KR101194795B1 - Lactic acid bacterium separated from kimchii and uses thereof - Google Patents

Abstract

PURPOSE: A Pediococcus pentosaceus strain isolated from Kimchi is provided to ensure high antibacterial activity and to be used in various industrial field. CONSTITUTION: A Pediococcus pentosaceus KFCC 11504P is isolated from Kimchi and has an antibacterial effect against Salmonella typhymurium, Micrococcus leteus, Bacillus subtilis, Bacillus cereus, Vibrio parahaemoliticus, and Escherichia coli. The strain also has acid resistance and bile acid resistance. An antibacterial composition contains the strain, culture liquid thereof, concentrate of the culture liquid, or dry material of the culture liquid as an active ingredient.

Description

Lactobacillus isolated from Kimchi and its use {LACTIC ACID BACTERIUM SEPARATED FROM KIMCHII AND USES THEREOF}

The present invention relates to a strain having antimicrobial activity against harmful microorganisms and viable in an acidic and intestinal environment and probiotics using the strain.

Lactic acid bacteria are bacteria that ferment sugars to produce more than 50% lactic acid, which is the most beneficial microorganism available to humans. It is widely distributed in the natural world such as the oral cavity, digestive tract, and soil of mammals as well as food, and it is a beneficial strain for human life that plays an important role related to food preservation and sensory characteristics. Lactic acid bacteria, widely used as foods as fermented foods containing lactic acid bacteria for a long time, have recently been recognized as stability as GRAS (Generally Recognized As Safe) microorganisms.

Based on the stability of lactic acid bacteria and the recognition that lactic acid bacteria are good for health, efforts to maintain health and prevent diseases have been increased through foods containing lactic acid bacteria. Recently, probiotics for evaluating the potential for use of lactic acid bacteria in functional foods have been increased. The research on tick lactic acid bacteria is extensively carried out worldwide.

Probiotic lactic acid bacteria should not only be recognized for their stability in origin and use, but also under acidic conditions in the upper gastrointestinal tract and in the presence of bile, should be able to adhere to the large intestine, and should have excellent strain stability or viability. Currently, probiotic lactic acid bacteria satisfying the above requirements are mostly Lactobacillus sp. ( Lactobacillus sp.) And Bifidobacterium strains ( Bifidobacterium sp.).

On the other hand, Kimchi is a food that can be manufactured by anyone at home without any special knowledge. More specifically, Kimchi is Korea's representative traditional fermented food that has been developed since the Three Kingdoms period. It is one of the representative Korean fermented foods that are aged through the fermentation process by microorganisms that exist around the living environment by mixing spices such as garlic and garlic.

Recently, a number of studies on the usefulness of kimchi have been published. For example, as physiological activities such as immune activity enhancement and anti-cancer effects have been revealed, it is recognized as a food necessary for maintaining health as well as nutrient supply, and soaking fresh vegetables without heat treatment. Therefore, it is reported that the content of lactic acid bacteria is higher than that of fermented milk products because all nutrients of vegetables are maintained to provide a good habitat for lactic acid bacteria.

The research on the lactic acid bacteria has been mainly conducted for dairy products, and the systematic and scientific researches on lactic acid bacteria involved in kimchi foot effect tablets have been conducted recently compared to the long history.

Therefore, the necessity of research on lactic acid bacteria that can act as antimicrobial against probiotic lactic acid bacteria or harmful microorganisms among kimchi bal effect tablet which is our traditional fermented food has emerged.

An object of the present invention is to provide a strain that is isolated from kimchi, has antimicrobial activity against harmful microorganisms, has acid resistance and bile resistance, and has excellent intestinal adhesion ability to become a probiotic lactic acid bacterium.

In addition, an object of the present invention is to provide an antimicrobial composition comprising at least one selected from the group consisting of the strain, its culture, the concentrate of the culture and the dried product of the culture as an active ingredient.

In addition, an object of the present invention is to provide a probiotic (probiotics) comprising at least one selected from the group consisting of the strain, its culture, the concentrate of the culture and the dried product of the culture as an active ingredient.

In order to achieve the above object, the present invention provides a Pediococcus pentosaceus strain ( Pediococcus pentosaceus ) which is isolated from kimchi and has antimicrobial activity against harmful microorganisms. Preferably the harmful microorganism is Salmonella typhimurium strain ( Salmonella typhymurium), the micro flow Proteus strains Rhodococcus (Micrococcus leteus), Bacillus subtilis strain (Bacillus subtilis), Bacillus cereus strains (Bacillus cereus), Vibrio para Molly Tee hee Syracuse strain (Vibrio parahaemoliticus ) and Escherichia coli ) may be a strain having antimicrobial activity.

In addition, an object of the present invention is to provide an antimicrobial composition comprising at least one selected from the group consisting of the strain, the culture solution of the culture solution and the dried product of the culture solution as an active ingredient.

In addition, an object of the present invention is to provide a probiotics (probiotics) comprising at least one selected from the group consisting of the strain, its culture broth concentrate of the culture broth and the dried product of the culture broth as an active ingredient.

In the present invention, the culture means that a specific microorganism is cultured in a culture medium or a culture medium, and the culture may include the specific microorganism. The culture is not limited in its formulation, and may be, for example, liquid or solid. In this aspect, the culture means the culture by inoculating a specific microorganism in a liquid medium. In addition, in this invention, the concentrated liquid of a culture liquid means the thing which concentrated the said culture liquid, and the dry matter of a culture liquid means that the water of the said culture liquid was removed or removed.

The culture medium means a solid or liquid containing nutrients necessary for growing animal cells, plant cells, or bacteria. The medium includes nutrients required by the microorganisms or cells to be cultured, that is, the culture, and may be added and mixed with a substance for a special purpose. The medium is also called an incubator or a culture medium, and is a concept that includes both natural medium, synthetic medium or selective medium.

In the present invention, the antimicrobial composition may mean an antimicrobial agent generically, and may mean the same as an antimicrobial agent, an antiseptic agent, a biological preservative or a natural preservative, and specifically, it is composed of Gram-positive bacteria, Gram-negative bacteria, and fungi (yeast and mold). It means a substance capable of inhibiting or inhibiting one or more developmental and life functions selected from the group, that is, an antibacterial and / or antifungal substance.

In the present invention, the probiotic means a probiotic having a beneficial effect on health by improving the intestinal microflora of humans and livestock. In order for the microorganism to function as a probiotic, certain bacteria must survive the passage through the stomach, and therefore, should be acid and bile resistant, and the intestinal viability should be good to maintain its activity for a certain period after ingestion.

The inventor of the present invention, while studying to identify probiotic lactic acid bacteria that can be used as a probiotic activator from our conventional lactic acid bacteria, especially kimchi, which is a representative traditional fermented food in Korea, the conventional biocide in Korea Lactobacillus Lactobacillus Compared to rhamnosus GG), not only has better acid and bile resistance, but also has good intestinal adhesion, antimicrobial activity against various harmful microorganisms, and comparable immune activity enhancement effect with Lactobacillus rhamnosus GG. F142-811 were separated strain, and identified the strain GRAS (Genrally regarded as safe) Phedi O Lactococcus pento sake mouse strain (microorganism belonging to the lactic acid bacteria Pediococcus pentosaceus ) to complete the present invention.

Hereinafter, the present invention will be described in detail.

The present invention is isolated from kimchi and Pediococcus pentosakeus strain having an antimicrobial activity against harmful microorganisms ( Pediococcus pentosaceus ), more specifically Pediococcus pentosakeus strain F142-811 ( Pediococcus pentosaceus F142-811).

Pediococcus pentosakeus strain F142-811 of the present invention has been isolated from kimchi, specifically, radish kimchi, has an antimicrobial activity against harmful microorganisms associated with food spoilage or food poisoning, and is a strain having the following microbial properties.

Specifically, the F142-811 strain isolated from the heat radish kimchi was identified as Gram-positive cocci, forming a circular milky-colored colony, and was negative as a result of a hemolysis test. It was confirmed that growth was possible in the medium. In addition, it can be grown using sugars, such as D-Xylose, Sucrose, Sucrose, Trehalose, Gentiobiose and D-Tagatose. It has been confirmed that there is a major action, lactose (lactose) is not available.

In addition, the F142-811 strain has a total of 1452 bp of 16S rDNA as shown in SEQ ID NO: 3, and the base sequence of the 16S rDNA is 99% sequenced with Pediococcus pentosaceus DSM20336T (AJ305321). It showed the same sex, finally identified as Pediococcus pentosakeus and named Pediococcus pentosakeus strain F142-811.

Pediococcus pentosakeus strain F142-811 ( Pediococcus pentosaceus F142-811) was deposited on February 14, 2011 at the Korea Microorganism Conservation Center located on the 2nd floor of Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea, and was given accession number KFCC 11504P.

The Pediococcus pentosakeus strain is preferably isolated from Pediococcus pentosakeus strain F142-811, more preferably kimchi and has antimicrobial activity against harmful microorganisms, Pediococcus pento with accession number KFCC 11504P Sakeus strain ( Pediococcus pentosaceu s KFCC 11504P).

The Pediococcus pentosakeus strain F142-811 has excellent acid resistance and bile resistance, and is also excellent in intestinal adhesion, it was confirmed that satisfies the requirements of probiotics or probiotic lactic acid (probiotics).

Specifically, the Pediococcus pentosakeus strain F142-811 was tested using an MRS liquid medium adjusted to pH 2.0, which is an acidic environment, and an MRS liquid medium containing 0.5% of bovine oxgall. Lactobacillus Lactobacillus , which has been reported to be an excellent probiotic and commercially available. rhamnosus GG) was found to have better or comparable acid and bile resistance.

In addition, the Pediococcus pentosakeus strain F142-811 is a gut using a galHT cell line (galactose-induced HT-29 cell line) induced by the mucosa of the small intestine using a Caco-2 cell line, a HT-29 cell line and a galactose. As a result of testing the adhesion ability, it was confirmed that it has an intestinal adhesion ability superior to Lactobacillus rhamnosus GG. More specifically, using a Caco-2 cell line, the Pediococcus pentosakeus strain F142-811 was found to have an adhesion rate 1.5 times better than that of Lactobacillus rhamnosus GG. It was confirmed that the adhesion rate was about 7.8 times better when using the HT-29 cell line and about 3.4 times, and in fact, when the Pediococcus pentosakeus strain F142-811 was ingested in the intestine, the mucous layer of the small and large intestine It was expected that with higher adhesion, it could be well utilized as a probiotic.

In addition, the Pediococcus pentosakeus strain F142-811 has antimicrobial activity against harmful microorganisms. The harmful microorganisms may be a food contaminating microorganism or a food poisoning causative bacterium, which is a microorganism causing contamination by contaminating the food with respect to the food, and causing food poisoning, and the bacterium of Bacillus as the food contaminating microorganism. sp.), Micrococcus sp.), Pseudomonas sp.), and the food poisoning agent that causes enteritis or food poisoning is Salmonella genus ( Salmonella). sp.), Vibrio sp.), Escherichia coli ). Among them, strains of the genus Micrococcus are Gram-positive cocci, and have a high proteolytic ability, and are pointed to cause contamination of foods of seafood, dairy products or noodles, and the micrococcus luteus strain ( Micrococcus). leteus ) is the most common species and is a skin flora that is isolated from the skin. The strain of the genus Salmonella is a harmful microorganism causing food poisoning, in particular the Salmonella typhimurium strain ( Salmonella typhymurium ) is known as the causative agent of typhoid fever.

The Phedi O Lactococcus pento sake F142-811 mouse strain is preferably the strain Salmonella typhimurium (Salmonella typhymurium ), Micrococcus strain leteus ), Bacillus subtilis strains ( Bacillus subtilis), Bacillus cereus strains (Bacillus cereus ), Vibrio parahaemoliticus strains and Escherichia coli ) may have an antimicrobial activity.

In addition, the Pediococcus pentosakeus strain F142-811 has immune enhancing activity or immune improving activity, specifically promotes the secretion of IL-1β and IL-12 in cytokines (cytokine) from immune cells, and produces NO. It has been confirmed to promote.

In the present invention, the cytokine is a trace bioactive substance in which immune cells or leukocytes are secreted by stimulation, and acts on various cells such as immune cells, leukocytes, hematopoietic cells, angiogenesis cells, neurons and endocrine cells Means a substance that induces various reactions, such as a reaction.

For example, IL-12 is a cytokine that acts as a switch for immunomodulatory mechanisms of T helper cell type 1 (TH1) and TH helper cell type 2 (TH2). Specifically, in relation to the TH1 immune response, which induces an inflammatory response, natural apoptosis cells are activated when a pathogenic microorganism is infected, thereby inducing immune action by natural apoptosis cells or activating cytotoxic T cells. In addition, IL-12 performs an antiallergic effect in relation to the allergic TH2 immune response and specifically decreases the amount of IL-4 secreted by the excess amount of IL-4 or IFN-gamma secreted by immune cells. The allergic response can be converted into a Th1 immune response that induces an inflammatory response.

The confirmed fact that the Pediococcus pentosakeus strain F142-811 promotes the secretion of IL-12, which reduces the amount of IL-4 secretion, antiallergic, activates phagocytosis of macrophages, cytotoxic It activates the immune function of T cells to carry out anticancer activity and, when infected by harmful microorganisms, promotes the secretion of cytokines, IL-12 or IL-1β, which are involved in the defense against harmful or pathogenic microorganisms. It is anticipated that by promoting NO production, it is possible to enhance the initial immune effect against pathogenic microorganisms.

In addition, the Pediococcus pentosakeus strain F142-811 was found to be harmless to the human body because hemolysis did not occur even when incubated with red blood cells.

According to the above results, the Pediococcus pentosakeus strain F142-811 has antimicrobial activity against harmful microorganisms, excellent acid resistance and bile resistance, excellent intestinal adhesion ability, immune improvement effect, and also secure safety It can be applied as an active ingredient or a probiotic of the antimicrobial composition.

In this aspect, the present invention is an antimicrobial agent for harmful microorganisms comprising at least one selected from the group consisting of the Pediococcus pentosakeus strain F142-811, a culture thereof, a concentrate of the culture, the dried product of the culture as an active ingredient It relates to a composition.

The Phedi O Lactococcus pento sake F142-811 mouse strain is preferably Phedi O Lactococcus pento sake mouse strain with the accession number KFCC 11504P (Pediococcus pentosaceus KFCC 11504P).

The antimicrobial composition may be used as a composition for preventing and improving microbial infections against the harmful microorganisms, and may be applied as a pharmaceutical composition or a food composition.

In addition, the Pediococcus pentosakeus strain F142-811 or the culture medium in which the strain is cultured has antimicrobial activity against harmful microorganisms. The culture medium in which the strain is cultured may be, for example, a culture medium in which the strain is cultured.

Specifically, the harmful microorganism is a strain of the genus Micrococcus which is a cause of food decay, for example ( Micrococcus sp.), preferably acids Proteus strains (Micrococcus leteus), the cause of food poisoning in Salmonella strains (Salmonella sp.), preferably from Salmonella typhimurium strain (Salmonella typhimurium), the Bacillus strain causative agents of food or deterioration (Bacillus sp.), preferably from the Bacillus subtilis strain (Bacillus subtilis) and Bacillus cereus strains (Bacillus cereus ), Vibrio sp., which causes enteritis, preferably Vibrio parahaemoliticus strain, and Escherichia coli causing diarrhea and enteritis. It can be coli).

The content of the active ingredient included in the antimicrobial composition may be appropriately selected in consideration of the purpose of use and the method of use, for example, 0.01 to 99 parts by weight, preferably 0.1 to 90 parts by weight based on the total composition. More preferably 1 to 75 parts by weight. The antimicrobial composition may further include an additive commonly used.

The present invention also provides a probiotic active agent comprising at least one selected from the group consisting of the Pediococcus pentosakeus strain F142-811, its culture solution, the concentrate of the culture solution, the dried product of the culture solution.

The Phedi O Lactococcus pento sake F142-811 mouse strain is preferably Phedi O Lactococcus pento sake mouse strain with the accession number KFCC 11504P (Pediococcus pentosaceus KFCC 11504P).

In the present invention, the probiotic means a probiotic having a beneficial effect on health by improving the intestinal microflora of humans and livestock. In order for the microorganism to function as a probiotic, the intestinal viability is good and its activity must be maintained for a certain period after ingestion. The Pediococcus pentosakeus strain F142-811 not only exhibits antimicrobial activity against harmful microorganisms, but also has excellent acid resistance and high resistance to artificial bile including bovine bile solution, and when hemoglobin reacts with red blood cells As it is confirmed that this does not occur, it is toxic to the human body, and thus satisfies all conditions for functioning as a probiotic.

The content of the antimicrobial composition included in the probiotic active agent may be appropriately selected in consideration of the purpose of use and the method of use, and preferably 0.01 part by weight to 99 parts by weight, more preferably 0.1 part by weight based on the total composition. To 90 parts by weight. The probiotic may further comprise an additive commonly used.

In addition, the present invention provides a food containing the probiotic. The probiotic has a microbial strain, which is a causative agent of food decay or decay, preferably micrococcus luteus strain, Bacillus sp. Strain, Bacillus subtilis strain and Bacillus cereus strain because it has an antimicrobial activity It can prevent or alleviate the deterioration, and has antibacterial activity against Salmonella spp., Salmonella typhimurium strain, which is a causative agent of food poisoning, and thus has an effect of preventing or improving food poisoning. In addition, the Vibrio genus strain, which is the cause of enteritis, preferably has antimicrobial activity against Vibrio parahemoliticus, and may have an anti-inflammatory or relieving effect on enteritis, and has an antimicrobial activity against E. coli causing diarrhea and enteritis. Therefore, it is effective in preventing or improving diarrhea and enteritis.

In the present invention, the food means a natural product or a processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process, for example, fruits, vegetables, and fruits. Dried or cut products of fruits, vegetables, fruit juices, vegetable juices, mixed juices or chips, noodles, livestock processed foods, fish processed foods, dairy foods, fermented foods, legumes foods, cereals, microbial fermented foods, confectionery , Seasonings, processed meats, acidic beverages, licorice, herbs, etc., but is not limited thereto.

The food of the present invention is not particularly limited to other ingredients except for containing the probiotic active agent as an essential ingredient in the indicated ratio, and may include various flavors or carbohydrates as additional ingredients, such as ordinary food, but is not limited thereto. It is not. The probiotic may be added to raw materials in the manufacture of foods, beverages, health drinks, gums, teas, vitamin complexes, and health functional foods, or by appropriate mixing with cooked foods, and food acceptable foods. It may be added together with auxiliary additives. At this time, the amount of the probiotic in the food or beverage may be added in an amount of 0.01% to 90% by weight of the total food weight, in the case of a beverage from 0.02 g to 20 g, preferably 0.5 g to 10 g based on 100 mL It can be added at the ratio of.

The food may be, for example, fermented food, health food or beverage.

Fermented food in the present invention means a food made by adding one or two or more microorganisms, such as lactic acid bacteria or yeast, using the fermentation action of the microorganisms, and in detail to add the fermented food spawn to the food base and to prepare Means food. The fermented food includes all non-sterile open fermented foods, such as liquor, bread, kimchi, milk brown, miso, soy sauce, cheese, butter, yogurt.

In addition, the probiotic may be used as a seed for fermented food, preferably used as a seed of kimchi. At this time, the amount of spawn is preferably adjusted according to the type of fermented food and the type of spawn. Preferably, it is inoculated at 0.0001 to 0.01 part by weight (wet part by weight) based on 100 parts by weight of the food main ingredient.

In the present invention, the functional food refers to a food group which is imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques and the like, the regulation of the biological defense rhythm of the food composition, Means a food which is processed and designed so that the body control function regarding the body is sufficiently expressed to the living body.

In the present invention, the drink refers to a generic term for drinking to quench thirst or enjoy the taste, and includes alcoholic beverages, soft drinks, water, syrup, tea, coffee, fruit drinks, and the like, and include lactic acid bacteria drinks. The lactic acid bacteria drink refers to a beverage in which lactic acid bacteria are cultured and lactic acid fermentation is added by diluting with sterilized water and adding sugar and flavoring. The beverage is not particularly limited to other ingredients except for containing the probiotic as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.

As described above, Pediococcus pentosaceus strain F142-811 ( Pediococcus pentosaceus F142-811) according to the present invention is excellent in acid resistance and bile resistance, excellent adhesion to intestinal cells, various harmful microorganisms, As an antimicrobial activity against Salmonella typhimurium strain, micrococcus luteus strain, Bacillus subtilis strain, Bacillus cereus strain, Vibrio parahemoliticus strain and E. coli can be used as a probiotic.

1 is an electron microscope photograph of the Pediococcus pentosakeus strain F142-811 according to an embodiment of the present invention.
2 is a nucleotide sequence of 16S rRNA of Pediococcus pentosakeus strain F142-811 according to an embodiment of the present invention.
Figure 2 is a diagram showing the phylogenetic relationship with other bacteria based on the nucleotide sequence of 16S rRNA of Pediococcus pentosakeus strain F142-811 according to an embodiment of the present invention.
Figure 3 is a photograph of Lactobacillus rhamnosus GG confirming the adhesion of the lactic acid bacteria to Caco-2 cell line according to an embodiment of the present invention.
Figure 4 is a photograph confirming the adhesion of the lactic acid bacteria to Caco-2 cell line according to an embodiment of the present invention, Figure 4a is a photograph confirming the cell adhesion of the Lactobacillus rhamnosus GG of a comparative example, Figure 4b is pedio It is a photograph confirming the cell adhesion ability of the caucus pentosakeus strain F142-811.
FIG. 5 is a graph showing the IL-1β production of Pediococcus pentosakeus strain F142-811 and Lactobacillus rhamnosus GG according to an embodiment of the present invention, and the vertical axis represents the secreted IL-1β production amount of 1 mL supernatant. FIG. The amount of sugar cytokines is expressed in ng, and the horizontal axis is the amount of each strain added in log CFU / mL, that is, the number of lactic acid bacteria contained per mL of the supernatant in log units.
Figure 6 is a graph showing the comparison of IL-12 production of Pediococcus pentosakeus strain F142-811 and other lactic acid bacteria according to an embodiment of the present invention, the vertical axis is the cytokine per 1 mL supernatant secreted IL-12 production The amount of is expressed in ng, the horizontal axis is the log CFU / mL of the added amount of each strain, that is, the number of lactic acid bacteria contained per mL of the supernatant in log units.
7 is a graph comparing NO production of Pediococcus pentosakeus strain F142-811 and other lactic acid bacteria according to an embodiment of the present invention, and the vertical axis represents the amount of cytokine per 1 mL supernatant secreted NO production. The horizontal axis shows log CFU / mL of the added amount of each strain, that is, the number of lactic acid bacteria contained per mL of the supernatant in log units.

Hereinafter, examples of the present invention will be described. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

< Example  1> Lactic acid bacteria  Isolation and identification

1-1 Lactic acid bacteria  detach

The edible portion and the liquid portion of yeolmu kimchi were mixed 1: 1, weighed 10 g, and then ground in sterile water of 90 mL. Serial dilution was performed on MRS (+ 0.5% CaCO3) solid medium and incubated at 30 ° C. for 2 days. Among the colonies, strains showing transparent rings for CaCO3 were selected, and lactic acid bacteria were isolated through gram staining and catalase test.

1-2 Lactic acid bacteria  Sympathy

F142-811, the lactic acid bacteria isolated in Example 1-1, was identified through morphological analysis using morphological characteristics including gram staining, biochemical analysis including glucose metabolism, and 16S rDNA sequence analysis.

Analysis of the morphological characteristics was observed the shape and color of the colony of F142-811, the isolated strain of Example 1-1, and after gram staining the strain, the shape was observed with an optical microscope (SEM) ( 1), hemolysis was also observed, and the results are shown in Table 1 and FIG. 1.

Characteristics F142-811 Source Yeolmu kimchi Morphology, Shape coccus Gram-stain + Catalase test - Acid production (pH) pH 4.01 Hemolysis - Colony color and transparency Transparent milk color

As shown in Table 1, the isolated strain F142-811 is a coliform form as a result of optical microscopic observation, forms a transparent milky colony, the pH of the growing medium was pH 4.01, was negative after hemolysis test, It was confirmed that growth was possible in LBS solid medium.

In addition, the identification was performed by the 16S rDNA sequencing. Specifically, after extracting 16S ribosomal RNA from the isolate strain F142-811, PCR was performed using the primer pairs shown in Table 2 below to perform 16S rRNA sequencing, to the 16S rRNA of the isolate strain F142-811. A total of 1,452 bp of nucleotide sequences were determined, and the results are shown in FIG. 2 and SEQ ID NO.

Primers Sequence (5 '-> 3') SEQ ID NO: 27F AGAGTTTGATCCTGG CTCAG One 1492R GGTTACCTTGTTACGACTT 2

Based on the performed nucleotide sequence, the nucleotide sequence was analyzed using a 3730 XL sequencer (Applied Biosystems, USA), and the homology of each strain was compared using the CLUSTRAL W program.

The phylogenetic relationship with other bacteria is shown in FIG. 3 based on the results of the homology search using the sequences determined through the 16S rRNA sequencing. As shown in FIG. 3, Pediococcus 99% (1452/1569), Pediococcus to pentosaceus DSM20336T (AJ305321) 98% (1452/1529) similarity to stilesii LMG23082T (AJ305320).

In addition, the sugar metabolism was reviewed and performed, and Pediococcus had the highest similarity as a result of 16S rDNA sequencing. Using pentosaceus DSM20336T (AJ305321) as a comparative example, the results are shown in Table 3 below. + In Table 3 means a positive reaction, and-means a negative reaction.

F142-811 DSM 20336 F142-811 DSM 20336 Glycerol - Salicin + Erythritol - Cellobiose + D-Arabinose - Maltose + + L-Arabinose + + Lactose - + Ribose + + Melibiose - D-Xylose + D Sucrose + L-Xylose - Trehalose + Adonitol - Inulin D MD-Xyloside - Melezitose - - Galactose + + Raffinose - Glucose + Amidon - Fructose + Glycogen - Mannose + Xylitol - Sorbose - Gentiobiose + Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose + Mannitol - - D-Fucose - Sorbitol - L-Fucose - α Methyl-D-mannoside - D-Arabitol - α Methyl-D-glucoside - L-Arabitol - N Acetyl glucosamine + Gluconate - Amygdalin + 2 keto-gluconate - Arbutin + 5 keto-gluconate - Esulin +

As shown in Table 3, the isolated strain Pediococcus eoteuna exhibit substantially similar contemporary saneung and pentosaceus DSM20336T, the F142-811 was divided in that, while using the D-xylose, lactose does not use.

As a result of the analysis, F142-811 strain is Pediococcus The strain was identified as pentosaceus , and the strain was deposited on February 14, 2011, at the Korea Microorganism Conservation Center located at 2F, Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea, and was given accession number KFCC 11504P.

< Example  2> Determination of antimicrobial activity against harmful microorganisms

Confirmation of the antimicrobial activity of the Pediococcus pentosakeus strain F142-811 identified in Example 1 was performed through a spot-array experiment for each of the indicator strains described in Table 4 below. The specific experimental method is as follows.

The Pediococcus pentosakeus strain F142-811 was inoculated in MRS liquid medium and incubated at 30 ° C. for 24 hours. The cultured cells were recovered by centrifugation, OD ₆₀₀ was adjusted to 1.0 with sterile water, and 10 μl was added to the MRS solid medium. The solid medium was inoculated with 0.1% inoculation of the harmful microorganisms described in Table 4 below in a top agar medium containing 0.5% agar formed with a specific medium for harmful microorganisms by drying for about 2 hours. The harmful microorganisms were cultured for 16 hours at the growth temperature of the lactic acid bacteria, and the diameters of the transparent rings, that is, the inhibitory rings formed around the lactic acid bacteria droplets were measured, and the results are shown in Table 4 above.

In relation to the antimicrobial activity of Table 4, '+++' means a strong growth inhibition, '++' means a moderate growth inhibition, '+' means a slight growth inhibition, '-' Means not to inhibit growth.

To microorganisms Antimicrobial Activity of F142-811 Bacillus cereus ATCC 27348 + Bacillus subtilis ATCC 6633 ++ Salmonella typhymurium KCTC 2515 ++ Micrococcus luteus ATCC 13513 +++ Lactobacillus plantarum ATCC 8014 - Listeria monocytogenes ATCC 19113 - Vibrio parahaemoliticus ATCC 27969 + Escherichia coli ATCC 25922 +

As shown in Table 4, Pediococcus pentosakeus strains F142-811 are Lactobacillus plantarum and Listeria Except for monocytogenes , the other strains showed antibacterial activity, especially Micrococcus High antimicrobial activity against luteus was confirmed.

The above results indicate that spore forming bacteria in canned foods are controlled by using lactic acid bacteria or its culture filtrate in the manufacture and processing of foods, or used as natural preservatives in various foods and feeds such as fermented milk, fermented alcoholic beverage and dairy products. As a result, it is expected to improve nutritional value, taste, and texture by reducing heat throughput, as well as to control storage of low temperature bacteria, pathogenic bacteria, and rot microorganisms.

< Example  3> Pediocaucus Pentosakeus  Of strain F142-811 Probiotic  Availability

The bacteria ingested through the oral cavity must survive through strong acidic gastric juice to reach the final target intestine. Therefore, the acid resistance test was performed on the Pediococcus pentosakeus strain F142-811 isolated from kimchi. In addition, the Pediococcus pentosakeus strain F142-811 passes through the pancreas and duodenum before it reaches the intestine through the stomach, where bile fluid is secreted. Therefore, in order to finally enter the intestine is an important characteristic that must also have resistance to bile juice. Therefore, a bile resistance test was performed on Pediococcus pentosakeus strain F142-811. In addition, since the Pediococcus pentosakeus strain F142-811 reaches the intestine and must be present in the intestine for a long time in order to function as a probiotic microorganism, a test for intestinal adhesion was performed.

3-1. Acid resistance  And My bile

The acid resistance and bile resistance test of the Pediococcus pentosakeus strain F142-811 of Example 1 was recovered to the same amount of pH 2.0 by recovering the cells of the Pediococcus pentosakeus strain F142-811 after the main culture was completed. Controlled MRS broth and bull's bile (oxgall) was performed using a medium containing 0.5%.

Specifically, the measurement of the biliary resistance was inoculated to 2.5 × 10 ⁶ CFU / ml of the Pediococcus pentosakeus strain F142-811, which had been pre-cultured to 5 ml of MRS, and then cultured at 30 ° C. for 24 hours, and the culture solution After centrifuging 5ml to recover the Pediococcus pentosakeus strain F142-811, and then washed twice in sterile water, inoculated in each medium and incubated for 24 hours at 30 ℃, live bacteria through stepwise dilution The number was measured by the method, and the experiment was repeated three times each, and the average value of each measured value is shown in Table 5 below.

In addition, as a comparative example of the Pediococcus pentosakeus strain F142-811, the bile resistance of L. rhamnosus GG ( L. rhamnosus GG) identified as a probiotic microorganism was confirmed and compared. The viable cell number was measured by incubating the Pediococcus pentosakeus strain F142-811 and Lactobacillus rhamnosus GG three times each to reduce the error in the experiment, the average value of each measured value is shown in Table 5 below. It was. The values listed in Table 5 below represent the number of surviving, i.e., measured, log CFU / well.

Strain name No treatment Viability (pH 2.5) 0.5% oxgall treatment L. rhamnosus GG 10.045 4.991 8.875 P. pentosaceus F142-811 9.964 5.079 8.778

As shown in Table 5, Pediococcus pentosakeus strain F142-811 was superior in acid resistance and bile resistance compared to Lactobacillus rhamnosus GG. More specifically, if no treatment, pediococcus pentosakeus strain F142-811 of the present invention was confirmed to have a slightly smaller cell number than Lactobacillus rhamnosus GG, but to pH 2.0 for acid or bile resistance test When cultured using a medium containing 0.5% of the bile lysed MRS liquid medium and ox bile, it was found that the number of cells after the incubation was rather high, compared to the conventional Lactobacillus rhamnosus GG used as a probiotic. It was confirmed that the property was excellent.

3-2. Intestines Attachment

Intestinal adhesion of the Pediococcus pentosakeus strain F142-811 of Example 1 using galcose, HT-29 and galactose galHT (galactose-induced HT-29 cell line) induced by the mucus composition of the small intestine It measured using.

More specifically, after culturing the Pediococcus pentosakeus strain F142-811, it is recovered by centrifugation, and the concentration of the cultured Pediococcus pentosakeus strain F142-811 was adjusted to an OD600 value of 1.0. . 100 μl of the adjusted strain was added to each well of a 24 well plate in which gallon cells, ie, galCo induced by mucosa composition of the small intestine by Caco-2, HT-29 or galactose, were cultured. Incubated.

After incubation in the plate, washed three times with PBS, using a 0.1% TritonX 100 to remove the pancreatic cells and lactic acid at the bottom of the well plate and diluted to a certain level, the number of viable cells was measured. As a comparative example, L. rhamnosus GG (L. rhamnosus GG), which was found to have excellent intestinal adhesion, and Lactobacillus asidophilus, a comparative control (negative control), which were found to have no intestinal adhesion, were used. The viable cell count was measured by the method, and the adhesion ability was confirmed.

The results of confirming the adhesion capacity is shown in Table 6 and Table 7 and FIG. The adherent cells described in Table 6 and Table 7 below show the number of adherent cells per cell line, that is, the number of lactic acid bacteria attached per cell line. It shows the adherent cell number against the number.

Initial measurement Number of adherent cells per cell line Adhesion rate L. rhamnsosus GG 8.3243 ± 0.5255 5.6288 ± 0.2475 1.2912 ± 0.2153 L. acidophilus 7.2715 ± 0.7385 3.3880 ± 0.6684 0.0118 ± 0.0137 F142-811 8.3111 ± 0.0800 5.696 ± 0.2166 1.9666 ± 0.9181

HT-29 Gal ht Initial cell count Number of adherent cells per cell line Adhesion rate Initial cell count Number of adherent cells per cell line Adhesion rate L. rhamnsosus GG 7.9938
± 0.2744 4.8745
± 0.5702 0.0784
± 0.0268 7.9212
± 0.6000 5.4327
± 0.2739 0.4357
± 0.0741 L. acidophilus 6.7285
± 0.6630 2.3211
± 0.9482 0.0007
± 0.0011 7.1774
± 0.4524 2.1809
± 0.4752 0.0003
± 0.0000 F142-811 8.5141 ± 0.0000 5.3174 ± 0.0548 0.2735 ± 0.0333 8.2699 ± 0.0393 6.1037 ± 0.3431 3.4177 ± 0.0270

As shown in Table 6 and FIGS. 3 and 4, the adhesion rate to Caco-2 was about 1.5 times higher than that of the Pediococcus pentosakeus strain F142-811 than Lactobacillus rhamnosus GG, and galHT and HT-29 It was confirmed that Lactobacillus rhamnosus GG is 3.4 times higher and 7.8 times higher, respectively, and the adhesion in the small intestine and large intestine mucosa is remarkably excellent.]

From the above results, pediococcus pentosakeus strain F142-811, which is a strain of the present invention, is excellent in acid resistance and bile resistance, and a large amount of live bacteria can arrive in the intestine by oral ingestion, which can be attached and act in the intestine. Therefore, it was confirmed that it can be usefully used as a probiotic microorganism or a probiotic.

< Example  4> Pediocaucus Pentosakeus  Determination of immunopotentiation activity of strain F142-811

In order to determine whether the Pediococcus pentosakeus strain F142-811 available as the probiotic has immunopotentiating activity, the effect of the strain on the secretion of cytokines, more specifically IL-1β and IL-12 Confirmed. The IL-1β and IL-12 are cytokines involved in the defense against pathogenic microorganisms. In addition, in relation to the sterilizing activity of immune cells against pathogenic microorganisms, the amount of NO secretion was measured.

In the experiment, RAW264.7 cell line, a male mouse macrophage distributed by KCLB, was added to DMEM high containing 10% FBS (Fetal Bovine Serum), 1 mM sodkum pyruvate, 100 IU penicillin / streptomycin, and 10 mM HEPES. In the glucose (Hyclone, USA) was changed to medium of the same composition every 2 days under conditions of 37 ℃ and 5% CO ₂ and incubated to confluent state.

The RAW264.7 cell line was dispensed at 5 X 10 ⁵ cells / mL per well in a 24 well plate (Falcon CO., USA), and then grown to 95% or more to form a monolayer, before mixing the lactic acid cells. Wash with PBS pH 7.4. The lactic acid bacteria suspension incubated in MRS liquid medium was centrifuged for 5 minutes under the condition of 12,000 rpm to recover the cells, and then washed with PBS (pH 7.4). The cells washed with PBS were suspended in RPMI medium without antibiotics so that the OD ₆₀₀ value was 1.0. 100 μl of the lactic acid bacteria suspension was mixed with 0.9 ml of antibiotic-free RPMI medium and incubated with a monolayered RAW264.7 cell line at 37 ° C. and 5% CO ₂ for 24 hours.

Only the supernatant was obtained from the culture medium. After removing the lactic acid bacteria by centrifugation for 5 minutes under conditions of 12,000 rpm to the supernatant, the supernatant was recovered. The content of cytokines secreted in the supernatant from which the cells were removed was measured using an ELISA kit (Enogen, USA), and the results are shown in FIGS. 5 to 7.

As shown in FIG. 5 to FIG. 7, the Pediococcus pentosakeus strain F142-811 is similar to Lactobacillus rhamnosus GG previously reported as a probiotic for IL-1β and IL-12 in cytokines. The secretion aspect was shown.

Korea Microbial Conservation Center (Domestic) KFCC11504P 20110214

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Claims (5)

It is separated from Kimchi, Salmonella typhimurium strain (Salmonella typhymurium ), Micrococcus strain leteus ), Bacillus subtilis strains ( Bacillus subtilis), Bacillus cereus strains (Bacillus cereus), Vibrio para Molly Tee hee Syracuse strain (Vibrio parahaemoliticus ) and Escherichia coli) Phedi O Lactococcus pento sake mouse strain has an antibacterial activity, having an accession number of KFCC 11504P having acid resistance and resistance to biliary for (Pediococcus pentosaceus KFCC 11504P). The method of claim 1,
The Pediococcus pentosakeus strains have the present-day capabilities for D-Xylose, Sucrose, Sucrose, Trehalose, Genentiobiose and D-Tagatose. Pediococcus pentosakeus strain having a 16S rRNA having a nucleotide sequence of SEQ ID NO: 3 pentosaceus KFCC 11504P). The method of claim 2,
The Phedi O Lactococcus pento sake mouse strain from interleukin immune cells -1β and interleukin -12 (Interleukin-12, IL- 12) Phedi O Lactococcus pento sake mouse strain (Pediococcus capable of promoting the secretion of pentosaceus KFCC 11504P). The antimicrobial composition comprising at least one selected from the group consisting of the strain of claim 1, the culture solution of the strain, the concentrate solution of the culture solution and the dried product of the culture solution. Probiotic active agent comprising at least one member selected from the group consisting of the strain of claim 1, the culture medium of the strain, the concentrated solution of the culture medium and the dried product of the culture solution.

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