KR20040083221A - Agrobacterium sp. beta 82 KCTC 10099BP producing water-soluble polysaccharide, water-soluble polysaccharide and producing method thereof - Google Patents

Abstract

PURPOSE: A novel mutant stain, Agrobacterium sp. beta 82(KCTC 10099BP), producing water-soluble polysaccharide, water-soluble polysaccharide produced thereby and a producing method thereof are provided. The water-soluble polysaccharide has immunological enhancement activity and anticancer activity. Therefore, it can be used in health foods, food additives, cosmetics and diet foods. CONSTITUTION: The mutant strain. Agrobacterium sp. beta 82(KCTC 10099BP), produces water-soluble polysaccharide. The method for producing the water-soluble polysaccharide comprises culturing Agrobacterium sp. beta 82(KCTC 10099BP) at pH 6.5 to 7.5 for 48 hours, wherein the water-soluble polysaccharide produced has immunological enhancement activity and anticancer activity, and is water-soluble glucan consisting of glucose, galactose and mannose in a ratio of 5.8:6.7:1.0.

Description

Agrobacterium strains producing water-soluble polysaccharides, water-soluble polysaccharides and methods for preparing the same {Agrobacterium sp. beta 82 KCTC 10099BP producing water-soluble polysaccharide, water-soluble polysaccharide and producing method

The present invention relates to a new strain produced by the Agrobacterium genus strain using a mutation method, and to a new strain that secretes the water-soluble polysaccharide material into the cell and to a water-soluble polysaccharide produced from the new strain. The present invention relates to a novel mutant strain that secretes a water-soluble polysaccharide material, a method for producing a new polysaccharide material using the same, and its application to the food, pharmaceutical and cosmetic industries.

Beta-glucan is a carbohydrate polymer polysaccharide present in mushrooms, yeasts, molds, plants, etc. It enhances the immune function of the human body, regulates the homeostasis and maintains blood pressure and blood sugar levels and anti-cancer effects. It has been widely studied because it has been widely known.

In Japan, as a medicine, polysaccharides of cloud mushrooms belonging to basidiomycetes (PSK, krestin), lentinan of shiitake mushrooms, schzophyllan, ganoderma lucidum mushrooms, and situation mushrooms (Korean Patent Application 1999-0081593, Korean Patent Application 1999-0081595), polysaccharides contained in various edible mushrooms, such as mulberry mushroom is used in clinical. The antiviral and anticancer action of the polysaccharides does not show direct cytotoxicity, but induces activation of lymphocyte cells and proliferation of macrophages, increases the number of cells with antibody-producing ability and promotes immune function in vivo (Biol. Pharm. Bull. 18 (10) 1320-1327, 1995; Immunology and Cell Biology 77: 395-403, 1999). In addition, anti-cancer drugs, radiation therapy due to white blood cell and platelet reduction, loss of appetite, hair loss, vomiting is also effective. However, even though beta-glucan obtained from mushrooms has a great effect on immunity, it is not easy to isolate and cultivate mycelium, and its yield is also very limited, so it is sold at an expensive price.

The structure of beta-glucans isolated from the cell walls of yeast is a structural backbone with glucose units linked by beta-glycosidic linkages at positions 1 and 3, with side chains at 4 or 6 carbons partially dependent on polysaccharide sources. (Carbohydrate Research 316: 161-172, 1999). In addition, the glucan structure isolated from the strains of the genus Agrobacterium is a single polysaccharide in which glucose units are linked by beta-glycoside bonds at positions 1 and 3 (Takeda Technical Report 1997). This structural difference is a factor influencing the properties of beta-glucans, and the other important property of beta-glucans is that they are insoluble in the aqueous solution. Insoluble microparticles (1-2 μm in size) beta-glucan are effective in regenerating damaged cells without toxicity when administered directly to the lesion, but when glucan microparticles are administered intravenously, It is known to be related to development and endotoxin strengthening, and it has been found to cause serious problems when used in clinical practice (Int. J. Immunopharmacol, 1986, 8, 313 .; Immunopharmacology, 1991, 22, 139). Therefore, in order to use beta-1,3 glucan for clinical use, studies are being actively made to make insoluble glucan obtained from yeast or bacteria into a substance soluble in water. Beta-glucans can be converted to soluble substances with bioactive carboxymethylation (Int. J. Biol. Macromol., 1995, 17, 323, Carbohydr.Polym. 1991, 15, 79), sulfuric acid esterification (sulfation). , Microbiol Immunol 1997, 41, 741), phosphatelation (phosphorylation, Carbohydr. Res., 1991, 219, 203), tetrahydrofurany etherification (Tetrahydrofuranylation, Cancer Treat. Reports, 1983, 67, 275) They are doing research to increase the solubility by making polysaccharides with low molecular weight by chemical method of making them and hydrolysis or enzyme treatment by acid or alkali treatment. The physiological activity of these glucan derivatives depends on the molecular weight (Mw), degree of brancing (DB), and conformation.The soluble glucan derivatives undergo a lot of hydrolysis during chemical reactions, resulting in low molecular weight or many substitutions. Soluble, but does not exhibit a biological activity has the disadvantage.

Beta-glucans produced by Agrobacterium strains were first discovered in 1962 by Professor Harada of Japan, and the glucans produced by these strains contain glucose monomers of beta-1,3, insoluble in water and heat. It has been named as curdlan because it has the characteristic of forming a gel (J. Ferment. Technol. 42, 615, 1964). Since the discovery of curdlan-producing strains, many studies have been conducted to develop mutant strains. These mutant strains focused on the development of high-productive strains that can mass-produce polysaccharides that are insoluble in water at low cost and high efficiency.

Accordingly, it is an object of the present invention to provide novel water-soluble polysaccharides having immunopotentiating activity.

It is also an object of the present invention to provide a novel microorganism producing the immunopotentiating polysaccharide material.

It is also an object of the present invention to provide such polysaccharides produced from new strains to anticancer agents, immunoactivators and acceptable industrial fields.

Figure 1 is a graph showing the trend of production of water-soluble polysaccharides from the Agrobacterium genus strains over time.

Figure 2 is a graph showing the effect of water-soluble polysaccharide isolated from mutant strains on the production of IFN-r in the presence of PHA as an immunostimulating agent in human peripheral blood mononuclear cells (PBMC-Periperal Blood Mononuclear Cell). A: water soluble glucan, LPS: Lipopolysaccharide

The present invention relates to a microbial strain that secretes water-soluble polysaccharides extracellularly, using a mutagen called MNNG (M-N-methyl-N'-nitro-N-nitroguanidine) by the mutation method to 99.9% A strain having excellent polysaccharide production ability has been developed, and the polysaccharide produced from this strain is separated and purified to investigate constituent monosaccharides and structural features, and to provide an immunostimulating polysaccharide material.

The present invention relates to the Agrobacterium sp.beta 82 new strain KCTC 10099BP to produce a water-soluble polysaccharide.

In one embodiment of the present invention used a new strain mutated by treatment with MNNG from the genus Agrobacterium strain.

In addition, the present invention is characterized by producing a water-soluble polysaccharide by fermenting, culturing the strain.

In addition, the present invention is characterized in that the fermentation step is performed within 48 hours at pH 6.5-7.5.

In addition, the present invention relates to a water-soluble polysaccharide having immuno-enhancing activity and anti-angiogenic effect, which is produced by the Agrobacterium sp.beta 82 new strain KCTC 10099BP.

In addition, the present invention is characterized in that the water-soluble polysaccharide is water-soluble glucan composed of glucose, galactose, mannose in a ratio of 5.8: 6.7: 1.0.

Furthermore, the present invention relates to a pharmaceutical composition comprising the water-soluble polysaccharide as an active ingredient.

Pharmaceutical compositions containing a water-soluble polysaccharide as an active ingredient can be formulated orally or injectable by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g., lactose, textose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants, if necessary : Silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols, the tablets also contain a binder (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or Polyvinylpyrrolidone), and preferably if desired, it contains a disintegrant (e.g. starch, agar, alginic acid or its sodium salt) or a boiling mixture and / or absorbents, colorants, antiseptics and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries such as preservatives, stabilizers, wetting or emulsifier solution promoters, salts and / or buffers for controlling osmotic pressure. In addition, they may contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally as desired, or parenterally, i.e., intravenously, subcutaneously, intraperitoneally, or topically. The dosage may be 0.001 μg-100 mg / kg daily. The amount can be administered in one to several portions. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

The pharmaceutical formulation of the present invention can be used for immuno-enhancing purposes and anti-cancer purposes.

Hereinafter, the specific configuration of the present invention through the embodiment will be described in detail. However, the scope of the present invention is not limited only to the description of these examples.

Example 1 Strain Mutation and Mutant Strain Selection

After culturing the parent strain Agrobacterium sp. ATCC 31750 and treating MNNG under conditions of 99.9% mortality, YP solid medium (0.005% Aniline Blue, yeast extract) added with aniline blue 1 g / L, bacto peptone 1 g / L, sucrose 20 g / L) and incubated at 30 ℃. Selection of strains were selected strains excellent in color, shape, polysaccharide production ability of the colony (colony) appear on the plate. The selected mutant strains were examined for the intensity of blue color and polysaccharide production in the medium to which aniline blue was added through the repeated passages. Were selected.

Example 2. Characteristics of Mutant Strains

Morphological and physiological characteristics of the strains used in the present invention are as follows. Morphological characteristics of both parent and mutant strains were Gram negative strains, which were rod-shaped and blue in medium supplemented with aniline blue. Strains were wrinkled and protruded into rough surfaces, but mutant strains were shiny. Selected mutant strains were deposited with the Korea Biotechnology Research Institute Gene Bank (KCTC), an international microbial depository institution, on November 3, 2001 as strain Agrobacterium sp. Beta 82 KCTC 10099BP. Physiological characteristics are shown in Table 1 of the experimental results using the API 20NE kit.

Test Agrobacterium sp. beta 82KCTC 10099BP OxidaseNO ₃ productionindole productionglucose acidificationarginine dihydrolaseureaseesculin hydrolysis (-glucosidase) gelatinasebeta galactosidaseglucose assimilationarabinose assimilationmannose assimilation + ---- ++-+++++++ --- +-

Example 3. Liquid Culture of Selected Strains

In the present invention, microorganisms were inoculated in a nutrient medium (pH 7.0) composed of sucrose, yeast extract and peptone, and incubated at 30 ° C. for 17 hours, and then the culture solution was aerated at 0.5 vvm, agitation speed was 600 rpm, and a carbon source. Phosphorus sucrose concentration is 50-100 g / L, nitrogen is the initial concentration of ammonium at 0.5-2.5 g / L, the initial concentration of phosphoric acid is 0.5-1.0 g / L, and the pH is increased in fermentation broth containing inorganic salts. The water-soluble polysaccharides were produced by culturing with 6.5-7.5. Incubation time is preferably incubated within 48 hours under the condition that the nitrogen source is not depleted.

Example 4 Separation and Purification of Water-soluble Polysaccharides

The cultured fermentation broth was diluted to an appropriate concentration and centrifuged at 18000 rpm, 30 ° C. for 30 minutes to obtain a supernatant. Ethanol was added to the supernatant so that the ethanol concentration was 80%, and the mixture was left at 4 ° C. for 24 hours, followed by centrifugation to obtain a supernatant and a precipitate. The precipitate was centrifuged by adding ethanol, and lyophilized to obtain a water-soluble polysaccharide. 6N hydrochloric acid was added to 1 mg / ml of the dried polysaccharide and hydrolyzed in a sterilizer at 121 ° C. for 1 hour, and the purity was measured using the DNS method. The purity was 85-90%.

Example 5 Constituents of Water-soluble Polysaccharides

In order to clarify the structure of the polysaccharide obtained in Example 4, the constituent monosaccharides forming the skeleton of the polysaccharide were identified using an acid hydrolysis method. According to the method reported in the literature (Manners et al ., 1973 JC Biochem. 135: 19-30), polysaccharides were made into alditol peracetate derivatives after complete acid hydrolysis. In order to compare and confirm the constituent sugars of the product, the polysaccharide known as succinoglucan produced by Alcaligenes faecalis var. Myxogenes 10C3 strain was reacted in the same manner, and then altitol peracetate. The product obtained after the derivative and the product obtained from the beta 82 strain were analyzed by GC-MS. The instrument used was Hewlett Packard (HP 5890 GC) and the column was DB-1 (0.32mm x 30m). As shown in Table 2, the monosaccharide that forms the framework of the water-soluble polysaccharide was identified as heteroglucan composed of glucose, mannose, and galactose in a ratio of 5.8: 6.7: 1.0.

Alditol peracetates Water Soluble Glucan (area%) D-Glucitol D-Galactitol D-Mannitol 43.07.449.6

Example 6 Immune Boosting Activity of Water-Soluble Polysaccharides

Beta-glucan stimulates humoral and cellular immunity in humans and animals to amplify the immune system and act as an immune modulator. The anti-cancer effect of beta-glucan is not to kill cancer cells directly, but to activate the macrophages, T cells, NK cells, etc. to restore the body's immunity, which has been suppressed. Human mononuclear cells were isolated and treated with water-soluble glucan, PHA (phytohemagglutinin), LPS (lipopolysaccharide), and IL-18 for 24 hours, and the effects of glucan on IFN-γ in human peripheral mononuclear cells were compared with those of control. γ was released, and when treated with PHA and when treated with LPS also increased, and when treated with cytokine IL-18 increased IFN-γ (Fig. 2).

In order to investigate the anticancer effect of the water-soluble polysaccharide of the present invention, Salcoma 180 was injected into the abdominal cavity of mice in vivo to generate cancer, and the water-soluble polysaccharide of the present invention was administered. Purified water was added to the negative control group. As a result of confirming the survival rate after 7 days, all of the treated water was dead at 3 weeks, but 2 water treated with soluble glucan survived after 9 weeks. Therefore, the anticancer effect is confirmed in clinical trials that the survival rate when the soluble polysaccharide of the present invention is treated after intraperitoneal injection of Salcoma 180 intraperitoneal cancer cells, and the soluble polysaccharide obtained from the mutant strain has anticancer effect (Table 3).

NO. of survivors / total <3 weeks <4 weeks <7 weeks <9 weeks Purified water (100 μl) 0/7 - - - Water-soluble polysaccharide (250 μg / mouse) of the present invention 0/7 2/7 2/7 2/7

The present invention can provide a new strain producing a water-soluble polysaccharide.

In addition, the present invention can produce and provide a novel polysaccharide material having immunopotentiating activity from a mutant strain mutated Agrobacterium genus strain.

In addition, the present invention can provide a method for separating and purifying immunopotentiating polysaccharides from a mutant strain mutated Agrobacterium sp.

In addition, the present invention is a polysaccharide material produced from the Agrobacterium strain exhibits a feature that is water-soluble as a substance having an immune enhancing ability can be utilized in a variety of uses, such as pharmaceutical fields, health supplements, food additives, cosmetics or diet foods.

Claims (6)

Agrobacterium sp.beta 82 New strain KCTC 10099BP that produces water-soluble polysaccharides. Method for producing a water-soluble polysaccharide using Agrobacterium ( Agrobacterium sp.beta 82 ) new strain KCTC 10099BP characterized in that the strain of claim 1 to ferment, culture to produce a water-soluble polysaccharide. The method of claim 2, wherein the fermentation step is performed at pH 6.5-7.5 within 48 hours. Agrobacterium ( Agrobacterium sp.beta 82 ) A method for producing a water-soluble polysaccharide using KCTC 10099BP. Water-soluble polysaccharide produced by the Agrobacterium sp.beta 82 new strain KCTC 10099BP of claim 1 having immuno-enhancing activity and anti-angiogenic effect. The water-soluble polysaccharide according to claim 4, wherein the water-soluble polysaccharide is water-soluble glucan composed of glucose, galactose, and mannose in a ratio of 5.8: 6.7: 1.0. A pharmaceutical composition comprising the water-soluble polysaccharide of claim 4 as an active ingredient.