KR20160082583A - The Functional Health Drink For Improving The Intestine Function and Environment and Enhencing Immunity - Google Patents

Abstract

The present invention relates to a nano-type kimchi lactic acid bacteria, and more specifically, to L. plantarum, which is isolated from kimchi and effective at improving intestinal health. According to the present invention, the nano-type kimchi lactic acid bacteria serves to suppress putrefactive microorganisms inside the intestines and enhances the immune function, thereby improving intestinal health, and preventing and treating colonic diseases including colitis and colon cancer. Also, the uniform size of constituent particles of the present lactic acid bacteria serves to facilitate absorption inside the intestines, and thus the degree to which intestinal health improves is proportional to the increase in concentration of the lactic acid bacteria. Therefore, the present nano-type kimchi lactic acid bacteria can serve as a food additive that is capable of imparting the benefit of lactic acid bacteria to a food product without altering the taste and aroma of the food product, and in which the present lactic acid bacteria is highly heat-resistant and thus can be added to thermally processed food products while maintaining the benefits thereof.

Description

The Functional Health Drink For Improving The Intestine Function and Environment and Enhencing Immunity}

The present invention relates to nano-type kimchi lactic acid bacteria, and more specifically, to a Lactobacillus plantarum (L. plantarum) lactic acid bacteria isolated from kimchi having excellent intestinal environment improvement effect.

Lactic acid bacteria (lactic acid bacteria, lactobacillus) are bacteria that produce lactic acid by decomposing sugars such as glucose, and are also called lactic acid bacteria, and have the property of inhibiting the growth of pathogens and harmful bacteria by lactic acid produced by lactic acid fermentation. It is used in food manufacturing such as brewed food. In addition, lactic acid bacteria live in the intestines of mammals and are used as intestinal agents by preventing abnormal fermentation by various bacteria. The lactic acid bacteria are Gram-positive bacteria, and have an anaerobic or anaerobic property, and several types are known in the genus Lactobacillus and the genus Streptococcus.

Kimchi is a Korean traditional food fermented by lactic acid bacteria, and the lactic acid bacteria classified in kimchi include Leukonostoc mesenteroides, Leukonostoc dextranicum, Lactobacillus brevis, Lactobacillus plantarum, Pediococcus pentosacues, and the like.

Patents related to lactic acid bacteria isolated from kimchi include Lactococcus lactis BH5 (Korean Patent No. 413335), which produces antibacterial peptide substances, Lactobacillus Plantarum NC (Korean Patent No. 771209), which has excellent antioxidant activity, and Bactero. There are lactic acid strains that produce gods (Korean Patent No. 1068531).

However, the conventionally disclosed kimchi lactic acid bacteria are all related to probiotics, and when ingested as food, the number of viable bacteria in the intestinal environment rapidly decreases, and there is a problem that the intrinsic effect is inferior because it is not absorbed in the intestine. Since strains that can grow in the culture medium are limited, there is a problem that the daily intake is limited.

Accordingly, an object of the present invention is to solve the above problems by identifying the lactic acid bacteria having the most excellent effect of improving the intestinal environment among lactic acid bacteria isolated from kimchi, and providing them as nano-type kimchi lactic acid bacteria.

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

In addition, the present invention provides a food additive comprising the Lactobacillus plantarum strain or a culture supernatant thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of colon diseases comprising the Lactobacillus plantarum strain or a culture supernatant thereof as an active ingredient.

In addition, the present invention provides a method of producing a nano-type kimchi lactic acid bacteria from the Lactobacillus plantarum strain.

In addition, the present invention provides a nano-type kimchi lactic acid bacteria prepared with the Lactobacillus plantarum strain.

In addition, the present invention provides a food additive or fermented food containing the nano-type kimchi lactic acid bacteria.

In addition, the present invention provides a pharmaceutical composition for preventing and treating colon diseases including the nano-type kimchi lactic acid bacteria.

In addition, the present invention provides a health functional food composition for preventing and treating colon diseases including the nano-type kimchi lactic acid bacteria.

The nano-type kimchi lactic acid bacteria according to the present invention has an inhibitory effect of intestinal decay bacteria and an effect of improving the intestinal environment through activation of immune functions, and has excellent preventive and therapeutic effects of colon diseases including colitis and colon cancer. In addition, since it is composed of uniform particles to facilitate absorption in the intestine, the effect of improving the intestinal environment increases proportionally depending on the concentration.

In addition, the nano-type kimchi lactic acid bacteria according to the present invention is a food additive that does not harm the natural taste and aroma of food, and can provide the effect of the lactic acid bacteria as it is. There are features that are maintained.

1 is a (a) colony observation image and (b) Gram staining result image of the kimchi lactic acid bacteria of the present invention.
Figure 2 is a molecular tree diagram showing the analysis of the 16S rRNA sequence of the kimchi lactic acid bacteria (SIID11558) of the present invention (the lower left line is the scale bar, the number located at the branch of the phylogeny is the bootstrap value, and T at the end of the main name is the species It represents the type strain of.)
Figure 3 is a manufacturing process chart of the nano-type kimchi lactic acid bacteria nF1 of the present invention.
Figure 4 is a microscopic photograph of the kimchi lactic acid bacteria of the present invention observed before and after nano-processing. (a) Before nano-processing: rod shape, (b) After nano-processing: spherical.
5 is a result of measuring the weight change of the experimental model of the present invention.
6(a) is a graph showing the change in the length of the colon of the colitis-induced mice treated with nF1, and (b) is a graph showing the change in the length of the colon of the colitis-induced mice to which kimchi to which nF1 powder was added. (a~d mean significant difference (p <0.05) according to Duncan's multiple range tests)
7 is a photograph showing the results of pathological observation in colon tissue.
8 is a graph showing changes in the expression level of inflammatory cytokines in colitis-induced mice treated with nF1. (a~d mean significant difference (p <0.05) according to Duncan's multiple range tests)
9 is a graph showing changes in the expression level of inflammatory cytokines in colitis-induced mice administered kimchi to which nF1 powder was added. (a~d mean significant difference (p <0.05) according to Duncan's multiple range tests)
10 is a graph showing changes in the expression level of inflammatory cytokine mRNA in colitis-induced mice treated with nF1. (a~d mean significant difference (p <0.05) according to Duncan's multiple range tests)
11 is a graph showing changes in the expression level of inflammatory cytokine mRNA in colitis-induced mice administered kimchi to which nF1 powder was added. (a~d mean significant difference (p <0.05) according to Duncan's multiple range tests)
12 is a graph showing the results of measuring the mRNA expression level of inflammation-related genes (iNOS, COX-2) in the colon of the mouse.
13 is a graph showing the results of measuring the number of intestinal lactic acid bacteria in a colitis-induced mouse.
14 is a graph showing the results of measuring the intestinal harmful enzyme activity of colitis-induced mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Lactobacillus plantarum strain isolated from kimchi.

The present inventors have been found that by separating the lactic acid bacilli from Kimchi intestinal environment unique effect that was selected the best microorganism is a result Gram-positive bacillus and forms of Lactobacillus Planta room (Lactobacillusplantarum) identified them. Shin Kyunju was deposited as "nF1" on November 8, 2012 to the National Institute of Technology and Evaluation (NITE; International Patent Organism Depositary, IPOD), a Japanese independent administrative corporation (NITE P-1462). It was confirmed that this strain has an inhibitory effect of intestinal spoilage bacteria and an effect of improving the intestinal environment through activation of immune function, and has the effect of preventing and treating colon diseases including colitis and colon cancer.

In addition, the present invention provides a food additive comprising the Lactobacillus plantarum strain or a culture supernatant thereof as an active ingredient.

When the kimchi lactic acid bacteria or the culture supernatant thereof of the present invention is used as a food additive, the kimchi-derived lactic acid bacteria may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). There is no particular limitation on the type of the food. Examples of foods to which the Lactobacillus plantarum strain of the present invention or the culture supernatant thereof can be added include meat, sausage, bread, chocolate, candy, confectionery, snacks, pizza, ramen, other noodles, gums, ice cream. It may include dairy products, various soups, beverages, teas, drinks, health drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the usual sense.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of colon diseases comprising the Lactobacillus plantarum strain or a culture supernatant thereof as an active ingredient.

The colon disease may include colitis or colon cancer, but is not limited thereto.

The Lactobacillus plantarum strain or its culture supernatant of the present invention can be administered orally or parenterally in the pharmaceutical composition for the prevention and treatment of colon disease, which is included as an active ingredient, and is the main component, the Lactobacillus plantarum strain. Alternatively, one or two or more pharmaceutically acceptable conventional carriers or additives may be selected in an effective amount of the culture supernatant thereof to prepare a composition of a conventional pharmaceutical formulation.

The carrier can be used by selecting one or two or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of flavors, vitamins, and antioxidants can be selected as additives. Can be used.

In the present invention, all pharmaceutically acceptable carriers and additives can be used, and specifically, lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol is good as a diluent, and magnesium stearate or talc is preferable as a lubricant. And, the binder is preferably selected from polyvinylpyrrolidone (PVP) or hydroxypropyl cellulose (HPC). In addition, the disintegrant is preferably selected from calcium carboxymethylcellulose (Ca-CMC), sodium starch glycolate, potassium polyacrylic or crospovidone, and the sweetener is selected from sucrose, fructose, sorbitol or aspartame, and is stable. Zero is selected from sodium carboxymethylcellulose (Ma-CMC), beta-cyclodextrin, white lead or xanthan gum, and as the preservative, it is preferable to select from methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate.

The pharmaceutical composition of the present invention is used for the purpose of efficiently treating or preventing colon diseases, and is not particularly limited in its formulation, and can be used as a beverage, powder, capsule, soft capsule, tablet, gum, adhesive type liquid composition. In addition, it can be used in transdermal dosage forms such as lotions, ointments, gels, creams, patches or sprays, and can be used as preparations such as baths, pills, tablets, powders, and granules.

In the pharmaceutical composition of the present invention, in addition to the above main ingredients, as auxiliary ingredients, mineral-containing compounds such as vitamins B, C, E, beta-carotene, Ca, Mg, Zn, phospholipids such as lecithin, or compounds such as maltol, oligosaccharides, amino acids, etc. can be used. Among these, a mixture of two to three components among vitamins C and E, beta-carotene, maltol, etc. is more preferable because it can reinforce the bioactive effect.

In addition, in addition to the above ingredients, natural flavors such as plum, lemon, pineapple or herbal flavors, natural fruit juices, and natural pigments such as chlorophyllin and flavonoids, as well as sweetening components, in order to enhance taste as well-known additives. , Honey, sugar alcohol, sugar, etc. can be used by mixing acidulants such as citric acid and sodium citrate.

In addition, the present invention provides a method of producing a nano-type kimchi lactic acid bacteria from the Lactobacillus plantarum strain.

In addition, the present invention provides a nano-type kimchi lactic acid bacteria prepared with the Lactobacillus plantarum strain.

The microbial cells of the nano-type kimchi lactic acid bacteria according to the present invention have a mode (particle size) of 1.0 µm or less in particle size distribution, and preferably 0.5 to 1.0 µm.

In the present invention, the "mode value in particle size distribution" is a value that serves as an index indicating the size of the bacteria, and refers to the particle diameter at which the relative frequency in the particle size distribution when the particle diameter of the cells is measured is the maximum.

The shape of the cells of the nano-type kimchi lactic acid bacteria of the present invention can be either live or dead, but in the case of live bacteria, since there is a possibility of causing a change in shape during delivery or display after product manufacture, it is a dead bacteria that does not cause any further change in shape. desirable.

It is known that lactic acid bacteria change their shape due to the stress when the growing environment during cultivation becomes poor. Therefore, in the present invention, by controlling the culture and processing conditions, the lactic acid bacteria are grown while maintaining the morphology of the lactic acid bacteria to be constant, thereby producing a nano-type kimchi lactic acid bacteria having a mode in the particle size distribution described above.

In addition, the "nano-type kimchi lactic acid bacteria cells" of the present invention is preferably dispersed.

The method of the dispersion treatment is not particularly limited, for example, a method of dispersing the culture medium of bacteria in a wet manner with a high-pressure homogenizer of about ^{150 kgf/cm 2 (1.5 MPa).}

In this case, it is preferable to add a known dispersant or excipient to the culture solution in advance, whereby the reaggregation of the cells can be efficiently prevented. The amount of the dispersant and excipient to be added varies depending on the properties of the cells, but in terms of mass, the amount is preferably 1 to 100 times, more preferably 2 to 20 times, with respect to the cells. Suitable dispersants and excipients include trehalose, dextrin, skim milk, and the like.

In addition, in order to finally obtain the "nano-type kimchi lactic acid bacteria cells" of the present invention as a powder, it is recommended to add a well-known dispersant or excipient to the culture solution and perform dispersion treatment so that the cells do not re-aggregate, and then freeze-drying or spray drying. desirable. Accordingly, it is possible to obtain a microbial powder having excellent dispersibility in water.

The microbial cells of the nano-type kimchi lactic acid bacteria of the present invention described above have a particle size of 1.0 μm or less to a nanometer (nm) size.

In addition, this cell body is made into a dry powder by the above method, and even when the powder is resuspended in a physiological digestive solution, the cell particle size is maintained at 1.0 µm or less. The physiological digestive fluid means an artificial gastric juice or intestinal fluid prepared by a known method.

The concentration of the Kimchi lactic acid bacteria Lactobacillus plantarum strain per 1 g of dry powder can be adjusted according to the amount of the dispersant or excipient added during the production of the nano-type kimchi lactic acid bacteria of the present invention. The present invention is characterized in that 100 million to 5 trillion kimchi lactic acid bacteria strains are included per 1 g of nano-type kimchi lactic acid bacteria powder, and preferably 1 to 5 trillion strains per 1 g of powder may be included.

Although the microbial cells of the nano-type kimchi lactic acid bacteria of the present invention may be commercialized as they are, in general, various additives or flavors may be added and blended to enhance the flavor, or may be prepared in a required shape to make a final product.

Specifically, the present invention provides a food additive or fermented food containing the nano-type kimchi lactic acid bacteria.

Specifically, various sugars, emulsifiers, sweeteners, acidulants, fruit juices, etc. are mentioned as ingredients added to and mixed with the nano-type kimchi lactic acid bacteria of the present invention. More specifically, sugars such as glucose, sucrose, fructose, and honey; Sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, and paratinite; And emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester, and lecithin. In addition, even if various vitamins such as vitamin A, vitamin B, vitamin C, and vitamin E, herbal extracts, grain components, vegetable components, milk components, and the like are blended, a nano-type kimchi lactic acid bacteria composition having excellent flavor can be obtained.

In addition, as flavoring, yogurt-based, berry-based, orange-based, quince-based, perilla-based, citrus-based, apple-based, mint-based, grape-based, pear, custard cream, peach, melon, banana, tropical, herbal, black tea, coffee And flavorings such as system, and these can be used alone or in combination of two or more. The amount of flavoring added is not particularly limited, but from the viewpoint of flavor, it is preferably about 0.05 to 0.5% by mass, particularly about 0.1 to 0.3% by mass, in the cells.

In addition, the present invention provides a pharmaceutical composition and a health functional food composition for preventing and treating colon diseases, including the nano-type kimchi lactic acid bacteria.

The cells of the nano-type kimchi lactic acid bacteria of the present invention described above may be made into any type of product, such as solid or liquid. Specifically, it is commercialized in various forms such as beverages, granules, tablets, capsules, etc. by a known method in the field of pharmaceutical or food production together with pharmaceutically acceptable salts, excipients, preservatives, coloring agents, flavoring agents, etc. can do.

In addition, the nano-type kimchi lactic acid bacteria cells of the present invention can be used as a food composition, in particular can be used in health food. Health food is more active than ordinary food, and refers to food for the purpose of health, maintenance and promotion of health, etc. The form may be liquid, semi-solid, or solid, and specifically, confectionery such as cookies, rice crackers, jelly, yokan, confectionery, bread, and cake; Beverages such as yogurt, soft drinks, nutritional drinks, sikhye, and fruit drinks; Soup, ice cream, milk powder, kimchi, ramen, etc. are mentioned.

In addition, the bacteria of the nano-type kimchi lactic acid bacteria of the present invention are lotion (toilet), cosmetic creams, emulsion, lotion, pack, skin milk (emulsion), gel, powder, lip cream, lipstick, under makeup, foundation, sun care, It can also be used for external skin preparations in various product forms, such as bath products, body shampoos, body rinses, soaps, cleansing foams, ointments, patches, jelly, and aerosols.

In addition, in the microbial cells of the nano-type kimchi lactic acid bacteria of the present invention, various ingredients or additives commonly used in cosmetics, quasi-drugs, and pharmaceuticals, such as those exemplified below, can be suitably blended as needed.

Specifically, moisturizing agents such as glycerin, petrolatum, urea, hyaluronic acid, and heparin; PABA derivatives (paraaminobenzoic acid, Escalol 507 (ISB Japan), etc.), cinnamic acid derivatives (Neo Heliopan, Parsol MCX (DSM Nutrition Japan, Ltd.), Sunguard B (Shiseido, Ltd.), etc. ), salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S ((Y) Shonan Chemical Service), etc.), dibenzoylmethane derivatives (parsol A, parsol DAM (DSM Nutrition Japan) (Note), etc.), heterocyclic derivatives (such as tinuvin), and ultraviolet absorbers and scattering agents such as titanium oxide; disodium edetate, trisodium edetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, Metal blockade agents, such as sodium polyphosphate, sodium metaphosphate, and gluconic acid; Sebum inhibitors such as salicylic acid, sulfur, caffeine, and tannin; sterilizing and disinfecting agents such as benzalkonium chloride, benzethonium chloride, and chlorohexidine gluconate; difenic acid hydrochloride Anti-inflammatory agents such as hydramine, tranexamic acid, guaazulene, azulene, allantoin, hinokitiol, glysarylic acid and its salts, glysarylic acid derivatives, and glycyletic acid; vitamin A, vitamin B group (B1, B2) , B6, B12, B15), folic acid, nicotinic acid, pantothenic acid, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, ubiquinone, vitamin K (K1, K2, K3, K4), etc. Vitamins; amino acids such as aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, pyrrolidonecarboxylic acid, and derivatives thereof; retinol, tocopherol acetate, magnesium ascorbate , Ascorbic acid glucoside, arbutin, kojic acid, ellagic acid, placenta extract, and other whitening agents; butylhydroxytoluene, butylhydroxyanisole, and propyl gallate; antioxidants such as zinc chloride, zinc sulfate, zinc phenol, zinc oxide, Astringents such as potassium aluminum sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, and lactitol; licorice, chamomile, maroni, pan's ear, peony, quince, gold, Various formulas such as Hwangbyeok tree, Hwangryeon, Sambaekcho, Ginkgo leaf In addition to water extracts and the like, oily components, surfactants, thickeners, alcohols, powder components, pigments, and the like can be appropriately blended.

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

However, it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the present invention and the scope of the technical idea only to illustrate the present invention, and it is natural that such modifications and modifications belong to the appended claims. will be.

And was the lowest in the nF1-H group (p <0.05). The expression levels of TNF-α, IL-1β, and IL-12 were not significant between the nF1-H and nF1-M groups, but were significantly decreased compared to the nF1-L group. The expression levels of nF1-H, nF1-M, and nF1-L were all significantly decreased compared to the control group, and the expression level of the nF1-H group was the least. In summary, it was found that the expression of cytokines decreased in the group administered with nF1 compared to the control group. In particular, the higher the concentration was, the lower the cytokine expression level was. Therefore, it was found that the effect of preventing colitis was highest when nF1 was ingested at a high concentration.

In addition, as shown in Figure 9, in the case of the mice administered kimchi, it was confirmed that the nF1-Kimchi group had significantly lower cytokine expression levels than in the N-Kimchi group (p <0.05), thus It could be confirmed that adding nF1 to kimchi increases the effect of preventing colitis.

3-5) Measurement of mRNA expression level of inflammatory cytokines in tissues

After washing the colon tissue of the experimental mouse with PBS, total RNA (total RNA) was isolated using trizol. Ss cDNA complementary to mRNA in 2 μg of RNA using oligo dT primer and AMV reverse transcriptase after quantifying the isolated RNA to make all mRNAs of the isolated RNA into cDNA. Was reverse transcribed. Using this cDNA as a template, amplify specific gene sites of TNF-α, IFN-γ, IL-6, IL-12, IL-17A, COX-2, and iNOS genes by PCR (polymerase chain reaction) method. I did. In this case, GAPDH, a housekeeping gene, was used as an internal control. Each PCR product was electrophoresed using a 2% agarose gel, stained with ethidium bromide (EtBr, Sigma, USA), and then confirmed under UV. The results of confirming the mRNA expression level of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, etc.) in the colon tissue through RT-PCR are shown in FIGS. 10 and 11.

According to Figures 10 and 11, the expression of TNF-α, IL-1β, IFN-γ, IL-6 and IL-17A was significantly increased in the control group than in the normal group (p <0.05), from which colitis was induced. It was found that the mRNA expression level of inflammatory cytokines increased.

On the other hand, in the case of the nF1 administration group, as shown in FIG. 10, the higher the concentration, the significantly decreased the mRNA expression levels of TNF-α, IFN-γ, and IL-6 (p <0.05). The mRNA expression levels of IL-1β and IL-17A were not significantly different between nF1-H and nF1-M, but were all lower than nF1-L (p <0.05).

In the case of the kimchi-administered group, as shown in FIG. 11, the inflammatory cytokine mRNA expression level of the F1-Kimchi group was significantly lower than that of the N-Kimchi group (p <0.05).

Therefore, it was found that the effect of preventing colitis was highest when nF1 was ingested at a high concentration, and it was confirmed that the effect of preventing colitis was significantly increased when nF1 was added to kimchi.

3-6) Measurement of mRNA expression level of inflammation-related genes (iNOS, COX-2)

The mRNA expression levels of the inflammation-related genes iNOS and COX-2 in the colon tissue were confirmed through RT-PCR, and the results are shown in FIG. 12, and the expression levels of iNOS and COX-2 were also described in Examples 3-4 and 3- It showed a similar pattern to the inflammatory cytokines of 5.

As shown in FIG. 12, the expression levels of iNOS and COX-2 in the control group were higher than that of the normal group, and it was confirmed that the expression levels of iNOS and COX-2 were significantly increased as ulcerative colitis was induced (p < 0.05).

On the other hand, in the case of the nF1 administration group, the higher the nF1 administration concentration, the lower the expression level, and the lower the administration concentration, the higher the expression level, indicating a significant difference in the expression levels of iNOS and COX-2 according to the nF1 administration concentration (p <0.05).

In addition, when comparing the nF1-Kimchi group and the N-Kimchi group, nF1-Kimchi's iNOS and COX-2 are 0.62 ± 0.00 and 0.60 ± 0.04, respectively, and N-Kimchi's iNOS and COX-2 are 0.83 ± 0.00, respectively. , 0.85 ± 0.01, confirming a significant difference between the two (p <0.05). In kimchi to which nF1 was added, the expression levels of iNOS and COX-2 were lower than that of general kimchi, indicating that the colitis prevention effect was significantly increased.

3-7) Measurement of the number of lactic acid bacteria in feces

It is known that lactic acid bacteria in the large intestine produce organic acids to lower the intestinal pH and suppress the growth of harmful bacteria, thereby improving the intestinal environment. Accordingly, the change in the number of lactic acid bacteria in the feces according to the administration of the test substance was measured, and the results are shown in FIG. 13.

The number of lactic acid bacteria was measured using a plate count technique. Mouse feces were diluted step by step with sterilized distilled water, heated and dissolved in advance, and then sprayed by 0.1 mL into MRS agar plates (Difco, Detroit, MI, USA) cooled to 43-45°C. Cultured in an incubator under anaerobic conditions at 37° C. for 2 days, and the total number of lactic acid bacteria was expressed as the number of colonies produced by the cultured bacteria (colony forming units per gram, CFU/g).

As shown in FIG. 13, as a result of measuring the lactic acid bacteria in the feces, it was found that the number of lactic acid bacteria was different depending on the test substance consumed by the mouse. There was no significant difference between the normal group and the control group, and the number of lactic acid bacteria in feces increased in both the nF1-treated group (nF1-H, nF1-M, nF1-L) and kimchi-treated group, of which the nF1-H group showed the greatest increase.

Specifically, after administration of the test substance for a total of 2 weeks, lactic acid bacteria in the feces of the nF1-H group were detected in a high amount of 8.47 ± 0.09 Log CFU/g, and the effect of increasing the lactic acid bacteria in the intestine when ingesting a high concentration of nF1 Appeared to be.

In addition, after 2 weeks, the lactic acid bacteria in the feces of the nF1-Kimchi group were 8.31 ± 1.10 Log CFU/g, and a higher amount of lactic acid bacteria was detected compared to general kimchi without nF1. It was confirmed that there is an increasing effect.

3-8) Measurement of harmful enzymes (β-glucosidase, β-glucuronidase) in feces

Beta-glucosidase and beta-glucuronidase, which are secreted through metabolism of intestinal bacteria, contain beta-glucoside glycoside compounds and glucuronic acid compounds. It is known as a harmful enzyme that transforms into harmful compounds. In particular, beta-glucuronidase, a representative harmful enzyme in the intestine, creates amines, toxic substances, mutants, etc., causing colorectal cancer by damaging the intestinal tract, and toxic substances absorbed into the intestine circulate in the body and damage various organs. It causes cancer, arteriosclerosis, liver failure, and immune function decline. Accordingly, changes in the activity of harmful enzymes in mouse feces according to administration of the test substance were measured, and the results are shown in FIG. 9.

Specifically, β-glucosidase activity is 0.1 M sodium phosphate buffer (pH 7.0) 0.3 mL, 2 mM p-nitrophenyl-β-D-glucopyranoside (p-nitropheyl-β-D- 0.1 mL of the enzyme solution was added to 0.2 mL of glucopyranoside) and reacted at 37° C. for 15 minutes, and then 0.3 mL of 0.5 N NaOH was added to terminate the reaction. 1 mL of distilled water was added thereto, centrifuged at 3,000 rpm for 20 minutes, and then the supernatant was taken and the absorbance was measured at 405 nm.

The β-glucosidase activity of mice before administration of the test substance (initial) was 1.11 ± 0.22 units/g, and as shown in FIG. 14, the activity of the normal group after administration of the test substance for 2 weeks was 1.48 ± The activity of the control group was 0.04 units/g and 2.76 ± 0.29 units/g.

On the other hand, in the case of the nF1-administered mouse group, the nF1-H group was 1.64 ± 0.06 units/g, the nF1-M group was 1.81 ± 0.06 units/g, and the nF1-L group was 2.57 ± 0.04 units/g. It was confirmed that the activity of β-glucosidase was lowered. In addition, the nF1-Kimchi group was 1.97 ± 0.23 units/g, and the N-Kimchi group was 2.09 ± 0.10 units/g, indicating significantly lower activity in kimchi added with nF1 (p < 0.05).

In addition, β-glucuronidase activity was 0.1 MS sodium phosphate buffer (pH 7.0) 0.38 mL, 2 mM p-nitrophenyl-β-D-glucuronide (p-nitropheyl-β-D-glucuronide) 0.02 0.1 mL of the enzyme solution was added to mL, followed by reaction at 37°C for 1 hour, and then 0.4 mL of 0.5 N NaOH was added to terminate the reaction. 1 mL of distilled water was added thereto, and after centrifugation at 3,000 rpm for 20 minutes, the supernatant was taken and the absorbance was measured at 405 nm.

Before administration of the test substance (initial), the β-glucuronidase activity of the mouse was 0.29 ± 0.01, and as shown in FIG. 14, after administration of the test substance for 2 weeks, the activity of the normal group was 0.27 ± 0.01 units. /g, the activity of the control group was 0.58 ± 0.04 units/g.

On the other hand, in the case of the nF1-administered mouse group, the nF1-H group was 0.44 ± 0.01 units/g, the nF1-M group was 0.44 ± 0.01 units/g, and the nF1-L group was 0.50 ± 0.05 units/g. The activity of β-glucuronidase was low in the medium concentration group. In addition, in the case of nF1-Kimchi group, 0.44 ± 0.01 units/g and in the N-Kimchi group were 0.47 ± 0.05 units/g, indicating significantly lower activity in kimchi added with nF1 (p < 0.05).

Therefore, when nF1 is ingested at high concentrations or when nF1 is added to kimchi, the activities of β-glucosidase and β-glucuronidase enzymes in feces decrease, producing toxic substances in the intestine (pro-carcinogen as carcinogen). Conversion) seems to have an effect of improving the environment in the colon.

Independent Administrative Corporation Product Evaluation Technology Infrastructure Organization Patent Microorganism Deposit Center nF1NITEP-1462 20121108

<110> BIOGENICS KOREA CO., LTD <120> Nano-Sized Lactic Acid Bacteria from Kimchi <130> 13P1169 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1492 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA of Lactobacillus plantarum gene: nF1 <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60 gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120 tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180 cgcatggtcc gagcttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240 ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300 taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420 gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480 ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 780 aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 840 ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 900 gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 1020 gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 1140 gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 1260 tcgcgagagt aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 1320 cctacatgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440 gtaacctttt aggaaccagc cgcctaaggt gggacagatg attagggtga ag 1492 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 gagtttgatc ctggctcag 19 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 tcgtaacaag gtagcc 16

Claims (2)

Is separated from Kimchi, and positive bacilli form gram, and the Lactobacillus Planta room (Lactobacillusplantarum) nF1 strain (accession number NITE P-1462), characterized in that having a 16S rRNA nucleotide sequence represented by SEQ ID NO: 1 treated nano, Wherein the mode of particle size distribution is in the range of 0.5 to 1.0 占 퐉. The health functional beverage for immune function activity comprising the nano-form Kimchi lactic acid bacteria composition. The method of claim 1, wherein the nano processing method comprising the Lactobacillus Planta room (Lactobacillusplantarum) 8 ~ heated 12 minutes sterilized nF1 strain step, the culture medium of the strain cultured under the conditions of pH 5 ~ 7 at 80 ℃ treatment , Adding an excipient to the culture solution, dispersing the mixture in a high pressure homogenizer at 140 to 160 kgf / cm 2, and lyophilizing the dispersed culture solution.

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