KR100808956B1 - Lactic acid bacteria separated from kimchi and exopolysaccharides produced thereby - Google Patents

Abstract

The present invention is isolated from Kimchi, produces exopolysaccharides, can survive in an acidic environment of pH 3.0 to pH 5.0, resistant to artificial gastric juice and artificial bile, and has probiotic activity ( Leuconostoc) sp.), the method for producing exopolysaccharides and exopolysaccharides produced by the strain, and 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 It relates to a probiotic comprising (probiotics).

The strain according to the present invention produces exopolysaccharides, and because it is excellent in intestinal viability, excellent as probiotics, the exopolysaccharides can be used in various industrial fields as a physiological functional material as well as a physical functional material. have.

Description

Lactic acid bacteria isolated from kimchi and exopolysaccharides obtained therefrom {LACTIC ACID BACTERIA SEPARATED FROM KIMCHI AND EXOPOLYSACCHARIDES PRODUCED THEREBY}

1 is a gram stained picture of Lukonostok kimchi eye GJ2, Lukonostok citrium C3 and Lukonostok mesenteroides C11 according to an embodiment of the present invention,

2 is a primer pair for amplifying 16S rDNA according to an embodiment of the present invention,

3 is a nucleotide sequence of 16S rDNA of Lukonostok kimchi eye GJ2 according to an embodiment of the present invention,

4 is a nucleotide sequence of 16S rDNA of ruconostock citrium C3 according to an embodiment of the present invention.

5 is a nucleotide sequence of 16S rDNA of Lukonostok mesenteroides C11 according to an embodiment of the present invention,

6 is a lineage with other bacteria based on the nucleotide sequence of 16S rDNA of Lukonostok kimchi eye GJ2 , Lukonostok citrium C3, and Lukonostok mesenteroides C11 according to an embodiment of the present invention. Is a table of developmental relationships,

Figure 7 is a growth curve measured by the absorbance of the growth time according to the incubation time of Lukonostok kimchi eye GJ2 , Lukonostok citrium C3 and Lukonostok mesenteroides C11 according to an embodiment of the present invention,

8 is a growth rate measured by using absorbance according to the NaCl concentration of the culture medium of Lukonostok Kimchi I GJ2 , Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 according to an embodiment of the present invention Curve,

9 is a growth of the growth medium according to the initial pH of the culture medium of Lukonostok Kimchi Eye GJ2 , Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 according to an embodiment of the present invention using absorbance Curve,

Figure 10 is a graph measuring the resistance to simple acid pH in the culture medium of Lukonostok kimchi eye GJ2 , Lukonostok citrium C3 and Lukonostok mesenteroides C11 pH 3.0 according to an embodiment of the present invention Is,

11 is a graph measuring resistance to artificial gastric juice of pH 3.0 of Lukonostok Kimchi Eye GJ2 , Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 according to an embodiment of the present invention.

12 is a graph measuring the resistance to artificial bile containing the 0.3% bull bile of Lukonostok kimchi eye GJ2 , Lukonostok citrium C3 and Lukonostok mesenteroides C11 according to an embodiment of the present invention. Is,

Figure 13 is a graph measuring the resistance to artificial bile containing 0.5% bull bile of Lukonostok kimchi eye GJ2 , Lukonostok citrium C3 and Lukonostok mesenteroides C11 according to an embodiment of the present invention ego,

Figure 14 contains 0.3% ox bile after pretreatment using artificial gastric juice of pH 3.0 of Lukonostok Kimchi I GJ2 , Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 according to an embodiment of the present invention. It is a graph measuring the resistance to artificial bile,

15 is a TLC analysis of the acid hydrolyzate of exopolysaccharides of Lukonostok Kimchi I GJ2 , Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 according to an embodiment of the present invention.

16 shows the results of HPLC analysis of acid hydrolysates of exopolysaccharides of Lukonostok Kimchi I GJ2 , Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 according to an embodiment of the present invention.

17 is a result of analysis of GPC (Gel Permeation Chromatography) of exopolysaccharide of Lukonostok kimchi eye GJ2 according to an embodiment of the present invention,

FIG. 18 shows Lukonostok Kimchi I GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 incubated for 48 hours in a sucrose solid medium containing 5% sucrose, according to one embodiment of the present invention. It is a photograph showing the form of each exopolysaccharide to generate,

FIG. 19 shows Lukonostok Kimchi I GJ2, Lukonostok Citrium C3, and Lukonosteck Mesenteroides C11, which were cultured for 48 hours in a sucrose solid medium containing 5% sucrose, according to an embodiment of the present invention. The form of each exopolysaccharide to be produced is shown schematically.

[TECHNICAL FIELD OF THE INVENTION]

The present invention is isolated from Kimchi and produces exopolysaccharides (EP), exopolysaccharides (EPS), can survive in acidic environment of pH 3.0 to pH 5.0, and has a viable probiotic activity ( Luconostoc sp) .), A method for producing exopolysaccharides and exopolysaccharides produced by the strain, and 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 It relates to a probiotics comprising.

[Private Technology]

Lactic acid bacteria (LAB) is a microorganism that is widely distributed in nature and uses carbohydrates anaerobicly to produce lactic acid and is used as a seed for various fermentation processing of dairy products, meat, vegetables, etc. It also contributes to sensory and nutritional value. In addition, some lactic acid bacteria contribute to the organization and viscosity of products by biosynthesizing exopolysaccharides [An overview of the functionality of exopolysaccharides produced by lactic acid bacteria. International Dairy Journal. 12: 163-171 (2002). Exopolysaccharides produced by lactic acid bacteria alleviate whey separation, increase viscosity and soften tissues, which are a major problem in lactic acid bacteria fermented milk products. and challenges. Trends in Biotechnology. 21 (6): 269-274 (2003).

Exopolysaccharide is a microbial polysaccharide that forms part of the cell wall around the cell wall or forms a slime type outside the cell wall during fermentation. It is a primary or secondary metabolite [Isolation of Exopolysaccharide Producing Enterobacter sp. and Physicochemical Properties of the Polysaccharide Produced by This Strain. Korean J. Biotechnol. Bioeng. 16 (4): 370-375 (2001). Exopolysaccharides are not used as energy sources and perform many functions related to the survival of microorganisms, such as protecting themselves from the external environment, such as drying, phagocytosis, protozoan wetness, antibiotics, toxic compounds, and osmotic stress. Studies on the Properties of the Stirred Yogurt Manufactured by Exopolysaccharide Producing Lactic Acid Bacteria. Korean J. Food Sci. Ani. Resour. 25 (1): 84-91 (2005)]. Exopolysaccharides are long chain polymers that dissolve or disperse in water and play an important role in thickening or gelling in food products. Exopolysaccharides are the most polysaccharides produced by microorganisms in practical terms, are the most commercially available polysaccharides because they are easy to recover from cultures and have low purification costs [Novel and established applications of microbial polysaccharides. Trends in Biotechnology. 16: 41-46 (1998).

Exopolysaccharides produced by lactic acid bacteria have already been reported to differ in the type and ratio of constituent sugars depending on the species.

Lactobacillus exopolysaccharides are classified into two groups, homopolysaccharides and heteropolysaccharides. Homopolysaccharide mainly refers to one type of monosaccharides such as fructose and glucose. Streptococcus salivarius and Streptococcus There are fructans type levan and inulin by mutans , and glucans type Leuconostoc mesenteroides subsp . with mesenteroides Leu . mesenteroides subsp . Dextran and Leu produced by dextranicum . Alteran and Streptococcus mutans and Streptococcus by mesenteroides The mutans produced by sobrinus belong to this [Novel and established applications of microbial polysaccharides. Trends in Biotechnology. 16: 41-46 (1998). Heteropolysaccharide consists of two or more different types of monosaccharides, the main constituent sugars of which glucose, galactose, fructose and rhamnose are combined in different proportions. Is fulfilling. In a few cases, non-sugar substances such as N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, and phosphate acetyl glycerol are bound to bind. It may be formed. Mainly Lactococcus lactis subsp . lactis , Lactobacillus mesophilic lactic acid bacteria such as casei and Lactobacillus sake and Lactobacillus acidophilus , Lactobacillus delbrueckii subsp . bulgalicus , Streptococcus Produced by thermophilic lactic acid bacteria such as thermophilus .

In recent years, exopolysaccharides have attracted attention as physiological functional materials as important biomaterials, not as simple physical functional materials in the past. Exopolysaccharide and Lactobacillus of Bifidobacteria among Lactic Acid Bacteria delbrueckii subsp . bulgalicus , Lactobacillus helverticus var. Exopolysaccharides derived from jugurti have been reported to have anticancer effects [Optimization of exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus RR grown in a semidefined medium. Appl. Environ. Microbiol. 64: 659-664 (1998). In addition, exopolysaccharides produced from Streptococci have been reported to have immunological effects, and anti-ulcer effects and cholesterol-lowering activity have been reported [Exocellular polysaccharide production by Streptococcus thermophilus . Biotechnol. Lett. 10: 255-260 (1998).

Moreover, such microbial exopolysaccharides can increase productivity by improving culture conditions and production conditions with high potential for industrial use, and can be mass-produced by continuous culture using a fermenter in a short period of time. It is currently attracting attention because it is easy to separate and recover saccharides.

Until now, many studies on exopolysaccharides produced from lactic acid bacteria have been conducted, but most of the studies are related to the use of spawn as a functional material of fermented milk products such as yogurt and cheese, or to search and characteristics of exopolysaccharide producing strains. Research has also been limited to lactic acid bacteria isolated from fermented milk products. If you removed from the fermented vegetable foods, especially kimchi Leuconostoc mesenteroides [(Strain Selection of generating a highly active dextran can Cray's (dextran sucrase) in Dongchimi Psychrotrophic Leuconostoc mesenteroides Producing a Highly Active dextran sucrase from Kimchi. Korean J. Food Sci.Technol. 34 (6): 1085-1090 (2002)] and Leuconostoc Generating Dextran from Kimchi Report on the separation of lactis [Isolation of dextran-producing Leuconostoc lactis from Kimchi. Journal of Microbiology. 39 (1): 11-16 (2001)].

The lactic acid bacteria involved in the Kimchi bal effect tablets had antibacterial activity [Physicochemical Characteristics of Yogurt Prepared with Lactic Acid Bacteria Isolated from Kimchi. Korean J. Food Culture. 20 (3): 337-340 (2005)], various health promoting functions have been reported, such as enhancing immune function, lowering blood cholesterol, anti- liver function, anti-cancer activity, antioxidant activity.

Recently, attention has been drawn to the functional aspects of probiotic actives among the characteristics of lactic acid bacteria. Probiotics are antibacterial agents that solve the residual and resistance problems caused by the use of antibiotics in terms of the intake of live strains, which can induce growth promotion by maintaining the flora of the intestinal microflora and reducing E. coli [Isolation and Characterization of Acid- and Bile-Tolerant Isolates from Strains of Lactobacillus acdophilus . Journal of Dairy Science. 82: 23-31 (1999)., Physicochemical Characteristics of Yogurt Prepared with Lactic Acid Bacteria Isolated from Kimchi. Korean J. Food Culture. 20 (3): 337-340 (2005)]. Therefore, the use of lactic acid bacteria having the functionality of the probiotic active as a spawn when immersing kimchi will increase the suitability.

The present invention is isolated from kimchi to produce exopolysaccharides and can survive in an acidic environment of pH 3.0 to pH 5.0, has a resistance to artificial gastric juice and artificial bile, and provides a bacterium of the genus Lukonostoc For the purpose of

Another object of the present invention is to provide an exopolysaccharide obtained from lactic acid bacteria isolated from kimchi and a method for producing exopolysaccharides using the lactic acid bacteria.

In order to achieve the above object, the present invention is separated from kimchi to produce exopolysaccharides, can survive in the stomach and intestine, in particular, can survive in acidic environment of pH 3.0 to pH 5.0, artificial gastric juice and artificial The present invention relates to a strain of Lukonostok, which has resistance to bile and has probiotic activity. As an example of the genus Lukonostok strain, the present invention provides Lukonostok Kimchi Eye GJ2, which is isolated from kimchi and produces exopolysaccharides.

The present invention also provides a method for producing exopolysaccharides and exopolysaccharides produced from the lactic acid bacteria.

Among the lactic acid bacteria isolated from kimchi, the present inventors have found that lactic acid bacteria that produce exopolysaccharides that act as self-defense substances in extreme survival environments can function as probiotics by living in gastric acid at low pH and bile secreted in the small intestine. The lactic acid bacteria producing exopolysaccharides were selected from lactic acid bacteria isolated from kimchi, and the present invention was completed by examining the presence or absence of a suit through an acid resistance, artificial gastric juice and artificial bile resistance test.

In addition, the present inventors have completed the present invention by separating and purifying the exopolysaccharide produced from the strain as a functional material and characterizing it.

In addition, the inventors have completed the present invention by developing a method for producing exopolysaccharide through the strain.

Hereinafter, the present invention will be described in detail.

Firstly, it is isolated from kimchi and produces exopolysaccharides, and can survive in the stomach and intestines, especially in acidic environments of pH 3.0 to pH 5.0, resistant to artificial gastric juice and artificial bile and having probiotic activity It provides a genus Lukonstock strain. The strain is preferably Lukonostok Kimchi GJ2 ( Leuconostoc kimchii GJ2), Leuconostoc citreum C3) and Leuconostoc mesenteroides C11, more preferably Lukonostok kimchi eye GJ2.

Leukonostoc kimchi eye GJ2 ( Leuconostoc kimchii GJ2) was deposited on March 14, 2006 with the Korea Research Institute of Bioscience and Biotechnology, located at 305-333, Ue-dong, Yuseong-gu, Daejeon, on March 14, 2006, and was granted accession number KCTC 10921BP.

Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 are lactic acid bacteria isolated from kimchi, which are Gram-positive and mucoid colony. The colonies of Lukonostok Kimchi Eye GJ2 and Lukonostok Citrium C3 are rounded, and the surface is smooth and milky, and the colonies of Lukonostok Mesenteroides C11 are rounded and the surface is smooth and white.

As a result of 16S rDNA sequencing analysis, isolated Lukonostok kimchi eye GJ2 of the present invention was Leuconostoc. kimchii ) shows high homology of 99% with AF179386. On the other hand, Luconostock Citrium C3 is Leuconostoc citreum 99% homology with AF111949, Lukonostek mecenteroides C11 is Leuconostoc 99% homology with mesenteroides AB023246.

The present invention, Lukonostok Kimchi I GJ2 produces exopolysaccharides, grows well in the range of 0% to 3% NaCl concentration, grows well in medium of pH 10.0 to pH 10.0, and especially the optimum pH is alkaline to pH 10.0. It grows best in the area. On the other hand, Luconostock Citrium C3 and Lukonostok mesenteroides C11 also grow well in the range of 0% to 3% NaCl concentration. In addition, Luconostock Citrium C3 grows well in a medium of pH 7.0 at pH 6.0, the optimum pH for growth is pH 7.0, and Luconostock Mesenteroides C11 grows well in a medium of pH 10.0 at pH 6.0, in particular Growth optimum pH is pH 7.0.

In addition, the present invention is a microbial activator comprising at least one selected from the group consisting of strains that produce exopolysaccharides, culturing medium, concentrated solution of culture medium or dried product of leucostock strain isolated from kimchi as an active ingredient. To provide. 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 of time after ingestion, and since the growth rate of many kinds of harmful bacteria is fast, the activation rate of the ingested live bacteria should be fast. In the present invention, the culture medium means a solid or liquid containing nutrients necessary for growing animal cells, plant cells, or bacteria, and the culture medium means cultured by inoculating a strain into a liquid medium. The concentrated solution of the culture solution refers to the concentration of the culture solution, and the dried product of the culture solution means that the water of the culture solution is removed.

In addition, the present invention provides a food, a personal care product or a medicine containing such probiotic.

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. Or dried or cut products of fruits, fruit juices, vegetable juices, mixed juices or chips, noodles, livestock processed foods, fish processed foods, dairy products, 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 to 0.01 to 90% by weight of the total food weight, in the case of a beverage may be added in a ratio of 0.02 to 20 g, preferably 0.5 to 10 g based on 100 mL. have.

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. It is preferably inoculated at 0.0001 to 0.01 parts by weight (wet parts by weight) based on 100 parts by weight of the food main ingredient.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food processed and designed to fully express the gymnastics function related 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.

In the present invention, the medicine may be used in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. It may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament. In the context of the pharmaceutical dosage forms of the probiotic according to the invention, the forms of their pharmaceutically acceptable salts can be used and can be used alone or in combination with other pharmaceutically active compounds as well as in suitable collections. Preferred dosages of the probiotic of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but will be appropriately selected by one of ordinary skill in the art. Can be. However, for the desired effect, the probiotic of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes, and all modes of administration can be expected. The medicine may also contain various supplemental herbal medicines, vitamins, minerals, dietary fiber and other medicinal and food supplements (eg, excipients, enhancers, coatings, etc.) as necessary.

Personal care products according to the present invention mean products for application to the body such as skin, hair and nails and include, but are not limited to, shampoos, conditioners, creams, lotions, cosmetics and soaps. The cosmetic means a product used by painting, spraying or other similar methods to cleanse or beautify the human body, and means less action on the human body, and is a flexible cosmetic water, nourishing cosmetic water, nutrition cream, massage cream, essence, pack, emulsifying Foundation, and the like.

In addition to the above, the probiotic of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the probiotic of the present invention may contain natural fruit juice, fruit juice and fruit juice for the preparation of vegetable juice beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the complex herbal extract of the invention.

In order for the lactic acid bacteria isolated from kimchi to be used as a probiotic, it must have resistance to gastric juice and bile solution, which are acidic environments, in order to balance the intestinal bacterial flora. Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 produce exopolysaccharide when inoculated in sucrose liquid medium. In the case of producing such exopolysaccharides, all three types of lactic acid bacteria did not die after 2 hours in 0.05 M sodium phosphate solution adjusted to pH 3.0 and artificial gastric juice adjusted to pH 3.0. It maintains the number of bacteria and shows high resistance. In addition, even in artificial bile, the initial bacterial count was maintained for 24 hours, and showed high bile resistance. As can be seen from these results, lactic acid bacteria that are isolated from kimchi and produce exopolysaccharides contain much more (3.0 g / L) bull's oxgall than intestinal bile (0.6 g / L). It was found to have the resistance to grow in the cultured medium, it was shown that it can actually function normally as a probiotic.

Luconosstock Kimchi-I GJ2 has antimicrobial activity in E. coli which is an indicator of fecal contamination, Pseudomonas causing sepsis, Listeria, Micrococcus, Salmonella, Staphylococcus, Streptococcus genus, etc. .

In addition, the present invention produces exopolysaccharides and exopolysaccharides obtained from strains which are separated from kimchi and can survive in an acidic environment of pH 3.0 to pH 5.0, and which have resistance to artificial gastric juice and artificial bile and have probiotic activity. Provide a way to.

The total sugar content of the exopolysaccharide obtained from the present invention Lukonostok Kimchi GJ2 is 95.4 to 97.2% w / w, the protein content is 0.7 to 0.8% w / w very small amount, constituent sugar As a result of analysis using TLC and HPLC, it was confirmed that all showed the same result as glucose used as a standard sugar, and is an exopolysaccharide in the form of a homopolysaccharide composed only of glucose to form a polymer. The average molecular weight (MW) of the exopolysaccharide obtained from Lukonostok Kimchi I GJ2 obtained through Gel Permeation Chromatography (GPC) was 240,000 to 560,000. On the other hand, in the case of exopolysaccharide separated and purified from Luconostock Citrium C3, the total sugar content was 88.0 to 90.00% w / w, and the total protein content was 0.8 to 1.0% w / w. Exopolysaccharides isolated and purified from Terroides C11 showed a total sugar content of 90.0 to 92.0% w / w and a total protein content of 0.85% to 1.05% w / w. Exopolysaccharides produced by Luconosestock Mesenteroides C11 are homopolysaccharides that are free of other constituent sugars and consist only of glucose to form polymers, like the exopolysaccharides produced by Luconosestock Kimchiai GJ2. It was found to be an exopolysaccharide in form.

Exopolysaccharides produced by previously reported lactic acid bacteria were only 1 to 2 g / L [Biodiversity of Exopolysaccharides Produced by Streptococcus thermophilus Strains Is Reflected in Their Production and Their Molecular and Functional Characteristics. Appl. Environ. Microbiol. 70 (2): 900-912 (2004)., Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595 M and R. Biochem. J. 263: 7-17 (2002). On the other hand, Lukonostok Kimchi I GJ2 produced crude exopolysaccharide with sucrose as the only carbon source, and its yield is 21.49 g / L, which is more than 10 times higher than that of conventional lactic acid bacteria. It can be said that the industrial use value is much larger.

In addition, the exopolysaccharides produced from Luconosstock Citrium C3 was 16.46 g / L, and the exopolysaccharides produced from Luconosstock Mesenteroides C11 were recovered to 22.98 g / L, which was higher than previously reported lactic acid bacteria. The production volume is more than 10 times higher, which means that their industrial use value is much higher.

It is isolated from the kimchi of the present invention and produces an exopolysaccharide, and can survive in an acidic environment of pH 3.0 to pH 5.0, and is resistant to artificial gastric juice and artificial bile, and has an exobacterial leukostock strain that has probiotic activity. The method for producing polysaccharide is iii) inoculate the lactic acid bacteria isolated from kimchi in sucrose liquid medium and incubated at 30 ° C. for 48 hours, ii) separating the polysaccharide from the culture medium, and iii) separating the polysaccharide from the culture medium. It characterized in that it comprises the step of purifying the exopolysaccharide in Lloyd. Separation of the polysaccharide was performed by centrifugation (9,950g, 25min) of the cell culture solution to remove the cells, and slowly added twice the cooled 95% ethanol to the recovered supernatant solution and precipitated at 4 ° C for 15 hours. After that, the precipitate may be recovered by centrifugation (9,950 g, 25 min, 4 ° C.), and lyophilized (Samwon freezing engineering Co., Korea). Purifying the exopolysaccharide is added to the cell culture broth trichloroacetic acid (trichloloacetic acid, Sigma chemical Co., USA) so that the final concentration is 4% (w / v) and after 2 hours at 4 ℃ Centrifugation (9,950g, 25min, 4 ℃) to remove the cells and precipitated protein and recover the supernatant, and the recovered supernatant was filtered with a 0.2 ㎛ filter (Milipore Co., USA) to remove the remaining protein , 2 times cooled 95% ethanol was added slowly to precipitate for 15 hours at 4 ℃, the precipitate was recovered by centrifugation (9,950 g, 25 min, 4 ℃) and the remaining ethanol dried on the recovered precipitate, The precipitate may be dissolved in tertiary distilled water, put in a dialysis-sack (MW cut off 10,000, Spectra / Por 6 membrane, Spectrum Laboratories, USA), dialyzed at 4 ° C. for 24 hours, and then lyophilized.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

EXAMPLE

< Example  1> Exopolysaccharides  Isolation and Identification of Production Strains

1-1. Exopolysaccharides  Isolation of Producing Strains

After collecting kimchi and grinding the entire contents, it is filtered aseptically and diluted with the appropriate magnification by adding sterile water, MRS (CaCO ₃ , Amersco Inc., USA) added 2% MRS (Difco Co., Plate) at 30 ° C. in a medium to form a transparent ring and tentatively identify colonies negative for Gram positive kit (Gram stain kit BD Co., USA) and catalase (Catalase Negative, Biomrieux Co., France) It was.

The kimchi lactic acid strain isolated from the kimchi and identified as lactic acid bacteria was sucrose solid medium (tryptone 10 g, yeast extract 5 g, dipotassium phosphate 5 g, diammonium citrate). (diammonium citrate) 5 g, sucrose 50 g / 1 L, pH 7.0) and incubated for 48 hours at 30 ℃, select the viscous bacteria showing viscous colonies, and again sucrose liquid medium (tryptone ) 10 g, yeast extract 5 g, dipotassium phosphate 5 g, diammonium citrate 5 g, sucrose 50 g / 1 L, pH 7.0) Confirmed secretion was selected as the exopolysaccharide-producing strain. The isolated strain was incubated in MRS liquid medium (Difco, France) and then added to 25% (v / v) of glycerol in the culture medium in the log phase and stored at -70 ℃, 5 mL when used in the experiment Inoculated in MRS liquid medium and incubated for 24 hours at 30 ℃, and then primary passage in MRS liquid medium, and then second passage in the sucrose liquid medium was used by incubating 48 hours at 30 ℃.

1-2. Exopolysaccharides  Identification of Production Strains

Isolated exopolysaccharide-producing strains were characterized by morphological characteristics including Gram stain kit (BD Co., USA), biochemical characteristics using API kit (50CHL, Biomrieux Co., France), and 16S rDNA sequence analysis ( Lee, HJ, Park, SY, and Kim, JH 2000. Multiples PCR-based detection and identification of Leuconostoc species.FEMS Microbiol. Lett. 193: 243-247). For primer pairs used for 16S rDNA sequencing, a forward primer and a reverse primer were shown in SEQ ID NO: 2, SEQ ID NO: 3, and Table 1, respectively. PCR was performed using Palmcycler (Corbett Reaserch, Australia).

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: Forward 5'- GCGGCGTGCCTAATACATGCAAGTCG-3 ' One Reverse 5'- GACCCGGGAACGTATTCACCGCGGC-3 ' 2

The homology test of the determined 16S rDNA sequence was performed by the Blast program (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) on the information registered in the GeneBank database.

Table 2 shows the results of the morphological analysis of the exopolysaccharide-producing isolate. In relation to the morphological analysis of the isolates, the isolates were inoculated in MRS medium (Difco, France) for 24 hours anaerobic incubation, Gram staining and observed by light microscopy. The results are shown in FIG. As can be seen in Figure 1 GJ2, C3 and C11 were all Gram-positive cocci. As can be seen in Figure 1 and Table 2, strains of GJ2, C3 and C11 were Gram-positive cocci, colonies were round, smooth surface, GJ2 and C3 was milky, C11 was white.

Characteristic GJ2 C3 C11 Gram stain (Gram-stain) + + + Colony morphology coccus & circular coccus & circular coccus & circular Colony surface smooth smooth smooth Colony color milk milk white Colony opacity opaque opaque Opaque

As for the analysis of biochemical properties, sugar metabolism was examined using API 50CHL kit (Biomrieux Co., France), and the results are shown in Table 3.

Reaction GJ2 C3 C11 Reaction GJ2 C3 C11 control - - - Esculine + + + Glycerol - - - Salicine + + + Erythritol - - - Cellobiose + + - D-Arabinose - - - Maltose - + + L-Arabinose + + + Lactose - - + Ribose + - + Melibiose - - + D-Xylose - - + Saccharose + + + L-Xylose - - - Trehalose + - + Adonitol - - - Inuline - - - β-Methyl-xyloside - - - Melezitose - - - Galactose - - + D-Raffinose - - + D-Glucose + + + Amidon - - - D-Fructose + + + Glycogene - - - D-Mannose + + + Xylitol - - - D-Fructose - - - β -Gentiobiose + + - Rhamnose - - - D-Turanose + - + L-Sorbose - - - D-Lyxose - - - Inositol - - - D-Tagatose - - - Mannitol - + + D-Fucose - - - Sorbitol - - - L-Fucose - - - α-Methyl-D-mannoside - - - D-Arabitol - - - α-Methyl-D-Glucoside + + + L-Arabitol - - - glucosamine + + + Gluconate + + + Amygdaline + + + 2 keto-gluconate + - + Arbutine + + + 5 keto-gluconate + - -

Incubate at 30 ° C. for 48 hours +: positive,-: negative

In connection with 16S rDNA sequencing of the isolate strain, a total of 1,306 bp was determined for the isolate strain GJ2, and the resulting nucleotide sequences are shown in SEQ ID NOs: 1 and 3. Sequencing of the determined 16S rDNA of GJ2 was carried out homology search by the Blast program, the results are shown in FIG. As shown in Figure 6, the isolated strain GJ2 is Leuconostoc kimchii ) showed a high homology of 99% with AF179386. For isolate strains C3 and C11, a total of 1,359 bp was determined, and the results are shown in SEQ ID NO: 4 and FIG. 4 (separated strain C3), and SEQ ID NO: 5 and FIG. 5 (separated strain C11). Fig. 6 shows the results of the homology search carried out using the 16S rDNA of the isolated strains C3 and C11. As shown in Fig. 6, the isolate strains C3 and C11 are Leuconostoc citreum) AF111949 and Lu Pocono stock mesen teroyi des (Leuconostoc mesenteroides) showed high homology with AB023246 and 99%, respectively. 6 shows a phylogenetic relationship with other bacteria based on the above results. 6 was end-identified base for GJ2 to Kono Stock kimchi children (kimchii Leuconostoc) as described above, in the case of C3 the base stock sheet Pocono Solarium (Leuconostoc citreum ), C11, indicates Leuconostoc mesenteroides .

In other words, isolated strain GJ2, a lactic acid bacterium that produces exopolysaccharide isolated from kimchi, was identified as Leuconostoc kimchii by analyzing 16S rDNA sequences and general characteristics including sugar metabolism. GJ2 to Lucono Stock Kimchi GJ2 ( Leuconostoc kimchii It was named GJ2) (KCTC 10921BP) and was deposited on March 14, 2006 with the Korea Research Institute of Bioscience and Biotechnology located at 305-333, Eui-dong, Yuseong-gu, Daejeon, Korea, and received the accession number KCTC 10921BP.

On the other hand, are identified as base stock sheet Pocono Solarium (Leuconostoc citreum) for C3 were named as base stock sheet Pocono Solarium C3, if the C11 base stock Pocono mesen teroyi des (Leuconostoc mesenteroides ) and named Lukonostek mesenteroides C11.

1-3. Exopolysaccharide Generation  Characterization of the strain

1) Growth according to incubation time

In order to examine the growth rate according to the culture time of the isolated strain of Example 1-2, 1% (v / v) of the isolated strain of Example 1-2 was inoculated into MRS liquid medium (Difco, France) at 30 ° C. Absorbance was measured at A ₆₀₀ every 3 hours while incubated for 36 hours at. Absorbance was measured by culturing the isolate three times to reduce the error in the experiment. The measurement results are shown in FIG. As shown in FIG. 7, Lukonostok Kimchi I GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 all increased logarithmically from 3 to 12 hours and peaked at 12 to 15 hours. Afterwards it was possible to observe entering the stationary phase.

2) badge NaCl  Growth rate by concentration

In order to investigate the effect of the initial NaCl concentration of the medium on the growth of the isolate strain, the above example was added to MRS liquid medium (Difco, France) in which NaCl was added at 0, 1, 3, 5 and 7% (w / v). Inoculated with 1% (v / v) of 1-2 strains were incubated for 24 hours at 30 ℃ and then confirmed the growth by measuring the absorbance at A ₆₀₀ . Absorbance was measured by culturing the isolate three times to reduce the error in the experiment and the measurement results are shown in FIG. As can be seen in Figure 8, Lukonostok Kimchi eye GJ2, Lukonostok citrium C3 and Lukonostok mesenteroides C11 all grown well in the range of 0% to 3% NaCl concentration. In case of GJ2, the optimal growth rate of NaCl was 0%, but the growth rate was similar to 0% at 1%, and gradually decreased thereafter. The concentration was 1% and NaCl was better grown when added to the proper concentration, and the optimal NaCl concentration of Lukonostek mesenteroides C11 was 0%.

3) initial of the badge pH Growth according to

In order to investigate the effect of the initial pH of the medium on the growth of the isolated strain, MRS liquid medium (Difco, France) adjusted to pH 4.0, 5.0, 6.0, 6.5, 7.0 and 10.0 with 1 N NaOH or 1 N HCl After inoculating 1% (v / v) of the isolated strain of Example 1-2 and incubated at 30 ° C. for 24 hours, the absorbance was measured at A ₆₀₀ to confirm growth. Absorbance was measured by culturing the isolate three times to reduce the error in the experiment and the results are shown in FIG. As can be seen in FIG. 9, Lukonostok Kimchi Eye GJ2 was well grown even in the range of pH 6.0 to pH 10.0, and the optimum growth pH was pH 10.0. C3 grew well in the range of pH 6.0 to pH 7.0, the optimum growth pH was pH 7.0, Lukonostok mesenteroides C11 grew well in the range of pH 6.0 to pH 10.0, the optimum growth pH Was found to be pH 7.0.

< Example  2> Isolate  Intestine Survivability

2-1. Acid resistance  And gastric juice resistant

The bacteria ingested through the oral cavity must survive through strong acidic gastric juice to reach the final target intestine. Therefore, Leuconostoc strain isolated from Kimchi The isolated strain of Example 1-2, which is sp.), was subjected to an acid resistance test and a test under artificial gastric juice.

The acid resistance test of the isolate strain of Example 1-2 was performed under 0.05 M sodium phosphate solution adjusted to pH 3.0 with 1 N HCl. Acid resistance was measured by comparing the number of viable cells, and the number of viable cells was measured by diluting three times at an appropriate ratio and expressed as an average value.

Resistance under gastric juice was prepared by preparing gastric juice to measure in an environment similar to the digestive tract conditions in the body. Preparation of artificial gastric juice is described by Kobayashi et al. [Kobayashi, Y., Tohyama, K. and Terashima, T. Studies on biological characteristics of Lactobacillus : Ⅱ. Tolerance of the multiple antibiotic resistance strain, L. casei PSR3002, to artificial digestive fluids. Jpn. J. Microbiol. 29: 691-698 (1974)] to 1000 units / mL of exopolysaccharides (Sigma Co., USA) in MRS liquid medium (Difco, France) adjusted to pH 3.0 using 1 N HCl. Was added. To produce exopolysaccharides, the isolated strain was inoculated with 1% (v / v) of the sucrose liquid medium of Example 1-1, incubated at 30 ° C. for 48 hours, followed by centrifugation (9,950 × g, 5 min). After removal of the cells, the cells were recovered, and then, 0.05 M sodium phosphate solution and artificial gastric juice of pH 3.0 were added to the supernatant removed in the same amount and incubated at 30 ° C. for 2 hours. After 2 hours of incubation, the number of viable cells was measured to compare the resistance against acid and artificial gastric juice. The number of viable cells was measured three times by diluting to an appropriate ratio. As a control, a isolate was used in which the isolated strain did not produce exopolysaccharide. These controls were pre-cultured (30 ° C., 24 hours) in an MRS liquid medium (Difco, France) without treatment to produce exopolysaccharides. The same procedure was followed and the results are shown in FIGS. 10 and 11. It was.

As can be seen in Figures 10 and 11, Luconosstock Kimchi Eye GJ2, Luconosstock Citrium C3 and Lucono, which were pre-cultured in the sucrose medium of Example 1-1 to produce exopolysaccharides. Stock Mesenteroides C11 did not die after 2 hours in 0.05 M sodium phosphate solution and artificial gastric juice and maintained high initial bacterial count (10 ⁸ CFU / mL) and showed high resistance. This suggests the possibility of surviving the stomach and moving to the intestine. Pure gastric juice has a pH of 1.4-2.0, and most of the microorganisms are killed here. However, considering the high pH of the stomach due to the buffering of the foods eaten, the survival rate is expected to be higher. On the other hand, the control group did not produce exopolysaccharides pre-cultured in MRS liquid medium (Difco, France) showed a 1 to 2 log cycle reduction.

From these results, when the present invention Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 are ingested orally, viability in the stomach is accompanied by acid resistance and bile characteristics of the isolates. It may be expected that the cell protective layer will depend on the exopolysaccharide production.

2-2. Artificial bile resistance

The high survival rate in the gastric juice of exopolysaccharide producing strains was predicted to be able to pass through the stomach and enter the intestine. In order to reach the intestine through the stomach, the pancreas and duodenum pass before it, 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. Gilliland et al. Reported that the resistance to the bile that probiotics used as probiotics can grow in a medium containing 0.3% of bile in bulls.However, in order to function normally as a probiotic, intestinal bile concentrations (0.6 g / L) must be resistant to growth in medium containing much more bile (3.0 g / L) of bull.

The preparation of artificial bile was 0.3% and 0.5% of oxgall (Sigma Chemical Co., USA) solution of sterile filtration filtered with MRS liquid medium (Difco, France) with 0.45 ㎛ filter (Milipore Co., USA). Added. Resistant to artificial bile of the isolated strain of Example 1-2 is considered to be moved to the intestine through the stomach when surviving in the preculture and gastric juice in the sucrose liquid medium of Example 1-1, After treatment for 2 hours, the culture solution was centrifuged (9,950 × g, 5min) to remove the supernatant, and the cells were recovered, and then artificial bile solution was added in the same amount as the supernatant removed above for 24 hours at 30 ° C. Incubated. After 24 hours of incubation, viable cell counts were measured to compare resistance to artificial bile. The number of viable cells was measured three times by diluting at an appropriate ratio, and the results are shown in FIGS. 12 to 14. As can be seen in Figures 12 and 13, Lukonostok Kimchi GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 were all 24 in artificial bile containing 0.3% and 0.5% bull bile. Maintaining the initial bacterial count for a period of time and showed high bile resistance, as shown in Figure 14, even when treated with 0.3% ox bile after 2 hours treatment in artificial gastric juice Lukonostok Kimchi eye GJ2, Lukonostok citrium Both C3 and Lukonostoke mesenteroides C11 maintained initial bacterial counts for 24 hours and showed high bile resistance. As a control, a isolate was used in which the isolated strain did not produce exopolysaccharide. These controls were pre-incubated in MRS liquid medium (Difco, France) (30 ° C., 24 hours) and treated in the same manner without producing exopolysaccharides and the results are shown in FIGS. 12 to 14. It was. As can be seen in Figures 12 to 14, when the pre-culture in the control MRS liquid medium did not produce exopolysaccharides showed a 1 to 2 log cycle reduction.

From these results, not only resistance to artificial gastric juices of the present inventors Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11, but also the viability of high concentrations of bile fluids were exo It can be expected that it will depend on the action of the cell protective film due to polysaccharide production.

In addition, the exopolysaccharide-producing strains isolated from this example, Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 survived in the environment treated with both strong acidic gastric juice and artificial bile. By doing so, it is expected to be able to function as a probiotic and reach the target site of the intestine, thereby informing the human body.

2-3. Antimicrobial Activity Verification

1) Determination of antimicrobial activity

Confirmation of the antimicrobial activity of Lukonostok Kimchi I GJ2 was carried out through the growth inhibition inhibition test for each of the indicator strains shown in Table 4 below. Experimental method is a direct method of adding cells directly [Kim, S.I., Kim, I.C. and Chang, H. C. Isolation and identification of antimicrobial agent producing microorganisms and sensitive strain from soil. J. Korean Soc. Food Sci. Nutr. 28 (3): 526-53 (1999).

Antimicrobial activity was classified as follows according to the formation of inhibitory ring, the experimental results are shown in Table 4. In relation to the antimicrobial activity of Table 4, '+' means that the inhibitory ring is detected, and '-' means that the inhibitory ring is not detected.

Indicative strain GJ2 Antimicrobial activity of Esherichia coli ATCC 25922 + Streptococcus faecalis ATCC 29212 + Streptococcus mutans ATCC 25175 + Micrococcus luters ATCC 9341 + Staphylococcus aureus ATCC 29213 + Salmonella typhimurium ATCC 19430 + Pseudomonas aeroginosa ATCC 27853 + Listeria monocytogenes ATCC 19111 +

Leuconostoc Kimchiai GJ2 ( Leuconostoc kimchii GJ2) shows that it has an antibacterial activity, etc. causing E. coli, sepsis, which is an index of fecal contamination Pseudomonas, Listeria a cause of food poisoning, Micrococcus, Salmonella, Staphylococcus, Streptococcus genus.

2) Effect of various enzymes of antimicrobial active substance

Trypsin (EC 3.4.21.4 type I, Sigma, Missouri, USA), lipase (EC 3.1.1.3 type VII, Sigma) and protease (type I, Sigma) were dissolved in 50 mM Tris-HCl buffer (pH 7.5), respectively. To prepare a solution in which alpha-amylase (EC 3.1.1.1 type Ⅷ-A, Sigma) was dissolved in 0.1 M sodium phosphate buffer (pH 7.0), and proteinase K (EC). 3.4.21.64, Sigma) was dissolved in a buffer containing 10 mM Tris-HCl, 50 mM NaCl and 5 mM EDTA, pH 7.5. The enzyme concentration was prepared at 20 mg / mL. Various enzyme solutions prepared in lyophilized Lukonostok Kimchi I GJ2 culture were added at a final concentration of 4 mg / mL and reacted at 37 ° C. for 12 hours to observe changes in antimicrobial activity. The experimental group was not treated with the enzyme under all the same conditions as above as a control.

The antimicrobial activity measurement results according to the enzyme treatment are shown in Table 5 below. In relation to the antimicrobial activity labeling of Table 5, '+' means that the inhibitory ring was detected, and '-' means that the inhibitory ring was not detected. When tested with trypsin, protease and proteinase K, the titer was lost, and when tested with lipase, alpha-amylase and alpha-chymotrypsin, antimicrobial activity was not affected. It was found that the substances related to the antimicrobial activity of Stock Kimchi GJ2 consisted of protein.

In other words, the antimicrobial substance from Lukonostok Kimchi Eye GJ2 is a proteinaceous substance that is decomposed by proteolytic enzymes in the body, so there is no fear of resistance of the antimicrobial substance due to the residual of the antimicrobial substance. Even when used, the stability can be said.

Enzyme (4mg / mL: at 37 ℃, for 12hr) GJ2 Antimicrobial activity of Control (non-enzyme treated sample) + Trypsin - Protease - Lipase + α-Amylase + Proteinase K - α-Chymotrypsin +

< Example 3 > Of exopolysaccharides  Characterization

3-1. detach

Separation of exopolysaccharides produced from the isolate strain was centrifuged (9,950 × g, 25min) cell culture at 4 ℃ to remove the cells, and two-fold cooling in the supernatant recovered to precipitate the exopolysaccharides 95% ethanol was added slowly and precipitated at 4 degreeC for 15 hours. The precipitate was recovered by centrifugation (9,950 × g, 25 min, 4 ° C.), and the remaining ethanol was dried and then lyophilized (Samwon freezing engineering Co., Korea), which was named crude exopolysaccaride [ Exopolysaccharide-producing lactic acid bacteria strains from traditional thai fermented foods: isolation, identification and exopolysaccharide characterization. International Journal of Food Microbiology. 51: 105-111 (1999).

The production amount of exopolysaccharide was expressed as the average value by quantifying the recovered exopolysaccharide by culturing the isolated strain three times each. Examining the results according to the above method, the exopolysaccharide produced from the previously reported lactic acid bacteria is only about 1 to 2 g / L while the exopolysaccharide recovered in the case of the present invention Lukonostok kimchi eye GJ2 At 21.49 g / L, the yield was more than 10 times higher than previously reported lactic acid bacteria, and 16.46 g / L of exopolysaccharide was recovered for Lukonostium Citrium C3, and 22.98 g for Lukonostoke Mesenteroides C11. Exopolysaccharide of / L was recovered and each showed a yield of 10 times higher than the previously reported lactic acid bacteria.

In addition, only sucrose was used as the carbon source in the case of Lukonostok Kimchi I GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11. Considering these results, the industrial use value of exopolysaccharides using Lukonostok Kimchi I GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 can be said to be much higher. .

3-2. refine

The purification process of exopolysaccharide is as follows. Trichloroacetic acid (Sigma chemical Co., USA) was added to the cell culture solution so that the final concentration was 4% (w / v), followed by 2 hours treatment at 4 ° C, followed by centrifugation (9,950 × g, 25min, 4 ℃) to remove the cells and precipitated protein. After removing the cells and the precipitated protein, the remaining supernatant was recovered and filtered through a 0.2 μm filter (Milipore Co., USA) to remove the remaining protein, followed by the slow addition of twice the amount of cooled 95% ethanol 4 It precipitated at 15 degreeC. The precipitate generated by the treatment was recovered by centrifugation (9,950 × g, 25 min, 4 ° C.), the ethanol remaining in the precipitate was dried, and the dried precipitate was dissolved in tertiary distilled water to remove dialysis-sack (MW cut off 10,000). , Spectra / Por 6 membrane, Spectrum Laboratories, USA) were dialyzed at 4 ° C. for 24 hours and then lyophilized [Separation, purification and characterization of extracellular polysaccharides produced by slime-froming Lactococcus lactis ssp. cremoris strains. International Dairy Journal. 9: 631-638 (1999).

Purified exopolysaccharide isolated from Lukonostok Kimchi Eye GJ2 showed 14.61 g / L, about 65% less than exopolysaccharide recovered by simple ethanol precipitation, and purified exo isolated from Lukonostok citrium C3 The polysaccharide was 7.73 g / L, about 40% less than the exopolysaccharide recovered by simple ethanol precipitation, and the purified exopolysaccharide isolated from Luconosstock Mesenteroides C11 was 4.77 g / L. The reduction was about 20% compared to exopolysaccharides recovered by simple ethanol precipitation.

However, these results showed that S. thermophilus produced 50-350 mg / L. Cerning et al., Lactic acid bacteria and human health. Korean J. Food Nutr. 6: 53-65 (1993)] and Lactococcus lactis ssp . Zhennai Yang showed that cremoris had an ability to produce exopolysaccharides of about 164-263 mg / L [Separation, purification and characterization of extracellular polysaccharides produced by slime-froming Lactococcus lactis ssp . cremoris strains. International Dairy Journal. 9: 631-638 (1999) and Lactobacillus ssp. Exopolysaccharide production capacity of SCU-M was reported to be 1680 mg / L [Isolation of Lactobacillus ssp. Producing Exopolysaccharide and Optimization of its Production. Korean J. Biotechnol. Bioeng. 17 (2): 169-175 (2002)].

In addition, in the case of Lukonostok Kimchi GJ2 and Lukonostok Citrium C3, T. Smitinont et al. [Exopolysaccharide-producing lactic acid bacteria strains from traditional thai fermented foods: isolation, identification and exopolysaccharide characterization. International Journal of Food Microbiology. 51: 105-111 (1999), reported by Pediococcus Compared with the production of 2.5 g / L and 6.0 g / L exopolysaccharides from pentosaceus , this is a high yield.

Therefore, the above results show that the industrial value is high when exopolysaccharides are produced by using Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11. In the case of using Lukonostok Kimchi GJ2, the industrial use value is very high.

3-3. Per total  And protein

Total sugar identification of purified exopolysaccharides is described by Dubois et al. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chemistry. 28: 350-356 (1956)] was measured by the phenol-sulfuric acid method. To 1 mL of exopolysaccharide solution dissolved in water, add 5% (w / v) phenol (phenol, Fluka Co., USA) in the same amount, immediately add 5 mL of concentrated sulfuric acid, and leave for 10 minutes. 10 minutes in a water bath to measure the absorbance at A ₄₉₀ . Absorbance was measured by reacting three times to reduce the error in the experiment. Glucose was used as a standard.

The total protein of exopolysaccharides is the method of Schacterle and Pollack [Method for the Quantitative Assay of Small Amounts of Protein in Biologic Material. Anal. Biochem. 51: 654-655 (1973). Add 1 mL of the same amount of alkaline copper solution to 1 mL of exopolysaccharide solution dissolved in water, allow to stand for 10 minutes, and immediately add 4 mL of phenol working solution and treat it for 5 minutes in a 55 ℃ hot water bath. Immediately after cooling, the absorbance was measured at A ₆₅₀ . Absorbance was measured by reacting three times to reduce the error in the experiment. Bovine albumin (Fraction V, Sigma) was used as a standard.

The total sugar and total protein content of the purified exopolysaccharides are shown in Table 6 as a percentage by averaging the results measured three times.

As can be seen in Table 6, Kimchi GJ2 ( Leuconostoc kimchii The total sugar content of the exopolysaccharide separated and purified from GJ2) was 95.4 to 97.2% w / w, and the total protein content was 0.7 to 0.8% w / w, and it was separated and purified from Lukonostium C3. The total sugar content of exopolysaccharides was 88.0 to 90.00% w / w, and the total protein content was 0.8 to 1.0% w / w, and leuconostoc mesentroid C11 ( Leuconostoc In the case of exopolysaccharide purified from mesenteroides C11), the total sugar content was 90.0 to 92.0% w / w, and the total protein content was 0.85 to 1.05% w / w.

Culture EPS (g / L) Total sugar  (g%) Total protein  (g%) One 2 Leu . kimchii GJ2 21.49 (± 0.46) 14.61 (± 0.09) 96.48 (± 0.49) 0.8 (± 0.26) Leu . citreum C3 16.46 (± 0.79) 7.73 (± 0.09) 89.38 (± 0.33) 0.9 (± 1.04) Leu . mesenteroides C11 22.98 (± 0.72) 4.77 (± 0.08) 91.05 (± 0.28) 0.95 (± 0.45)

1. Crude exopolysaccharide separated by ethanol precipitation

2. Purified Exopolysaccharide

3-4. Check per configuration

Exopolysaccharide hydrolysates were analyzed using TLC and HPLC to identify the constituent sugars of the exopolysaccharides [Effect of Reaction Time on the Rheological Properties of dextran Formed Solution Produced by Crude dextran sucrase from Leuconostoc mesenteroides Sikhae. Kor. J. Appl. Microbiol. Biotechnol. 20 (3): 316-323 (1992). Hydrolysis of the exopolysaccharide was performed for 3 to 6 hours at 100 ℃ with 2 N sulfuric acid, neutralized with 1 N NaOH and filtered with a 0.45 ㎛ filter (Milipore Co., USA) was used as a sample.

TLC was prepared by dropping the prepared sample in a TLC plate (TLC plate) (Whatman K5F, 150Å) in 1 μL and then using a developing solvent having acetone: butanol: distilled water ratio of 4: 5: 2. After the development, the solution was immersed in an ethanol solution containing 0.5% (w / v) α-naphthol and 5% (v / v) sulfuric acid, dried, and color developed in an oven at 120 ° C. for 10 minutes. Strain Selection of Psychrotrophic Leuconostoc mesenteroides Producing a Highly Active dextransucrase from Kimchi. Korean J. Food Sci. Technol. 34 (6): 1085-1090 (2002). The results by the above method are shown in FIG. 15.

As can be seen in Figure 15, exopolysaccharides produced in Lukonostok Kimchi Eye GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 are one at the same development rate as glucose used as standard sugar. It can be estimated as a homopolysaccharide consisting primarily of glucose by showing a spot.

Acid hydrolysates of purified exopolysaccharides were analyzed by HPLC for more accurate analysis. HPLC used a carbohydrate analysis column (3.9 mm x 300 mm. Waters) as an analytical column and 80% (v / v) acetonitrile as a mobile phase. The flow rate of the mobile phase was 1.5 mL / min, and 20 μL of the sample was injected using an auto-sampler (MIDAS 830) and detected by a RI (Regractive Index) detector. The results are shown in FIG. 16.

As can be seen in Figure 16, the exopolysaccharides produced in Lukonostok Kimchi I GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 have the same retention time as glucose used as standard sugar. It was found that only a single peak of) produced only exopolysaccharides of the phosphopolysaccharide type consisting of glucose only. Thus, the exopolysaccharides produced by Lukonostok Kimchi I GJ2, Lukonostok Citrium C3, and Lukonostok Mesenteroides C11 are homopolysaccharides that contain no other constituent sugars and consist only of glucose to form polymers. Was determined.

3-5. Viscosity and molecular weight

1) Measurement of viscosity

For viscosity, a small sample adapter was attached to a Brookfield viscometer (LVDVII + Pro, USA) and measured using spindle No. 18. The viscosity of crude exo recovered from the sucrose culture medium of Example 1-1, the supernatant and the supernatant of the sucrose culture medium in which the isolated strains of Example 1-2 were cultured through ethanol precipitation as in Example 3-1. Polysaccharide and the supernatant were carried out on purified exopolysaccharide purified in the same manner as in Example 3-2. Crude exopolysaccharides and purified exopolysaccharides recovered through ethanol precipitation were measured by dissolving in 20 mL and 10 mL of tertiary distilled water, respectively. Viscosity was measured by preparing each exopolysaccharide three times to reduce errors in the experiment. In addition, in contrast to the difference in viscosity according to the production amount of the exopolysaccharide was measured for the same concentration is shown in Figure 18 and 19 and Table 7. In other words, The result of measuring the viscosity of each of the exopolysaccharides produced from 50 mL of the sucrose culture medium in which the isolated strains of Example 1-2 were cultured in the sucrose culture medium of Example 1-1, exopoly In order to exclude the influence of the production amount of saccharide, the result of measuring the viscosity by adjusting the crude exopolysaccharide and purified exopolysaccharide to the same concentration (4% w / v) from the isolated strain is shown in Table 7 .

As can be seen in Figure 18 and 19, when culturing Lukonostok Kimchi eye GJ2 in a sucrose solid medium, a exopolysaccharide in a transparent form having a flow of about syrup was produced.

The exopolysaccharides of Luconosstock Citrium C3 and Luconosstock Mesenteroides C11 are similar to the exopolysaccharides of Luconosstock Kimchi GJ2 in that they are glucan-type homopolysaccharides containing glucose as a component. However, different forms of exopolysaccharides were produced. That is, as can be seen in Figure 18 Luconosstock Citrium C3 produced an opaque and sticky form of exopolysaccharides, Luconosstock Mesenteroides C11 produced an opaque and thinly spreading exopolysaccharides It was.

In the sucrose culture medium of Example 1-1, 20 mL and 10 mL of crude exopolysaccharide and purified exopolysaccharide, respectively, produced from 50 mL of the sucrose culture medium in which the isolated strain of Example 1-2 was cultured It was dissolved in primary distilled water, the viscosity was measured, and the result is shown in Table 7. These results indicate that the difference in the amount of exopolysaccharide production from each isolate may have influenced the viscosity, so that the crude exopolysaccharide and purified exopolysaccharide from each isolate were the same concentration (4% w / Also about the thing adjusted to v), the viscosity was measured and the result is shown in Table 7.

As can be seen in Table 7, the culture supernatant of 13.3 cp of the Lukonostok Kimchi I GJ2 showed 482.15 cp of crude exopolysaccharide and the viscosity of 4,662 cp of purified exopolysaccharide. . As a result of measuring crude exopolysaccharide and purified exopolysaccharide by adjusting to the same concentration (4% w / v), in the case of Lukonostok Kimchi Eye GJ2, 110 to 130 cp and 240 to 280 cp, respectively. Preferably 119.4 cp, 266.3 cp, the crude exopolysaccharides and purified exopolysaccharides in Luconosstock Citrium C3 were 126-146 cp, 3,620-3,820 cp, preferably 136.3 cp, 3,719 cp, respectively. The high viscosity, Lukonostok mesenteroides C11 was 3 to 4 cp, 5.5 to 6.5 cp, preferably 3.57 cp, 6.07 cp respectively showed a relatively large difference and the lowest viscosity.

Culture Supernatant (centipoise) Exopolysaccharides (centipoise) One 2 3 4 Leu . kimchii GJ2 13.03 (± 0.22) 482.15 (± 7.25) 119.4 (± 10.06) 4,662 (± 9.07) 266.3 (± 20.5) Leu . citreum C3 7.31 (± 0.04) 139.25 (± 0.25) 136.3 (± 10.03) 3,633 (± 10.97) 3,719 (± 100.3) Leu . mesenteroides C11 2.21 (± 0.15) 5.39 (± 0.05) 3.57 (± 0.5) 3.67 (± 0.06) 6.07 (± 0.5)

1. Dissolve crude crude exopolysaccharide from 50 mL of culture in 20 mL of tertiary distilled water.

2. Crude exopolysaccharide with 4% (w / v) concentration of produced exopolysaccharide

3. Dissolved purified exopolysaccharide from 50 mL of culture in 10 mL of tertiary distilled water.

4. Purified exopolysaccharide with 4% (w / v) concentration of produced exopolysaccharide

2) measurement of molecular weight

The average molecular weight of exopolysaccharides produced by Lukonostok Kimchi Eye GJ2 was measured by GPC (Gel Permeation Chromatography). A GPC system (Agilent 1100 series, Agilent, U.S.A.) was used, and the column (colunmn) was used as the experimental conditions Zorbax bioseries GFC 250 (MW range: 500 to 400,000) and the mobile phase was water. The GPC was carried out at 35 ° C. at a flow rate of 1 mL / min and the dose was 100 μL. The concentration of the sample was 10 mg / mL. Molecular weight measurement results of the GPC are shown in FIG. 17.

As can be seen in Figure 17, the molecular weight measurement results showed that the total peak was distributed between 4.48 minutes and 7.93 minutes, the highest peak was 5.37 minutes, the distribution index (D) is 1.14 and the weight average (MW) is 240,000 to 560,000, preferably 248,000 to 551,500, more preferably 248,856 to 551,122 and most preferably 360,606. In order to calculate the weight average, the slice was calculated by dividing into 70 pieces.

As described above, the genus Luconostok strain according to the present invention ( Leuconostoc sp.) is used as a probiotic active agent to produce exopolysaccharides, which have a higher survival rate under acidic conditions and higher resistance to artificial gastric juice and artificial bile as well as antibacterial activity when producing exopolysaccharides. Can be. In addition, Luconostok Kimchi Eye GJ2, Lukonostok Citrium C3 and Lukonostok Mesenteroides C11 according to the present invention are lactic acid bacteria that produce other exopolysaccharides reported so far in a medium containing sucrose as the only carbon source. The production of exopolysaccharides using the leukostock strains is very high in industrial use since the production of exopolysaccharides is 10 times higher than that of the leukostock strains.

Attach sequence list electronic file

Claims (10)

delete Leuconostoc kimchii GJ2 strain (KCTC 10921BP), isolated from kimchi, producing exopolysaccharides, resistant to artificial gastric juice and artificial bile, surviving in the stomach and intestine, and having probiotic activity . Probiotics comprising at least one selected from the group consisting of the strain of claim 2, the culture of the strain, the concentrate of the culture and the dried product of the culture as an active ingredient (probiotics). Food comprising the probiotic of claim 3. The food of claim 4, wherein the food is fermented food or a beverage. A fermented food comprising the probiotic active agent of claim 3 as a seed. delete delete delete delete

Images