed as means ± SD (n = 3).

As shown in Table 7, weissella according to Example 1 cibaria The strain (KCCM 12211P) has excellent self-cohesion, and it is believed that the colonization in the intestines will effectively colonize the colon. Further, as shown in Tables 6 and 8, the Weissella according to Example 1 cibaria The strain (KCCM 12211P) inhibits the growth of pathogenic bacteria including pathogens such as Staphylococcus, Listeria, Salmonella and Escherichia coli, and has excellent co-cohesion with food-borne pathogens. It seems to be possible.

(3) Intestinal attachment of pathogenic causative bacteria of strain Low performance  evaluation

Weissella according to Example 1 cibaria (KCCM 12211P) were evaluated for their ability to inhibit intestinal adhesion. At this time, Lactobacillus rhamnosus GG strain (KCTC 12202BP) was used as a control.

Specifically, for the evaluation of inhibition of intestinal adhesion of food poisoning causative organisms, HT-29 (human colon adenocarcinoma cell line) cell line inoculated with 2 × 10 ⁵ cells / well was cultured until a single layer was formed and then mixed bacteria (strain of causative agent of food poisoning and strain (10 ⁷ cells / well, respectively) and cultured at 37 ° C for 2 hours. The cells were washed three times with phosphate-buffered saline to remove unattached microorganisms. After removing the cell line using 1% Triton X-100 solution, the number of bacteria attached to the cell line was measured using a selective medium according to each strain. Are shown in Table 9.

A causative strain of food poisoning Number of bacteria
(CFU / mL) Intestinal adhesion inhibition of foodborne pathogens (%) Initial number of bacteria After 2 hours Control group Listeria monocytogenes 9.0 × 10 ⁶ 5.4 × 10 ⁶ 40.00 + 4.80 ^a Salmonella Enteritidis 4.5 × 10 ⁶ 2.4 × 10 ⁶ 47.41 ± 9.99 ^a Escherichia coli 7.3 × 10 ⁶ 4.4 × 10 ⁶ 39.70 ± 8.47 ^a Example 1 Listeria monocytogenes 9.0 × 10 ⁶ 3.7 × 10 ⁶ 59.02 + - 9.23 ^b Salmonella Enteritidis 4.5 × 10 ⁶ 1.9 × 10 ⁶ 56.76 ± 10.84 ^b Escherichia coli 7.3 × 10 ⁶ 3.5 × 10 ⁶ 52.27 ± 9.07 ^b

* Different superscripts in the same row represent statistical differences between strains (p <0.05). Values are expressed as means ± SD (n = 3).

As shown in Table 9, weissella according to Example 1 cibaria The inhibitory effect of strain (KCCM 12211P) on the intestinal adhesion of foodborne pathogens such as Listeria, Salmonella and Escherichia coli was significantly higher than that of the control group.

Experimental Example  4: Organic acid of the strain Generation  evaluation

Weissella according to Example 1 cibaria The organic acid production ability of the strain (KCCM 12211P) was evaluated. At this time, Lactobacillus rhamnosus GG strain (KCTC 12202BP) was used as a control.

Specifically, the strain was inoculated into the MRS medium, cultured at 37 ° C for 24 hours, and then the supernatant was taken using a centrifuge. The supernatant was filtered through a 0.2 μm cellulose membrane filter and analyzed by HPLC. HPLC analysis was carried out using Agilent 1100 series and Bio-Rad Aminex HPX-87H columns, and a peak was observed at 210 nm using a UV detector. The results are shown in Table 10.

Organic acid (g / L) Lactic acid
(Lactic acid) Acetic acid
(Acetic acid) Citric acid
(Citric acid) Pyroglutamic acid
(Pyroglutamic acid) Control group 22.89 ± 3.54 ^b 0.41 ± 3.11 ^a 0.13 ± 2.12 0.08 + 4.85 ^a Example 1 13.08 ± 2.45 ^a 1.08 ± 2.45 ^b Not detected 0.11 + - 2.34 ^b

* Different superscripts in the same row represent statistical differences between strains (p <0.05). Values are expressed as means ± SD (n = 3).

As shown in Table 10, weissella according to Example 1 cibaria Strain according to the result in Example 1, an organic acid producing ability in accordance with the growth of a verified by HPLC of (KCCM 12211P) Weissella cibaria Unlike the control, the strain KCCM 12211P did not produce citric acid, and lactic acid and acetic acid were produced at 13.08 ± 2.45 g / L and 1.08 ± 2.45 g / L, respectively. This organic acid production ability is expected to affect the intestinal environment.

In addition, pyroglutamic acid acts as a precursor of pramanicin, which acts as an intermediate metabolite of glutathione, which plays an important role in the antioxidant action against toxic substances or oxidative stress in the body, or exhibits antimicrobial activity , Weissella according to Example 1 cibaria The production ability of the strain (KCCM 12211P) by pyroglutamic acid was significantly higher than that of the control group.

Experimental Example  5: Evaluation of Antioxidative Activity of Strain

Weissella according to Example 1 cibaria The antioxidative activity of the strain (KCCM 12211P) was evaluated by various methods through the following (1) to (4) experiments. At this time, Lactobacillus rhamnosus GG strain (KCTC 12202BP) was used as a control.

For the following experiments (1) to (4), the strain was cultured in MRS medium for 24 hours, and the cells were recovered using a centrifugal separator and washed with PBS. Thereafter, the cells were finally suspended in PBS.

(1) DPPH  (1,1- 피덴 -2- picrylhydrazyl ) Radical Scatters  evaluation

The DPPH radical scavenging activity was verified by measuring the absorbance at 517 nm after mixing 1 mL of the DPPH solution prepared in 100 μM and 200 μL of the suspension, reacting in the dark at room temperature for 20 minutes.

(2) ABTS ⁺  (2,2'- azino -bis (3- ethylbenzothiazoline -6- sulphonic  acid)) radical Scatters  evaluation

The ABTS ⁺ radical scavenging ability was obtained by mixing ABTS reagent of 7.4 mM and potassium persulfate of 2.6 mM in the same amount, reacting for 24 hours, diluting with water to obtain absorbance of 0.70 ± 0.01 at 732 nm, taking 1 mL and mixing with 100 μL of the suspension Respectively. After incubation at room temperature for 20 minutes, the absorbance was measured at 732 nm.

(3) Hydroxyl radical of the strain Scatters  evaluation

Hydroxyl radical scavenging activity was determined by dissolving ferric chloride, EDTA, deoxyribose, ascorbic acid, and hydrogen peroxide at a concentration of 0.1 mM, 0.1 mM, 2.8 mM, 1 mM, and 20 mM in a potassium phosphate buffer at pH 7.4, After incubation at 37 ° C for 90 minutes, 1 ml of 60 mM TCA (trichloroacetic acid) and 0.3 ml of 200 mM TBA were added and incubated for 15 minutes. After cooling to room temperature, absorbance was measured at 532 nm.

The activity of the antioxidant experiments by the radical scavenging activity of the above experiments (1) to (3) was calculated using the following equation.

Radical scavenging ability (%) = [1- (A _s / A _c )] x 100

A _s , absorbance of the sample; A _c , absorbance of negative control

(4) Fatty acid oxidation of the strain Inhibition  evaluation

In the case of β-carotene bleach inhibiting ability, 2 mg, 44 μL and 200 μL of β-carotene, linoleic acid and Tween 80 were added to 10 mL of Chloroform, and the mixture was concentrated under reduced pressure at 50 ° C. and dissolved by adding 100 mL of distilled water . 4.5 mL of the prepared β-carotene aqueous solution was mixed with 0.5 mL of the suspension, and the absorbance at 470 nm was measured at intervals of 2 hours while reacting at 50 ° C. The inhibitory activity of β-carotene bleaching was calculated as follows.

β-Carotene bleaching inhibition (%) = [As / AI] × 100

As, absorbance of sample after incubation; AI, initial absorbance of the sample

Experimental Example  6: Evaluation of anti-inflammatory / immunomodulatory activity of strains

Weissella according to Example 1 cibaria The anti-inflammatory / immunomodulatory activity of the strain (KCCM 12211P) was evaluated by RAW 264.7 cells in the rat macrophage using the following experiments (1) and (2). At this time, Lactobacillus rhamnosus GG strain (KCTC 12202BP) was used as a control.

For the following experiments (1) and (2), the strains cultured in the MRS medium were recovered using a centrifuge and subjected to heat treatment at 85 ° C for 30 minutes. After washing three times with PBS, the cells were suspended in DMEM (Dulbecco's Modified Essential Medium) at a concentration of ¹ × 10 ⁶ and 1 × 10 ⁷ CFU / mL, respectively.

(1) Nitric oxide formation inhibitory activity of the strain

RAW 264.7 cells were inoculated into 96-well plates at a concentration of 2 × 10 ⁵ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C. After 2 hours, the strains were suspended in 1 × 10 ⁶ and 1 × 10 ⁷ CFU / mL, and cultured for 2 hours. LPS (lipopolysaccharide), an endotoxin, was treated at a concentration of 1 μg / Were induced and cultured for 24 hours. The inhibitory effect of nitric oxide on LPS induction was verified using Griess reagent, and the results are shown in FIG.

As shown in FIG. 3, treatment of LPS induced significant expression of nitric oxide in RAW 264.7 cells, and the expression of Weissella The cibaria strain (KCCM 12211P) inhibited the production of nitric oxide in a dose dependent manner and showed significant inhibitory effect on nitric oxide production as compared with the control. Therefore, it is expected that the overexpression of endotoxin-induced inflammatory reaction of Gram-negative bacteria can be mitigated.

(2) Inhibitory activity on the production of inflammatory mediators by the strain

RAW 264.7 cells were inoculated in a 6-well plate at a concentration of 5 × 10 ⁵ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours. After incubation for 2 h, the cells were treated with LPS (lipopolysaccharide) at a concentration of 1 μg / mL to induce inflammation. The cells were cultured for 24 h at a concentration of 1 × 10 ⁶ and 1 × 10 ⁷ CFU / Time. The inhibitory activity of the inflammatory mediators on the induction of inflammation of LPS was confirmed by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and the results are shown in FIG.

As shown in FIG. 4, treatment of LPS significantly induced the expression of inflammatory mediators such as iNOS, COX-2 and inflammatory mediators such as IL-1β, IL-6, and TNF-α, Weissella according to Example 1 The cibaria strain (KCCM 12211P) treatment significantly inhibited the expression of inflammatory mediators such as iNOS and COX-2 and inflammatory mediators such as IL-1β, IL-6, and TNF-α compared to the control do. Therefore, it is expected that inhibition of inflammatory mediator and cytokine expression by LPS-induced endotoxin-induced inflammation will effectively inhibit inflammatory diseases and toxin accumulation by inhibiting the production of inflammatory metabolites such as NO and PGE2 do.

Hereinafter, formulation examples of the composition containing the strain of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1: Preparation of powder

Weissella cibaria The strain 20 mg

Lactose hydrate 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight container to prepare powders.

Formulation Example 2: Preparation of tablets

Weissella cibaria The strain 10 mg

Corn starch 100 mg

Lactose hydrate 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation Example 3: Preparation of capsules

Weissella cibaria The strain 10 mg

Microcrystalline cellulose 3 mg

Lactose hydrate 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the usual preparation method of capsules.

Formulation Example 4: Preparation of injection

Weissella cibaria The strain 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Sodium dihydrogenphosphate 26 mg

After the above components were mixed, they were prepared with the above-mentioned component contents per 1 ampoule (2 mL) according to the usual injection preparation method.

Formulation Example 5: Preparation of a liquid preparation

Weissella cibaria The strain 10 mg

10 g per isomer

5 g mannitol

Purified water quantity

Lemon incense quantity

The components are dissolved in purified water according to the usual preparation method, and the lemon flavor is added in an appropriate amount. Then, purified water is added to adjust the total volume to 100 mL, sterilized and filled in a brown bottle to prepare a liquid preparation.

Formulation Example 6: Preparation of Health Functional Foods

Weissella cibaria The strain 10 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed And then granules are prepared and used in the manufacture of health functional foods according to conventional methods.

Formulation Example 7: Preparation of health drinks

Weissella cibaria The strain 10 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Purified water quantitation

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Konkuk University Industrial Cooperation Corp <120> NOVEL STRAIN OF WEISSELLA CIBARIA AND USE OF THE SAME <130> 1064729 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1499 <212> RNA <213> Weissella cibaria <400> 1 ctcaggatga acgctggcgg cgtgcctaat acatgcaagt cgaacgcttt gtggttcaac 60 tgatttgaag agcttgctca gatatgacga tggacattgc aaagagtggc gaacgggtga 120 gtaacacgtg ggaaacctac ctcttagcag gggataacat ttggaaacag atgctaatac 180 cgtataacaa tagcaaccgc atggttgcta cttaaaagat ggttctgcta tcactaagag 240 atggtcccgc ggtgcattag ttagttggtg aggtaatggc tcaccaagac gatgatgcat 300 agccgagttg agagactgat cggccacaat gggactgaga cacggcccat actcctacgg 360 gaggcagcag tagggaatct tccacaatgg gcgaaagcct gatggagcaa cgccgcgtgt 420 gtgatgaagg gtttcggctc gtaaaacact gttgtaagag aagaatgaca ttgagagtaa 480 ctgttcaatg tgtgacggta tcttaccaga aaggaacggc taaatacgtg ccagcagccg 540 cggtaatacg tatgttccaa gcgttatccg gatttattgg gcgtaaagcg agcgcagacg 600 gttatttaag tctgaagtga aagccctcag ctcaactgag gaattgcttt ggaaactgga 660 tgacttgagt gcagtagagg aaagtggaac tccatgtgta gcggtgaaat gcgtagatat 720 atggaagaac accagtggcg aaggcggctt tctggactgt aactgacgtt gaggctcgaa 780 agtgtgggta gcaaacagga ttagataccc tggtagtcca caccgtaaac gatgagtgct 840 aggtgtttga gggtttccgc ccttaagtgc cgcagctaac gcattaagca ctccgcctgg 900 ggagtacgac cgcaaggttg aaactcaaag gaattgacgg ggacccgcac aagcggtgga 960 gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcccttgaca 1020 actccagaga tggagcgttc ccttcgggga caaggtgaca ggtggtgcat ggttgtcgtc 1080 gt; gccagcattt agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg 1200 atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggcgtat 1260 acaacgagtt gccaacccgc gagggtgagc taatctctta aagtacgtct cagttcggat 1320 tgtaggctgc aactcgccta catgaagtcg gaatcgctag taatcgcgga tcagcacgcc 1380 gcggtgaata cgttcccggg tcttgtacac accgcccgtc acaccatgag agtttgtaac 1440 acccaaagcc ggtggggtaa ccttcgggag ccagccgtct aaggtgggac agatgatta 1499

Claims (10)

Antimicrobial, antioxidant, anti-inflammatory and immunomodulating activity,
The antimicrobial agent may be selected from the group consisting of Micrococcus flavus , Staphylococcus aureus , Listeria monocytogenes , Streptococcus mutans , Escherichia coli , Salmonella From the group consisting of Salmonella Typhimurium , Salmonella Enteritidis , Campylobacter jejuni , Campylobacter coli , and Vibrio parahaemolyticus . Weissella cibaria strain D30 (KCCM 12211P), which is for one or more selected.
delete The method according to claim 1,
Wherein said strain further comprises a strain of Weissella cibaria D30 (KCCM 12211P) which further has at least one characteristic selected from the group consisting of the following i) to iv):
I) Acid resistance, biliary properties, alcohol resistance and intestinal adherence;
Ii) inhibition of? -Glucuronidase activity;
Iii) inactivated hemolytic activity; And
Iv) Antibiotic resistance to Ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline. Antibiotic resistance to ampicillin, Gentamicin, Kanamycine, Streptomycin, Tetracycline, Ciprofloxacin, Chloramphenicol or Doxycycline.
The method according to claim 1,
Wherein said strain is at least one selected from the group consisting of self-coagulant and Staphylococcus aureus , Listeria monocytogenes , Salmonella Enteritidis and Escherichia coli , Has cohesive force of;
At least one intestinal adhesion inhibitor selected from the group consisting of Listeria monocytogenes , Salmonella Enteritidis and Escherichia coli .
Weissella cibaria strain D30 (KCCM 12211P).
The method according to claim 1,
Said strain producing at least one selected from lactic acid, acetic acid and pyroglutamic acid but not producing citric acid, such as Weissella &lt; RTI ID = 0.0 &gt; cibaria strain D30 (KCCM 12211P).
The method according to claim 1,
The strain is a strain of Weissella cibaria D30 (KCCM 12211P) comprising the 16S rRNA base sequence of SEQ ID NO: 1.
A strain of Weissella cibaria D30 (KCCM 12211P) as an active ingredient,
Micrococcus flavus , Staphylococcus aureus , Listeria monocytogenes , Streptococcus mutans , Escherichia coli , Salmonella typhimurium, Streptococcus spp ., Staphylococcus aureus , Listeria monocytogenes , Streptococcus mutans , Escherichia coli , At least one selected from the group consisting of Salmonella Typhimurium , Salmonella Enteritidis , Campylobacter jejuni , Campylobacter coli , and Vibrio parahaemolyticus . / RTI &gt;
The method of claim 7, wherein
Wherein the strain is a live cell, a dead cell, or a culture filtrate.
delete A strain of Weissella cibaria D30 (KCCM 12211P) as an active ingredient,
Inhibiting the production of inflammatory mediators comprising i) NO production inhibition, ii) inhibition of inflammatory mediator production comprising iNOS or COX-2, or iii) inflammation mediated cytokines including at least one selected from the group consisting of IL-1 ?, IL-6 and TNF-? A health functional food for preventing or ameliorating an inflammatory disease or an immune disease through inhibition of production.

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