KR20110052808A - A polysaccharide producing lactobacillus paracasei and a use thereof - Google Patents

Abstract

PURPOSE: A novel Lactobacillus sp. is provided to ensure polysaccharide production, immunity enhancement, anticancer(breast cancer, lung cancer), and antibacterial activity. CONSTITUTION: A Lactobacillus paracasei KB28(deposit number: KCAA91506P) has a polysaccharide production ability. A composition for enhancing immunity contains the Lactobacillus paracasei KB28 or culture thereof. A functional food composition for suppressing cancer cell proliferation contains the Lactobacillus paracasei KB28 or culture thereof. A pharmaceutical composition for suppressing cancer cell proliferation contains Lactobacillus paracasei KB28 or culture thereof. The Lactobacillus paracasei KB28 is cultured in a medium containing carbon source, nitrogen source, vitamin, and mineral.

Description

A novel polysaccharide producing Lactobacillus paracasei having a health functional effect and producing extracellular microsaccharides

The present invention relates to a novel microorganism of the genus Lactobacillus, because the microorganism of the present invention exhibits polysaccharide production, immunopotentiation, anticancer (breast cancer, lung cancer), antihypertensive, antimicrobial activity, Can be used in the manufacture of health functional materials and fermented products, such as immune boosters, food additives, probiotics.

The present invention relates to a novel Lactobacillus genus microorganism, and more particularly, to Lactobacillus paracasei KB28 having immuno-enhancing, anticancer and antibacterial activity derived from kimchi.

Lactic acid bacteria decompose carbohydrates and use them to make lactic acid. The lactic acid bacteria are divided into five genera, and are divided into Streptococcus, Lactobacillus, Leukonostoc, Bifidobacteria, and Pediococcus. Lactobacillus as lactic acid bacteria of milk system (milk) delbrueckii subsp. bulgaricus, Lb. acidophilus , Streptococcus thermophilus , Lactococcus lactis and the like. They have already been studied a lot in Europe. Plant-based lactic acid bacteria were Lb. plantarum , Lb. brevis , Leuconostoc Mesenteroides and Pediococcus pentosaceus are known and are known as the major lactic acid bacteria of fermented foods in East Asia including Korea.

Lactic acid bacteria produce lactic acid to maintain the pH of the intestinal acid to inhibit the reproduction of harmful bacteria, improve diarrhea and constipation, and also play a role in vitamin synthesis, anticancer action, serum cholesterol lowering. In particular, lactic acid bacteria have a specific protein that can bind strongly to the intestinal mucosa and epithelial cells to prevent the growth of harmful bacteria, help a lot of work. In addition, lactic acid bacteria are known to exhibit an immune enhancing effect, and promotes the proliferation of macrophages play a role in strengthening the cognitive ability, sterilization ability, etc. of harmful bacteria in the intestines of macrophages. (Saavedra, JM 2001. Am . J. Clin . Nutr . 73: 1147S-1151S: Sashihara, T., Sueki, N. and Ikegami, S. 2006. J Dairy Sci . 89: 2846-55.) Peptide-based antimicrobial compounds are also produced among the metabolites of lactic acid bacteria, which are called bacteriocin, and research is being conducted to improve the intestinal health and food preservation. In addition, oligosaccharides and extracellular polysaccharides produced by various sugar transfer enzymes of lactic acid bacteria are indigestible carbohydrates, which have been reported to have immune and anticancer activity as well as to support the growth of beneficial bacteria.

In recent years, studies have been actively conducted to separate lactic acid bacteria and develop the lactic acid bacteria as probiotics, food additives, suits, and the like, in order to settle the lactic acid bacteria in the intestine in the human intestine. Korean Patent No. 226495 discloses "A novel Bifidobacterium longum MK-G7 bifidus strain having its physiological activity suitable for Koreans and its use", and Korean Patent No. 158049 describes "moisturizing Bifidobacterium longgum HS90 ", which produces good amounts of viscous polysaccharides, is disclosed. In addition, Korean Patent No. 2000-316517 discloses "New Lactobacillus ashidophilus YD9904 strain having excellent acid producing ability and acid resistance and a product containing the same", and Korean Patent No. 2001-70689 discloses "acid resistance and Lactobacillus ashdophilus 30SC which has excellent anti-biliary resistance and has antibacterial ability to inhibit the growth of pathogenic microorganisms and perishable microorganisms is disclosed, and Korean Patent Application No. 2001-11797 discloses "Rotavirus and harmful microorganisms." Novel acid resistant Lactobacillus luteri probio-16 and inhibitors containing probiotic actives having inhibitory activity are disclosed.

On the other hand, the polysaccharides produced by lactic acid bacteria are involved in the physical properties of various foods, in the case of yogurt increases the viscosity and greatly affects the feeling of mouth feel in the mouth when edible. This polysaccharide is known to promote the process of settling in the gut when ingested with lactic acid bacteria, whereas the anti-cancer effect (Kitazawa, H. et al. (1998) Int. J. Food Microbiol. 40, 169-175 ), Immune enhancing effect (Hosono, A. et al. (1997) Biosci. Biotechnol. Biochem. 61, 312-316), cholesterol lowering effect (Nakajima, H. et al. (1992) J. Food Sci. 57, 1327-1329), various physiological activity functions such as enhanced macrophage activity has been reported.

In addition, the conventional method of separating polysaccharide-producing lactic acid bacteria by analyzing polysaccharides is phenol sulfuric acid method (Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC, (2005) .Anal Biochem. 339 , 69-72), but the process of completely removing monosaccharides, disaccharides, and oligosaccharides contained in the medium was time-consuming and inaccurate.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel lactic acid bacterium that produces extracellular polysaccharide and has health functional properties.

Another object of the present invention is to provide an industrial application using the same.

In order to achieve the above object, the present invention has a Lactobacillus paracasei ( Lactobacillus) having an extracellular polysaccharide production ability paracasei ) provides KB28 (Accession Number: KACC91506P).

The present invention also provides an immunoactivating composition comprising the Lactobacillus paracasei KB28 of the present invention or a culture thereof.

The present invention also provides a functional food composition for inhibiting cancer cell proliferation containing Lactobacillus paracasei KB28 of the present invention or a culture thereof.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting cancer cell proliferation containing Lactobacillus paracasei KB28 or a culture thereof.

In another aspect, the present invention provides a composition for antihypertensive blood containing Lactobacillus paracasei KB28 or a culture thereof.

In another aspect, the present invention provides a lactobacillus paracasei KB28 or a formal composition containing the culture thereof.

The present invention also provides a probiotic composition containing Lactobacillus paracasei KB28 of the present invention or a culture thereof.

The present invention also provides a feed composition containing Lactobacillus paracasei KB28 of the present invention or a culture thereof.

The present invention also provides a composition for food addition containing Lactobacillus paracasei KB28 of the present invention or a culture thereof.

In another aspect, the present invention provides a fermentation product containing Lactobacillus paracasei KB28 or a culture thereof.

In another aspect, the present invention provides a method for culturing Lactobacillus paracasei KB28, characterized in that it is cultured in a medium consisting of carbon source, nitrogen source, vitamins and minerals.

In one embodiment of the present invention, the culture conditions are preferably 30-45 ℃, 10-40 hours incubation is not limited thereto.

Hereinafter, the present invention will be described.

In order to overcome the limitations of the conventional strain selection, the present invention primarily uses strains that produce polysaccharides in large quantities using thin-film chromatography mass screening method (TLC High-through put screening). Excellent strains were selected by secondarily analyzing various health functions for the selected and selected strains.

Polysaccharide-producing microorganism selection method using thin layer chromatography in the present invention is a method of image analysis by dropping 20 samples on a thin film in a short time to develop the oligosaccharides including monosaccharides and disaccharides are separated from the development process polysaccharides remaining at the origin As it was selectively detectable, there was an advantage that it is suitable for the first selection by the high-through put method, compared to the polysaccharide analysis method using phenol sulfate.

Probiotics in the present invention are a group of microorganisms defined as probiotic organisms that have a beneficial effect on animal and human hosts. Advantageous effects include improving the microbial balance of the intestinal microflora or improving the properties of the native microflora. The beneficial effects of probiotics can occur through direct antagonistic effects on specific groups of organisms that reduce numbers, effects on their metabolism, or stimulation of immunity. Probiotics can inhibit the number of viable organisms of undesirable organisms by producing antimicrobial compounds or by competing for nutrients or attachment sites. It may also stimulate the immune system by increasing or decreasing enzyme activity to alter microbial metabolism, increase antibody levels, or increase macrophage activity.

Foods to which the composition of the invention may be added are products based on (or derived from) milk and daily industrial products (milk, dairy products, milk derivatives) and plant products (particularly fruits).

The composition of the present invention to be used as the food additive is in the ratio of 0.1 to 3.0 g per 100 g of food, more preferably in the ratio of 0.2 to 1.0 g per 100 g of food, most preferably 0.4 to 0.7 g per 100 g of food It is preferable to add to the food in the ratio of.

For example, the compositions of the present invention may be in the form of milk, yoghurt (natural or fruit-added) or other fermented milk, milk based desserts (eg chocolate, coffee or vanilla), milk based beverages, milk whey beverages, or fruits. Concentrates can be added to beverages, fruit juices (fruits, for example, apricots, peaches, pears, apples, bilberries, tropical fruits), tomato juice, carrot juice, tea (natural or peach tea) or plant-based beverages. have.

The composition of the present invention can be commercialized separately from foods that can be mixed.

As used herein, the term "anticancer" is the broadest meaning encompassing all of the prevention, treatment, alleviation and alleviation of symptoms.

In addition, the term "cancer prevention" in this specification is the broadest meaning including inhibiting any one or more of the initiation stage, promotion stage, progression stage and metastasis stage of cancer. Among them, it refers to inhibiting cancer development at the onset and promotion stage of cancer. In addition, blocking to inhibit the carcinogen from reacting with or reaching the target within the cell

prevention of cancer through blocking mechanisms. It also includes preventing cancer through a suppressing mechanism that inhibits the processes in which cells exposed to carcinogens appear as neoplasia. It also includes preventing cancer by facilitating ex vivo release of carcinogenic or precarcinogenic substances. It also includes metabolizing precancerous substances to prevent cancer.

The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention can be used alone or in combination with other pharmaceutically active compounds, as well as in suitable collections.

The pharmaceutical compositions according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, ointments, creams, external preparations, suppositories, and sterile injectable solutions. It may be used in formulated in any form suitable for pharmaceutical formulations.

In the compositions according to the present invention, the preferred dosage of the compound of the present invention depends on the subject's age, sex, weight, symptoms, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art. have. However, for the desired effect, the strain composition of the present invention is preferably administered at 0.001 to 10 g / kg body weight per day. Administration may be administered once a day or may be divided several times. In addition, the dosage may be increased or decreased depending on age, sex, weight, degree of disease, route of administration, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as parenteral, oral, and all modes of administration can be expected, for example, oral, rectal Or by intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

On the other hand, the composition according to the present invention, there is no serious toxicity and side effects can be used with confidence even for long-term use for the purpose of prevention.

On the other hand, in the case of formulating the strain of the present invention into tablets, capsules, chewing tablets, small bags, granules, powders, liquid solutions, suspensions, dispersions, emulsions, syrups for the purpose of oral administration, gum arabic, Corn starch, binders such as microcrystalline cellulose or gelatin, excipients such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sweeteners such as sucrose or saccharin and Flavoring agents such as peppermint, methyl salicylate or fruit flavors may be included, and when the dosage unit form is a capsule, liquid carriers such as polyethylene glycol or fatty oil may be included in addition to the above components.

The present invention also provides a health functional food composition comprising the strain of the present invention and a food acceptable additive. The food composition according to the present invention may be, for example, a variety of food products such as dairy products, chewing gum, candy, ice cream, confectionery, beverage products such as soft drinks, mineral water, alcoholic beverages, and health functional foods including vitamins and minerals. have.

At this time, the amount of the strain of the present invention in the food may be added at 0.001 to 99.9% by weight of the total food weight, in the beverage in a ratio of 0.001 to 0.1 g, more preferably 0.05 to 0.1 g based on 100 ml Can be added.

The beverage containing the strain of the present invention is not particularly limited to other ingredients except for containing the strain as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, such as ordinary drinks. .

In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the functional food compositions of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As the culturing method of the strain of the present invention, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, reactor type, or the like can be used.

The carbon source is required for the growth of microorganisms, and for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid can be used.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as those derived from natural products such as peptone, meat juice, yeast extract, malt extract, caseinate and corn steep liquor.

Moreover, as an inorganic substance, 1 potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. are mentioned, for example. You may add antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin, to a culture liquid.

As can be seen from the above description and the following examples, the microorganism of the present invention exhibits polysaccharide production, immunopotentiation, anticancer (breast cancer, lung cancer), antihypertensive, and antimicrobial activity. In addition, it can be variously used in the manufacture of health functional materials and fermented products such as immune enhancers, food additives, probiotics.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  One: Extracellular  Selection of lactobacillus produced in large amount of sugar

<1-1> Extracellular Polysaccharide Producing Lactic Acid Bacteria

Fermentation broth consisting of various fruits and vegetables was prepared and used as a sample. This sterile physiological saline (0.85% (w / v) ) to 10 ^{-1, -3, -5, -} diluted to ^7, then, spread on BL agar, MRS agar and incubated aerobically and anaerobically at 37 ℃. First, extracellular polysaccharide-producing bacteria were selected for the colonies grown.

Analysis of the production of exopolysaccharides was performed by chemically defined medium [CDM, 25 g casamino acid per liter, 2 g K ₂ HPO ₄ , 1 g Tween 80, 2 g ammonium citrate, added Casamino acid, glucose, sucrose and lactose, etc. 20 g glucose (G medium), or 20 g lactose (S medium) or 20 g with 5 g sodium acetate, 0.1 g MgSO ₄ H ₂ O, 0.05 g MnSO ₄ H ₂ O, 0.5 g L-cysteineHCl sucrose (containing S medium)]. After inoculating the isolates with CDM and incubating for 48 hours at 37 ° C., the cells and the media were separated by centrifugation, and the extracellular free extracellular polysaccharides were separated from the media fraction, and the cell binding and extracellular polysaccharide contents were precipitated. Was analyzed. Cell-linked extracellular polysaccharide was analyzed for polysaccharide content by thin layer chromatography by dissolving the precipitated cells in physiological saline after crushing.

<1-2> Analysis of Extracellular Polysaccharide Production Using Thin Film Chromatography

The free extracellular polysaccharide fraction and the cell binding extracellular polysaccharide fraction obtained above were analyzed using thin layer chromatography. Each fraction (1 μl) was added to thin layer chromatography (Whatman K6F) and developed with acetonitrile / water (85:15) solution, followed by color development solution (into 0.3% (w / v) N- (1- Naphthyl) -ethylenediamine, 5% (v / v) H ₂ SO ₄ in MeOH) was used to develop the sugar content. The high-throughput extracellular polysaccharide-producing high-throughput polysaccharide produced by TLC analysis was isolated from the polysaccharide fraction by the same culture method, followed by precipitating the polysaccharide by ethanol precipitation and measuring the yield after drying. Polysaccharide-producing microorganism selection method using thin layer chromatography according to the present invention is a method of dropping 20 samples in a thin film and dozens of thin films at the same time in a short time in the chamber image separation method oligosaccharides including monosaccharides, disaccharides are separated in the development process As a result, the polysaccharide remaining at the origin can be selectively detected, which is suitable for the primary selection by the high-through put method compared to the polysaccharide analysis method using phenol sulfate.

As a result, KB28 strain was selected as an excellent strain. This strain produced a total of 318.1 mg / L by producing 42.9 mg / L of cell-bound extracellular polysaccharides and 275.3 mg / L of free extracellular polysaccharides when cultured in glucose medium, as shown in FIG. Extracellular polysaccharides of 46.3, 368.3, and 414.6 mg / L were generated, and 25.6, 114.6, and 140.1 mg / L of extracellular polysaccharide were produced when cultured in lactose medium (FIG. 1).

<1-3> Extracellular Polysaccharide Composition Analysis

The polysaccharide fraction purified by ethanol precipitation and dialysis of the isolated extracellular polysaccharide was added, followed by addition of 2M trifluoroacetic acid (TFA), heating at 121 for 1.5 hours, followed by hydrolysis to obtain the alditol acetate method using gas chromatography. Jones and Albersheim, 1972). As a result, the constituent sugars of KB28-produced extracellular polysaccharides were Glc (68.7 mol.%), Gal (16.9 mol.%), Man (5.5 mol.%), Rha (2.1 mol.%), Ara (3.2 mol.%) And Consists of Xyl (3.6 mol.%).

Example  2: Lactobacillus  Isolation and Identification of Genus Microorganisms

(1) Lactobacillus isolation using selective medium

Fermentation broth consisting of various fruits and vegetables was prepared and used as a sample. This sterile physiological saline (0.85% (w / v) ) to 10 ^{-1, -3, -5, -} diluted to ^7, then, spread on BL agar, MRS agar and incubated aerobically and anaerobically at 37 ℃. Morphological observation of the cultured colony was performed, and cell types were distinguished by optical microscopy (× 1000). In addition, biochemical characteristics were confirmed through catalase, oxidase test, 15 ° C, 45 ° C culture, and gram staining test.

(2) Classification using SDS-PAGE Analysis

After 2 × sample dye buffer was added to the pure strain isolated from the selection medium, the sonicator (homogenizer / Stirrer-Sonicator GE 130PB, Sonics and Materials Inc., Newtown, CT, USA) was treated for 2 minutes to elute the protein. Heat for 5 min at 100 ° C. and centrifuge for 5 min at 13,000 rpm for protein denaturation. The supernatant was analyzed at 140 V for 90 minutes using an electrophoresis device (Bio-Rad, Hercules, CA, USA) using a 10% polyacrylamide separating gel (Dongin Bio., Korea). Whole cell protein pattern by staining with 0.25% Coomassie brilliant blue R-250 (Sigma, St. Louis, MO, USA) for 30 minutes, destaining with destaining solution and scanning with high-performance camera (Olympus, Tokyo, Japan) Analyzed. Protein pattern analysis was performed by visually observing the clarity, width, position and number of the bands. Broad range protein molecular markers (Promega, Madison, Wi., USA) and commercial strains were compared and analyzed.

(3) 16S rRNA Sequencing Analysis

Universal bacterial primer pairs for PCR amplification of the 16S rRNA gene (ca. 1500 bp product); 27F (5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 1) and 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2)) were used. 16s rRNA gene analysis was commissioned by SolGent Co. Ltd. (Daejon, Korea) The results were analyzed using DNA Data Bank of Japan (.ddbj.nig.ac.jp).

As a result, the selected strain showed 99.9% homology with Lactobacillus paracasei. Therefore, the strain was named Lactobacillus paracasei KB28, and was deposited in Lactobacillus paracasei KB28 (Accession Number KACC91506P) on November 09, 2009 at the National Institute of Agricultural Science, National Institute of Agricultural Science, Suwon, Korea.

Characteristics Contents Growth temperature 15 ℃, 37 ℃, 45 ℃ Gram stainning positivity Catalase test voice Oxidase test voice

Table 1 is a table showing the characteristics of the strain of the present invention.

Example  3: KB28 of Enzyme activity  Measure

<3-1> Enzyme Activity Measurement

The activity of β-galactosidase, α-amylase, Pullulanase, Dextransucrase, and Levansucrase enzymes was measured using selective medium, respectively. β-galactosidase was inoculated and grown in X-gal-added MRS, followed by blue colony as enzyme secretion bacteria. α-amylase and Pullulanase were inoculated in MRS medium containing Starch and pullulan, respectively, followed by Iodine test. The production of transparent halo around the colony was selected as the enzyme secreting bacteria.

As a result, the Lactobacillus paracasei strain showed activity only on β-galactosidase.

Example  4: KB28 of Immune activity

1) Preparation of experimental animals

Experimental animals were used to divide the 8 weeks old BALB / c male mice in Orient (Korea) divided into control and experimental groups. Solid feed and water were supplied for 12 hours of day and night rhythms, with unlimited supply, and adapted for 1 week under constant temperature and humidity.

2) Preparation of Lactic Acid Bacteria

Lactic acid bacteria are inoculated in MRS medium and incubated for 48 hours at 37 ° C. It is washed twice with sterile physiological saline. Distilled water was added thereto, followed by heat treatment at 100 ° C. for 30 minutes using an autoclave and freeze-drying. Before the heat treatment, some samples are taken and the number of live bacteria is measured to determine the number of lactic acid bacteria per gram.

3) Preparation of Splenocyte Suspension for IFN-γ Measurement and Cultivation of Splenocytes

The spleens of the mice were extracted and washed with RPMI-1640 (Invitrogen, USA) containing 10% fetal bovine serum (FBS). The spleen was crushed with a micro slide glass and filtered with a 0.40 μm nylon cell strainer. After centrifugation at 1000 rpm for 10 minutes, erythrocytes were destroyed with erythrocyte lysis buffer. After centrifugation twice, splenocytes were resuspended in 10% FBS RPMI-1640. Measure the concentration of spleen cells through phase contrast microscopy. The resuspended splenocytes in a 48 well plate were seeded (500 uL) at a concentration of 1 × 10 ⁶ cells / ml, and then inoculated with lactic acid bacteria and incubated in a 37 ° C. 5% CO ₂ incubator for 48 hours. After incubation, the supernatant was centrifuged at 1000 rpm for 10 minutes and used as a sample while being stored at -20 ° C until analysis.

* 500 uL seeding solution: For example (300 uL: Spleen cell suspension + 200 uL: lactic acid bacteria suspension), each prepared and mixed to 500 uL.

* Lactobacillus concentration: Prepare 3 points of the same concentration (1 × 10 ⁶ ), 10 times less concentration (1 × 10 ⁵ ) and 10 times more concentration (1 × 10 ⁷ ) as Spleen cells per well.

4) Cytokine measurement method (ELISA method and Quantative real time RT-PCR)

Measurement of interferon-γ (IFN-γ) was performed using the OPT EIA set (Pharmingen). Each well of a 96 well plate was coated with capture antibody at 4 ° C. overnight. 200 μl / well of PBS containing 10% FBS was added and blocked at room temperature for 1 hour. After washing three times to completely remove the blocking buffer, the standard IFN- [gamma] and the sample were diluted appropriately, 100 μl were dispensed and left at room temperature for 2 hours. After 5 washes, 100 μl of biotinylated detection antibody (IFA-γ, Pharmingen, USA) and avidin (avidin) for IFN-γ were dispensed and left at room temperature for 1 hour. After washing seven times, 100 μl of TMB substrate reagent was added, and after 30 minutes, 50 μl of 1M H ₂ SO ₄ was added. Optical density (OD) was measured at 450-570 nm with a microplate reader. Cytokines of TNF-α, IL-6, and IL-12 were measured by Quantative Real Time RT-PCR and expressed relative to GAPDH expression levels.

As a result, in the case of INF-γ, only lactic acid bacteria showed high secretion capacity of 10 pg / mL, and lactic acid bacteria and extracellular polysaccharide complexes were 2500 pg / mL.

INF-γ is a cytokine that activates Th2 (cellular immune response), and antagonistically reduces Th1 (antibody immune response) to balance to inhibit IgE production. In addition, the results of examining the activity of various inflammatory cytokine showed a tendency to increase in a concentration-dependent manner.

Example  5: KB28 of Anticancer activity

Microorganisms and extracellular polysaccharides cultured on MRS agar plate containing 2% sucrose, glucose and lactose were heat treated and then destroyed using an ultrasonic crusher. The samples were lyophilized and stored at -80 ° C. The anticancer effect of the cytoplasmic fraction of lactic acid bacteria was measured by MTT method against cancer cell lines of H460 (lung cancer cell line) and MCF-7 (breast cancer cell line).

As a result, it showed high anticancer activity in a concentration-dependent manner and showed more than 90% anticancer activity at a concentration of 40 ug / mL for both cancer cells.

Example  6: KB28 of Antihypertensive ability

ACE inhibition was measured by Hman and Cheng.

Microorganisms and extracellular polysaccharides cultured on MRS agar plate containing 2% sucrose, glucose and lactose were heat-treated and destroyed using an ultrasonic crusher, and then lyophilized and stored at -80 ° C.

100 μl of substrate 5 mM hippuryl-histidyl-leucine solution, 80 μl of ACE (0.2 unit / ml) solution and 100 μl of inhibitor solution were mixed with 0.1 M potassium phosphate buffer (pH 8.3) containing 0.3 M NaCl. 100 μl of distilled water was added and reacted at 37 ° C. for 30 minutes. 250 μl of 1N HCl was added to stop the reaction, followed by addition of 1.25 mL of ethyl acetate (Ethylacetate).

After vortexing and centrifugation, 1 ml of ethyl acetate layer was taken and volatilized. Then, 1 ml of distilled water was added to measure the absorbance of hydroxy acid separated from the substrate by enzyme at 280 nm. ACE inhibition was calculated by the following equation.

ACE inhibition (%) = (absorbance of 1-sample / absorbance of control) × 100

As a result, the control (medium) showed a inhibition rate of 10%, whereas in the case of Lactobacillus paracasei KB28 showed a inhibition rate of 77%.

Example  7: KB28 Antimicrobial effect

Escherichia coli KCTC1476, Salmonella typhimurium KCTC2515, Listeria monocytogenes ATCC19115 and Staphylococcus The antibacterial effect of aureus KCTC1916 against food poisoning bacteria was measured by Agar spot assay.

As a result. KB28 strain Lactobacillus paracasei showed antimicrobial effect against four food poisoning bacteria.

1 is a table measuring the extracellular polysaccharide production performance. EPS production analysis was performed using a chemically defined medium containing glucose, sucrose and lactose.

2 shows Lb in mouse macrophages . It is a graph showing the mRNA expression of TNF-a, IL-6 and IL-12 by paracasei (LP). The mRNA expression was measured by real time RT-PCR. (A) shows TNF-a, (B) shows IL-6, and (C) shows mRNA expression of IL-12. Data is mean ± standard deviation of three independent experiments.

3 is Lb of the present invention . stimulated with paracasei It is a graph showing IL-6 production of mouse macrophages. Hayeoseo pooling the mouse macrophage cell a 2 ug / mL, 20 ug / mL and 200 ug / mL of pre-treated Lb. Incubated with paracasei . IL-6 levels were measured by ELISA after 48 hours of incubation of pretreated cells. (A) untreated, (B) heat treated, sonicate and UV-treated cells. Data is mean ± standard deviation of three independent experiments.

4 Lb of the present invention. stimulated with paracasei It is a graph showing IL-12 production of mouse splenocytes. Hayeoseo pooled mouse spleen cells in the cell of 2 ug / mL, 20 ug / mL and 200 ug / mL of the pre-treatment Lb. Incubated with paracasei . IL-12 levels were measured by ELISA after 48 hours of incubation of pretreated cells. (A) untreated, (B) heat treated, sonicate and UV-treated cells. Data is mean ± standard deviation of three independent experiments.

5 is a graph showing anticancer activity against lung cancer, and b is breast cancer.

6 is a table showing that the Lactobacillus paracasei KB28 strain has an antimicrobial effect against four food poisoning bacteria.

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Claims (12)

Lactobacillus paracasei KB28 (accession number: KACC91506P) having extracellular polysaccharide production. An immunoactivating composition comprising the Lactobacillus paracasei KB28 of claim 1 or a culture thereof. A functional food composition for inhibiting cancer cell proliferation comprising Lactobacillus paracasei KB28 of claim 1 or a culture thereof. The pharmaceutical composition for inhibiting cancer cell proliferation containing lactobacillus paracasei KB28 of claim 1 or a culture thereof. The anti-hypertensive composition containing Lactobacillus paracasei KB28 of Claim 1 or its culture. Claim 1 lactobacillus paracasei KB28 or a formal composition containing the culture thereof. A probiotic composition comprising the Lactobacillus paracasei KB28 of claim 1 or a culture thereof. A feed composition comprising the Lactobacillus paracasei KB28 of claim 1 or a culture thereof. A food additive composition comprising the Lactobacillus paracasei KB28 of claim 1 or a culture thereof. A fermentation product comprising the Lactobacillus paracasei KB28 of claim 1 or a culture thereof. The method of culturing Lactobacillus paracasei KB28 according to claim 1, wherein the Lactobacillus paracasei KB28 is cultured in a medium consisting of a carbon source, a nitrogen source, vitamins and minerals. The method for culturing Lactobacillus paracasei KB28 according to claim 11, wherein the culture conditions are 30-45 ° C., or 10-40 hours or a combination thereof.

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