KR101245730B1 - A method of depolymerizing Grifola frondosa Exo-polysaccharides, Grifola frondosa Exo-polysaccharides obtained therefrom, and cosmetic composotion and food product containing them - Google Patents

Abstract

The present invention relates to a low molecular weight mycelium mycelium extracellular polysaccharide, a method for preparing the same, a cosmetic composition containing the same, and a food. The physiological function can be improved by reducing the molecular weight of the leaf mycelium mycelium extracellular polysaccharide by irradiation. . In particular, since the radiation protection effect and the antioxidant effect is improved, it can be included in the cosmetic composition to exhibit excellent skin aging prevention. In addition, when included in food, it may exhibit an immune enhancing effect and an anticancer effect.

Description

A method of depolymerizing Grifola frondosa Exo-polysaccharides, Grifola frondosa Exo-polysaccharides obtained therefrom, and cosmetic composotion and food product containing them}

The present invention relates to a low molecular weight method of leaf mushroom extracellular polysaccharide, to a leaf mushroom extracellular polysaccharide obtained therefrom, to a cosmetic composition and a food comprising the same, and more specifically to lower molecular weight of leaf mushroom mycelium extracellular polysaccharide by irradiation The present invention relates to a method for reducing the molecular weight of a leaf mycelium mycelium extracellular polysaccharide, thereby improving a physiological function, a leaf mycelium mycelium extracellular polysaccharide obtained therefrom, and a cosmetic composition and food containing the same.

The mushroom belongs to the taxonomic fungus of the basidiomycetes (some of which are asymptococci), 15,000 species are known worldwide, and about 2,000 of them can be developed for food.

Mushrooms consist of mycelia and fruiting bodies accumulating metabolites obtained from their metabolism. Compared to normal plants, mycelia correspond to roots, stems, and leaves, and fruiting bodies correspond to flowers. Mycelia contain more than four times the nutrients of fruiting bodies and about 50 to 60 times more medicinal ingredients. Sometimes referred to as entity. Recently, extracts of fruiting bodies and mycelium and mycelium culture have been found to be effective in improving the constitution and preventing and treating various diseases, and thus the use as health foods and medicines has been greatly increased.

In particular, unlike the conventional chemotherapeutic agents, the polysaccharide of a specific structure produced by mushrooms has been reported to activate the immune function in the host to exhibit an anticancer effect as an immunotherapy agent. Mushroom polysaccharides can be divided into three: cell wall polysaccharide constituting the cytoskeleton, intracellular polysaccharides present in the form of nutrient storage in the cell and extracellular polysaccharides secreted extracellularly. Polysaccharides that make up cell walls and membranes are α-glucan, β-glucan, heteroglucan and chitin.

Extracellular polysaccharides (EPs) are one of the biotechnological materials and depend on imports. Most of the extracellular polysaccharides are applied to food additives, health supplements, etc. Recently, they are actively applied to cosmetics to promote cell activity and collagen production to prevent skin aging, and to damage, irritate, and irritate skin by UV rays. It is known to alleviate the immune system.

Recently, the development of various functional cosmetics has been actively promoted. Natural cosmetic products are commercialized in various applications such as wrinkle improvement, whitening and UV blocking substances, removal of harmful oxygen, promotion of collagen synthesis, skin wrinkle prevention, immunity enhancement and anti-inflammatory, hair growth agent, etc. It is becoming.

In this regard, various mushroom-derived materials are applied to the cosmetic composition, and examples thereof include situation mushrooms, agaricus, cordyceps, ganoderma lucidum mushroom, shiitake mushrooms, and the like. However, these mushroom-derived materials are mostly applied in small amounts as fruiting body extracts. In addition, beta glucan derived from yeast that is commonly used has a problem of poor solubility of the material itself, and water-soluble derivatives developed by some multinational companies have a problem of high price.

Leafy mushrooms (Grifola frondosa) are taxonomically belonging to the genus Democratic Mushrooms, Tortillas and Leafy Mushrooms. Treated as a high-quality edible mushroom, it is known to be excellent in immune enhancement and anti-cancer effects, and was developed as a variety of health supplements, but it is very expensive and its use is limited. In addition, the chemical structure of beta glucan, the main polysaccharide of leaf mushrooms, is reported to be the most potent ingredient that stimulates cellular immune responses, unlike the structure of beta glucan found in other medicinal mushrooms.

Republic of Korea Patent Application No. 10-2008-0129473 "Mushroom mycelium culture and mass production method of immuno-active polysaccharide" describes a method for mass production of mycelium culture and polysaccharide of leaf mushroom using liquid culture technology,

Republic of Korea Patent No. 10-0438009 "Cosmetic composition having an anti-aging effect and whitening effect containing the extract of leaf mycelium mycelium" includes the extract obtained after removing the low-molecular substances from leaf mycelium mycelium extract or leaf mycelium mycelium culture It describes about the cosmetic composition characterized by containing it in the quantity of 0.00001-30 weight% with respect to weight.

In addition, the Republic of Korea Patent Application No. 10-2009-0116193 "Pharmaceutical composition for the treatment and prevention of inflammatory bowel disease containing leaf mushroom extract as an active ingredient" is a pharmaceutical for treating and preventing inflammatory bowel disease containing leaf mushroom extract as an active ingredient The composition is described.

Thus, although the research on leaf mushrooms has been recently made, the correlation between the molecular weight of leaf mushroom extracellular polysaccharide and its physiological function has not been studied yet. MEANS TO SOLVE THE PROBLEM The present inventors came to this invention as a result of earnestly researching and paying attention to this.

Water-soluble polysaccharides derived from mushrooms do not have poor solubility problems, but their physiological functions have not been sufficiently demonstrated in human application. Accordingly, the present invention intends to propose a solution to the above by making the polysaccharide low molecule.

According to the present invention, there is provided a method of low molecular weight mycelium mycelium extracellular polysaccharide by irradiation.

According to the present invention, there is also provided a low molecular weight leaf mushroom mycelium extracellular polysaccharide obtained from the above method.

In addition, according to the present invention, it provides a cosmetic composition comprising a low molecular weight leaf mushroom mycelium extracellular polysaccharide.

In addition, according to the present invention, there is provided a food composition comprising a low molecular weight leaf mushroom mycelium extracellular polysaccharide.

The low molecular weight mycelium mycelium extracellular polysaccharide according to the present invention has been shown to exhibit improved physiological function. Therefore, when used in the cosmetic composition it can exhibit an excellent skin anti-aging effect by the radiation protection effect and antioxidant effect, and when used in addition to food may exhibit an immune enhancing effect and an anticancer effect.

1 shows a leaf mushroom.
2 is a graph showing the change in molecular weight according to irradiation.

According to the present invention, leaf mycelium mycelium can be obtained by culturing the mycelium obtained by germinating spores obtained from the leaf mushroom fruiting body in a solid medium or a liquid medium. However, in the case of the solid medium, it is preferable to use the liquid medium because the culture period is long, the mycelia yield is low, the mycelium culture is not uniform, and the media components may be mixed during the mycelium extraction.

According to the present invention, the radiation to be irradiated is cobalt 60, but is not limited thereto, and any type of radiation generally used in the art may be used. In addition, the radiation may be irradiated in an amount in the range generally irradiated with a low level of radiation dose, preferably in the range of 1 to 50 kGy, more preferably in an amount in the range of 40 to 50 kGy.

The polysaccharide according to the present invention may be included in the cosmetic composition. The formulation of the cosmetic composition which may include the polysaccharide is not particularly limited. In addition, the polysaccharide may be included in an amount commonly used in cosmetic compositions in the art. Preferably, the polysaccharide may be included in an amount of 1 to 15% by weight based on the total weight of the cosmetic composition.

The cosmetic composition of the present invention may include, in addition to the polysaccharide, components commonly used in cosmetic compositions, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

The polysaccharide according to the present invention may be included in food. The food which can contain the said polysaccharide is not specifically limited in the kind and form. Preferably, the polysaccharide may be added in an amount of 0.0001 to 30% by weight, preferably 0.1 to 20% by weight, based on the total weight of the food, based on the total weight of the food, respectively, based on the total weight of the food. You can adjust as much as you like.

Hereinafter, the present invention will be described in more detail below. However, the present invention is not limited thereto.

1. Materials and Methods

(1) Strain and Medium Composition

The leaf mushroom strain ( G. frondosa KCTC6726) used in this study was distributed from Korea Gene Bank. The preservation medium was potato agar medium (PDA, potato dextrose agar: pH 5.5), incubated at 25 for 7 days and then preserved at 4, and used with subculture every 4 weeks. The seed and main culture medium was used as a basic medium by applying the composition of Lilly-Barneet medium (glucose 20.0g, peptone 2.0g, MgSO ₄ · 7H ₂ O 0.5g, KH ₂ PO ₄ 1.0g, 1.0 liter of water). . The spawn culture solution was incubated at 120 rpm for 3 days at 25 ° C with 100 mL of basic medium in 500 mL Erlenmeyer flask.

(2) Mycelial Growth and Polysaccharide Production

The mycelium obtained by centrifuging the culture broth collected at 8000xg for 20 minutes was washed 2 ~ 3 times with distilled water, dried at 60 ° C for 24 hours, and then weighed by measuring dry weight to separate the extracellular polysaccharide. The supernatant was added four times of ethanol (95%) to the filtrate filtered with filter paper (Whatman No. 2) and then precipitated at 4 ° C. for 24 hours. The precipitated extracellular polysaccharide was centrifuged at 8000xg for 10 minutes, and the dried product of the lyophilized extracellular polysaccharide was quantified.

(3) Separation and Purification

After adding 4 times the amount of ethanol (95%) to the supernatant from which the mycelium was separated from the fermentation broth, the precipitate was centrifuged and lyophilized to obtain crude polysaccharide. 50 g of the dry weight of the isolated mycelium was added to 50 mL of distilled water, followed by 100 ° C. Hot water was extracted for 4 hours at to obtain a filtrate. This was used as mycelium extract containing extracellular polysaccharides.

2. Analysis of Polysaccharide Characteristics of the Present Invention by Irradiation

Leaf fungus mycelium extracellular polysaccharide was dissolved in PBS at a concentration of 0.1 g / mL and irradiated with cobalt 60 gamma-irradiator (10 kGy / h). Irradiation doses varied within 1, 10, 20, 30, 40 and 50 kGy. After irradiation, the samples were stored refrigerated.

The molecular weight of the polysaccharide investigated was measured by gel permeation chromatography (GPC). The measurement conditions were PLaquagel-60 columns, 40 columns, 30 columns (300 X 7.5mm, 8 mm, Polymerlaboratories, Ltd., UK), the mobile phase was 0.1M sodium nitrate, flow rate was 1 mL / min.

The results showed that the higher the radiation dose, the smaller the size of the molecular weight. That is, the molecular weight of the extracellular polysaccharide of the leaf mycelium was 210 kDa, and showed a molecular weight distribution of 20 kDa at 50 kGy starting from about 115 kDa after radiation 1 kGy (FIG. 1).

3. Evaluation of the Physiological Function of the Polysaccharide of the Present Invention

(1) radiation protection effect

Ten 10-week-old C57 black mice were used per group to determine survival before radiation and post-differentiated polysaccharide treatment. Irradiation to the mouse was 10 Gy.

The sample preparation was prepared by dissolving the polysaccharide in PBS at a concentration of 0.1 g / mL and irradiating the same to prepare a low molecular weight polysaccharide. Sample treatment was injected into the abdominal cavity of mice 6 hours prior to irradiation. Mortality of the control and experimental groups was assessed 40 days after irradiation. The results are shown in Table 1 below.

In the case of the protective effect against irradiation, the smaller the molecular weight, the greater the protective effect against irradiation. That is, in the sample which did not control the molecular weight size, after the irradiation, the population began to die, and two survived until the 10th day, and all died on the 11th day. However, the survival rate after irradiation was increased in the sample having reduced molecular weight size, and the survival population was observed even at 40 days after irradiation in the samples of molecular weight size 48, 32 and 20 kDa. Therefore, it was evaluated that the protective effect against irradiation was the best at the molecular weight size of 48 kDa or less.

(2) Antioxidant ability  evaluation

In order to evaluate the mechanism of radiation protection effect, antioxidant activity was measured. Antioxidant activity was determined by DPPH and ABTS radical scavenging activity.

A. Measurement of DPPH free radical scavenging activity

2,2-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging activity was measured. That is, 2 mg, 0.2 mg, and 0.02 mg of samples were dissolved in 4 mL of methanol, 1 mL of 1.5 × 10 ⁻⁴ M DPPH solution (methanol) was added, and the absorbance was measured at 517 nm after being left for 30 minutes. At this time, ascorbic acid was used as a reference material for the activity comparison, and the absorbance ratio of the added group to the sample-free group was expressed.

In the case of DPPH radical scavenging ability, the scavenging ability of the sample without controlling the molecular weight was 11%. However, when the molecular weight was adjusted, the smaller the molecular weight, the higher the scavenging ability. The results are shown in Table 2 below.

B. Determination of ABTS radical scavenging activity

Total antioxidant activity was measured by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) cation decolorization assay method. That is, ABTS + ions were formed by leaving ABTS 7.4 mM and potassium persulfate 2.6 mM in a dark place for 12 hours, and then the solution was diluted with distilled water to have an absorbance of 0.7 at 734 nm. Sample 500 was added to 1 mL of diluted ABTS solution and reacted for 10 minutes at room temperature. The change in absorbance was measured using a spectrophotometer (GE healthcare, Pischaway, NJ, USA). In the case of ABTS radical scavenging ability, the scavenging ability was also 10% in the sample which did not control the size of the molecular weight, but when the molecular weight was reduced, the smaller the molecular weight, the higher the scavenging ability. The results are shown in Table 3 below.

Both assays showed high radical scavenging activity at molecular weight sizes of up to 48 kDa. These results are consistent with the results of the survival test using mouse, and the leaf mushroom mycelium extracellular polysaccharide was able to increase the antioxidant efficiency of the sample by controlling the size of the molecular weight small, and this increased antioxidant capacity ultimately induced after irradiation It is believed that the survival rate is increased by contributing to the elimination of radicals in the body.

4. Low molecular weight  Containing polysaccharides Cosmetics  Of composition and food Manufacturing example

(1) Production Example of Cosmetic Composition

Cream compositions were prepared according to the methods commonly used in cosmetic compositions using the ingredients and their contents set forth in the tables below.

ingredient content
(weight%) ingredient content
(weight%) Leaf mushroom mycelium extracellular polysaccharide 10 Disodium ethylenediaminetetraacetate 0.02 Allantoin 0.2 Dipropylene glycol 3.0 Diglycerin 5.0 Methyl Paraben 0.2 Polyacrylamide & C13-14 Isoparaffin Laureth 0.8 Met 2.0 Glyceryl Polymethacrylate & Propylene Glycol 1.5 Biosaccharide Gum-1 0.5 Cetostearyl alcohol 2.7 Profile paraben 0.1 Buchil Paraben 0.1 Tocopherol Acetate 0.05 Isopropyl myristate 2.0 Carboxyvinyl polymer 0.05 Squalane 3.0 Sorbitan cisquioleate 0.8 Glyceryl Stearate & Fiji-100 Stearate 1.5 Polysorbate 80 1.6 Shea butter 3.0 Dimethicone & Polysilicon-11 2.0 Cyclomethicone 2.0 Stearyl Dimethicone 2.0 Calcium hydroxide 0.05 Hyaluronic Acid (1%) 5.0 Imidazolidinylurea 0.3 Benzophenone-5 0.02 Combination incense Proper amount Purified water Add 100%

The cosmetic composition containing the low molecular weight mycelium mycelium extracellular polysaccharide of the present invention is not limited to the above formulations and compositions, and may be prepared in any formulations and compositions according to methods commonly used in the art.

(2) Production example of food

The leaf fungus mycelium mycelium of the present invention can be made of a product that can be administered through various routes as follows, but the use is not limited to the following form and composition.

A. Capsule

Leaf fungus mycelium extracellular polysaccharide 20.0% (w / w), lactose 79.5% (w / w), 0.5% magnesium stearate (w / w) components are mixed and dispersed in a capsule containing about 100 mg each.

B. Tablet

Leaf fungus mycelium extracellular polysaccharide 20.0% (w / w), magnesium stearate 0.5% (w / w), croscarmellose sodium 2.0% (w / w), lactose 76.5% (w / w), PVP (polyvinyl) Pyrrolidine) 1.0% (w / w) components are combined and granulated with a solvent such as methanol. Thereafter, the formulation is dried and tablets are formed with a suitable tablet press.

C. Beverage

9% by weight of the mycelium mycelium extracellular polysaccharide, 10% by weight fructose, 1% by weight per white sugar, 0.2% by weight of sodium citrate was added to 100% by weight of purified water and stirred to prepare a conventional beverage production method.

Claims (4)

After centrifugation of mycelial culture of leafy mushroom (Grifola frondosa), 4 times of ethanol (95%) was added to the filtrate, and the supernatant was precipitated for 24 hours, and then centrifuged again to remove the supernatant, freeze-dried, The obtained extracellular polysaccharide is irradiated with cobalt 60 radiation (γ) at 10 kGy / h to lower the extracellular polysaccharide. A polysaccharide having a molecular weight of 48 kDa or less derived from a leaf fungus mycelium extracellular polysaccharide.
delete A homeostasis functional cosmetic composition comprising an extracellular polysaccharide derived from leaf mushroom mycelium having a molecular weight of 48 kDa or less as an active ingredient.
A homeostasis functional cosmetic composition comprising an extracellular polysaccharide derived from leaf mushroom mycelium having a molecular weight of 48 kDa or less as an active ingredient.

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