KR20050060725A - Method for producing mycelium of agaricus blazei murrill containing organic selenium - Google Patents

Abstract

The present invention relates to a method for producing an organic selenium-containing Agaricus blazei Murrill mycelium, wherein the inorganic selenium is incubated at 48 to 72 hours from the start of culture or 20 to 30 hours after the end of the culture. It is characterized in that it is added at the previous point, and the obtained mycelia powder as it is or by enzymatic treatment to obtain an extract having high water solubility and absorption, and applied to a variety of foods or cosmetics, such as the effect of Agaricus itself, such as anticancer, immune enhancement Together, the effect of selenium, which is essential for biological antioxidant activity, can act synergistically.

Description

Method for producing mycelium of Agaricus blazei Murrill containing organic selenium}

The present invention relates to a method for producing Agaricus blazei Murrill mycelium containing organic selenium. More specifically, inoculate Agaricus mushroom mycelium into a sterilized liquid medium and incubate by adding sterilized inorganic selenium to obtain Agaricus mushroom mycelium containing a large amount of organic selenium, or enzymatically treating the obtained mycelium to obtain a water-soluble extract. It is about.

Agaricus blazei Murrill is known to have the best anticancer activity among various mushrooms, and research on its biologically active components and effects has been continuously reported. Some of the studied effects are as follows.

(1) immune enhancing effect: activate human macrophages, complement and the like and induce various immune related cytokines;

(2) antitumor effect: inhibition of cancer progression and metastasis by activation of immune cells;

(3) anti-circulatory disease effects: improvement of hypertension, hypotension, heart disease, angina pectoris, myocardial infarction, arteriosclerosis, etc .;

(4) anti-endocrine disease effect: improving effect of diabetes, hyperlipidemia, allergy, etc .;

(5) anti-urinary diseases and anti-skin diseases: improving effects of cystitis, prostate hypertrophy, atopic dermatitis, dermatitis.

Such physiological activity was found to be due to the glucose polysaccharides of beta-1,3- and beta-1,6- structures, which are particularly present in Agaricus mushrooms (Mizuno, T. et al. Agri. Biol. Chem. , 54 (11), 2889-2896, 1990; Dong Q. et al. Carbohydrate Research , 337, 1417-1421, 2002). There is an increasing demand for and consumption of Agaricus mushrooms, and a large amount of beta glucan is obtained as a practical active ingredient. The design of the method became necessary. Algorithms for the production of fruiting bodies for Agaricus mushrooms (Korean Patent Laid-Open Patent Publication No. 2000-0011645), glucan-based liquid culture method (Korean Patent Laid-Open Publication No. 1999-0060704) have been devised.

Selenium is a physiological component of glutathione peroxidase (GSH-Px), a cytoplasmic antioxidant (Rotruck, JT et al. Science , 179, 588-590, 1973), which is an essential trace mineral for organisms. It has been reported to exhibit various physiological activities such as.

(1) antioxidant effect: removal of free radicals in the human body;

(2) immune enhancing effect: prevention of inflammatory healing and various diseases as an essential nutrient source;

(3) anticancer effect: prostate cancer, colon cancer, lung cancer, liver cancer, breast cancer, pancreatic cancer, etc .;

(4) antiviral effect: stop the progression of AIDS virus, hepatitis virus disease.

Selenium can usually be obtained from meat, fish and shellfish, and differs depending on the selenium content in the local soil. Particularly, in Korea, granite with low selenium content accounts for more than 70% of the whole country, so the selenium content in the soil is only 1/2 to 1/3 of the optimal area. In other words, there is a limit to the intake of selenium in the diet, the situation is necessary to supplement the additional selenium.

Currently, selenium is obtained by adding inorganic selenium salt in yeast culture in the form of yeast containing organic selenium having a high absorption rate in the body and safe for the human body (Korean Patent Laid-Open Patent No. 1997-0001530). The method of obtaining in the form of an edible mushroom containing organic selenium by adding (Korean Patent Laid-Open Patent Publication No. 2002-0059554) has been published. In addition, recently, a method of organically obtaining trace minerals such as chromium, selenium, and germanium through liquid culture of mushroom mycelium, which can effectively obtain beta glucan, has been devised (Korea Patent Publication No. 2002-0096575).

Therefore, in the present invention, by developing a method for producing organic selenium using the agaricus mushroom mycelium, selenium-containing agaricus mushroom mycelium which increased anticancer, antioxidant and various disease prevention effects of organic selenium based on the physiological activity of beta glucan, as it is, or By enzymatic treatment to obtain a high water-soluble and water-absorbent extract to be applied as a material of health food and functional food.

An object of the present invention is to inoculate agaricus mushroom mycelium in a liquid culture solution, and then, by adding and incubating inorganic selenium, to provide an organic selenium-containing agaricus mycelium, or a method for producing an extract having high water solubility and high absorption through enzymatic treatment thereof will be. In the present invention, while culturing the agaricus mushroom mycelium through the liquid culture mainly beta glucan to obtain the selenium in the organic form essential for the action of biological antioxidant enzymes to use the effects of beta glucan and organic selenium at the same time.

In order to achieve the above object, the production method of the organic selenium-containing Agaricus mushroom mycelium according to the present invention, liquid culture of the Agaricus mushroom mycelium, mycelium culture step of introducing the inorganic selenium into the medium during the culture; And a mycelium recovery step of removing the inorganic selenium remaining after the mycelium culture step and recovering the organic selenium-containing mycelium.

The production method of the organic selenium-containing Agaricus mushroom mycelium according to the present invention, the seed culture step of culturing the seed of Agaricus mushroom mycelium before the mycelium culture step; And a medium preparing step of preparing a medium of Agaricus mushroom mycelium.

The production method of the organic selenium-containing Agaricus mushroom mycelium according to the present invention, after the step of recovering the mycelium, further comprises the step of preparing a water-soluble extract of the mycelium by adding an enzyme to the recovered mycelium to react with the mycelium. It is characterized by.

In the method for producing an organic selenium-containing Agaricus mushroom mycelium according to the present invention, the mycelium culturing step is characterized in that the inorganic selenium is added to the beginning or end of the culture.

In the method for producing an organic selenium-containing Agaricus mushroom mycelium according to the present invention, the inorganic selenium is added at a time point 48 to 72 hours after the start of the culture or 20 to 30 hours before the end of the culture. In the method for producing the organic selenium-containing Agaricus mushroom mycelium according to the present invention, the inorganic selenium is sodium salt, calcium salt or selenium oxide of selenium.

In the method for producing an organoselenium-containing Agaricus mushroom mycelium according to the present invention, the inorganic selenium is characterized in that at least one selected from the group consisting of Na ₂ SeO ₃ , Na ₂ SeO ₄ , CaSeO ₃ and SeO ₂ .

In the method for producing an organic selenium-containing Agaricus mushroom mycelium according to the present invention, the inorganic selenium is added to the medium in an amount of 5 to 100 mg / L by weight of selenium.

In the method for producing an organic selenium-containing Agaricus mushroom mycelium according to the present invention, the step of preparing a water-soluble extract of the mycelium, any one or more mycelium cell wall selected from the group consisting of cellulase, protease, glucanase, chitinase and lysozyme The degrading enzyme is treated with purified water and then extracted simultaneously with sterilization with hot water. The organic selenium-containing Agaricus mushroom mycelium or the water-soluble extract of the mycelium according to the present invention is characterized in that it is prepared by the above-described manufacturing method.

The health food composition according to the present invention is characterized by containing the above-mentioned organic selenium-containing Agaricus mushroom mycelium or a water-soluble extract of the mycelium as an active ingredient.

The cosmetic composition according to the present invention is characterized by containing the above-mentioned organic selenium-containing Agaricus mushroom mycelium or a water-soluble extract of the mycelium as an active ingredient.

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

The mycelium production method according to the present invention includes the following steps to obtain an organic selenium-containing agaricus mycelium and a water-soluble extract thereof capable of simultaneously exerting effective physiological activities of selenium and agaricus.

1) spawn culture of Agaricus mushroom mycelium;

2) media preparation and sterilization of Agaricus mushroom mycelium;

3) culture of Agaricus mushroom mycelium and introduction of inorganic selenium;

4) recovery of organoselenium-containing Agaricus mushroom mycelium;

5) Preparation of water soluble extract of organo selenium-containing Agaricus mushroom mycelium.

If this is described in detail by process,

Step 1: cultivate spawn of Agaricus mushroom mycelium

Agaricus mushroom mycelium is cultured in agar medium or inoculated in liquid medium to shake culture.

Second Process: Media Preparation and Sterilization of Agaricus Mushroom Mycelium

The medium for the liquid culture of Agaricus mushroom mycelium is composed of glucose 1.0 ~ 10%, yeast extract and malt extract 0.2 ~ 1.0% and a small amount of inorganic salt, preferably glucose 4%, yeast extract 0.5%, malt extract 0.5% , KH ₂ PO ₄ 0.1%, MgSO ₄ 0.05% sterilized for 30 minutes at 121 ℃ and cooled to room temperature.

Step 3: Cultivation of Agaricus Mushroom Mycelium and Introduction of Inorganic Selenium

While maintaining the temperature of the incubator at 25 ~ 30 ℃, aseptic inoculation of Agaricus mushroom mycelium seedlings and incubated for 5 to 10 days. Preferably it is incubated for a total of 7 days at 28 ℃. At this time, the introduction of inorganic selenium is 48 to 72 hours after the start of the growth of stable Agaricus mycelium, or 20 to 30 hours before the end of the culture of the mycelium to minimize the effect of selenium on the mycelial growth, continuously or discontinuously Split the total dose over 10 to 20 hours. Inorganic selenium is dissolved in a small amount of purified water in advance, 0.2 to 0.45um membrane filtration, and then aseptically added while stirring the incubator. At this time, as the source of selenium, sodium and calcium salts of selenium such as Na ₂ SeO ₃ , Na ₂ SeO ₄ , CaSeO ₃ , or SeO ₂ may be used, and 5 to 100 mg / L is added by weight of selenium. Preferably, Na ₂ SeO ₃ is added to the medium at a concentration of 60 mg / L.

When the amount of inorganic selenium added in the production of Agaricus mycelium containing organic selenium is 5 to 100 mg / L as described above and the amount is less than 5 mg / L, it is difficult to expect the effect of selenium upon ingestion, and when it is more than 100 mg / L, Selenium inhibits the growth of mycelium and organicization of inorganic selenium is no longer achieved. Figure 2 shows the degree of organication of the added selenium by measuring the methylene blue reduction test (Methylene blue reduction test; US Patent No. 4,530,846).

4th step: recovery of organo selenium-containing Agaricus mushroom mycelium

After the completion of the culture, in order to remove the remaining inorganic selenium, 1 to 3 times purified water was added to the mycelium culture and stirred to recover the mycelium by compression filtration, centrifugation or the like. Alternatively, 1 to 3 times alcohol is added to the mycelium culture, and the precipitate produced by stirring is recovered by compression filtration, centrifugation, etc., and reprecipitated 2 to 3 times to recover the mycelium. Alternatively, a method of obtaining mycelium by alcohol precipitation of the primary mycelium obtained by washing with purified water is also possible.

When recovering mycelium, any of the above methods can be used, and the obtained mycelium is dried at 40 to 50 ° C. for 12 to 24 hours to obtain a final mycelium. The mycelia obtained at this time are rich in various nutrients, such as beta glucan and organic selenium, as well as proteins, carbohydrates, trace minerals, and by-products produced in culture.

Step 5: Preparation of Water-Soluble Extract of Organic Selenium-containing Agaricus Mushroom Mycelium

In order to increase the usefulness of the selenium-containing mycelium obtained in the third step, the mycelium cell wall degrading enzymes such as cellulase, protease, glucanase, chitinase, and lysozyme were purified to 35-55 ° C. and 12-24. Immediately after the time treatment is extracted with sterilization simultaneously with hot water of 80 ~ 100 ℃ to obtain a water-soluble selenium-containing Agaricus mushroom mycelium extract. It can be obtained by filtration into a liquid phase or drying to form a powder.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the present invention is not limited only to these examples.

Example 1: Preparation of organoselenium-containing Agaricus mycelium added with selenium at the beginning of culture

The culture medium was sterilized and cooled by adding 10 L of purified water, 4% glucose, 0.5% yeast extract, 0.5% malt extract and trace mineral salts, and then inoculated with agaricus mushroom mycelium spawn and incubated at 28 ° C. for a total of 7 days. 66 hours after the start of the culture, the inorganic selenium as Na ₂ SeO ₃ was administered in 10 divided doses at a concentration of 60 mg / L. After incubation, the recovered mycelium was washed twice with purified water and filtered to remove inorganic selenium. The same amount of ethanol was added thereto, the mycelium precipitate was collected, filtered and recovered by vacuum drying at 45 ° C. for 24 hours, followed by crushing to obtain about 250 g of organic selenium-containing Agaricus mycelium powder.

Growth of Agaricus mycelium by the culture is as shown in FIG. At this time, the inorganic selenium was introduced 48 to 72 hours after the growth of the mycelium is relatively stable culture to minimize the inhibition of mycelium growth by selenium, stimulate the protein synthesis of the mycelium to increase the amount of selenium can be substituted protein. Afterwards, supplemented with only medium and minimized external stimulation to maximize the production of beta glucan. The culture was terminated when the growth of mycelium slowed down.

The amount of selenium was compared using the mycelia by selenium-free culture as a control. At this time, selenium is measured by oxygen flask digestion method, Inductively Coupled Plasma Spectrometer, UV / VIS spectrophotometry, Fluorescence spectrophotometry, High Performance Liquid Chromatography, Atomic Absorption Spectophotometry (Atomic Absorption Spectophotometry) can be used in any method. The results are shown in the table below.

Control group (ppm) Experimental group (ppm) 0.15 414.28

On the other hand, beta glucan of the agaricus mycelium obtained by the present culture is known enzyme analysis method (McCleary, BV Journal of the Institute of Brewing , 91, 285-295, 1985; Manzi, P. Food Chemistry , 68, 315-318, 2000 ) Was quantified by pretreatment according to the prepared powder. The results are shown in the table below.

Control group (w / w%) Experimental group (w / w%) 31.16 30.38

As shown in Table 1 and Table 2, when the agaricus mycelium was cultured by adding inorganic selenium, the selenium content of the experimental group was much higher than that of the control group, and the content of beta glucan was about 30% in both the control group and the experimental group.

Example 2 Preparation of Organic Selenium-containing Agaricus Mycelium Added with Selenium 24 Hours Before End of Culture

The culture medium was sterilized and cooled by adding 10 L of purified water, glucose 4%, yeast extract 0.5%, malt extract 0.5%, and a trace inorganic salt, and then inoculated with agaricus mushroom mycelium spawn and incubated at 28 ° C. for 7 days. Twenty four hours before the end of the culture, the inorganic selenium as Na ₂ SeO ₃ was divided into ten doses of 30 and 60 mg / L, respectively. The recovered mycelium was washed twice with purified water and filtered to remove inorganic selenium. Half of the volume of the recovered mycelium was taken, the same amount of ethanol was added, the mycelium precipitate was collected, collected by filtration, vacuum-dried at 45 ° C. for 24 hours, and then ground to obtain about 130 g of organic selenium-containing Agaricus mycelium powder.

Growth of Agaricus mycelium by the culture is as shown in FIG. At this time, the inorganic selenium is introduced at the time when the growth of mycelium is slowed down, and there is no fear of inhibiting the growth of mycelium by selenium, and selenium may be added in a state in which beta glucan is stably cultured. In addition, there is an advantage that can be added by adjusting the amount of selenium according to the culture state.

The amount of selenium was measured and compared by fluorescence spectroscopy using the mycelium by selenium-free culture as a control. The results are shown in the table below.

Control (ppm) Experimental group 30mg / L (ppm) Experimental group 60mg / L (ppm) ` 0.20. 243.74 510.35

On the other hand, beta glucan of the agaricus mycelium obtained by the present culture is known enzyme analysis method (McCleary, BV Journal of the Institute of Brewing , 91, 285-295, 1985; Manzi, P. Food Chemistry , 68, 315-318, 2000 ) Was quantified by pretreatment according to the prepared powder. The results are shown in the table below.

Control group (w / w%) Experimental group 30mg / L (w / w%) Experimental group 60mg / L (w / w%) 30.10 29.10 31.06

When the agaricus mycelium was cultured by adding inorganic selenium as described above, the selenium content of the experimental group was increased in proportion to the amount of selenium administered, and the content of beta glucan was about 30% in both the control group and the experimental group.

Example 3 Preparation of Water-soluble Organic Selenium-containing Agaricus Mycelium Extract by Enzyme Treatment

Take half of the volume of mycelium recovered in Example 2 and sterilize mycelial cell wall degrading enzymes such as cellulase, protease, glucanase, chitinase, and lysozyme with hot water at 90 ° C immediately after treatment for 45 hours and 16 hours with purified water. And extract at the same time to obtain a water-soluble selenium-containing Agaricus mushroom mycelium extract. After filtration and lyophilization, 100 g of powdery extract was obtained.

The amount of selenium was compared with the extract treated with the mycelia by selenium-free culture as a control. The results are shown in the table below.

Control (ppm) Experimental group 30mg / L (ppm) Experimental group 60 mg / L (ppm) 0.12 250.20 495.37

On the other hand, beta glucan of the agaricus mycelium obtained by the present culture is known enzyme analysis method (McCleary, BV Journal of the Institute of Brewing , 91, 285-295, 1985; Manzi, P. Food Chemistry , 68, 315-318, 2000 ) Was quantified by pretreatment according to the prepared powder. The results are shown in the table below.

Control group (w / w%) Experimental group 30mg / L (w / w%) Experimental group 60mg / L (w / w%) 25.21 24.30 26.10

The mycelium obtained in Example 2 was used as a control to compare the water solubility before and after the enzyme treatment. The results are shown in the table below.

Control Experimental group 30 mg / L Experimental group 60 mg / L - +++ +++

The extract obtained by enzymatically treating the Agaricus mycelium cultured by adding inorganic selenium as described above had a selenium content similar to that before the enzyme treatment, and the beta glucan content was slightly decreased. This is because the insoluble portion of the mycelium glucan was removed in the extract manufacturing process, the resulting extract has a very good solubility in water has a variety of advantages in terms of absorption and forming.

Through the present invention, the agaricus mushroom mycelium containing the organic selenium can be produced in high yield, and the selenium-containing agaricus mushroom mycelium and the enzyme that increase the anticancer, antioxidant and various disease prevention effects of the organic selenium based on the physiological activity of betaglucan It can be used as a material for health foods and functional foods by obtaining an extract with high water solubility and water absorption, and further expand the scope of application to functional cosmetics. Specifically, the organic selenium-containing agaricus mushroom mycelium according to the present invention can be used in health foods and functional foods as a new material that can be expected in the efficacy of selenium and agaricus at the same time by formulating various tablets, capsules, pills, and the like. In addition, the water-soluble extract obtained through the enzymatic treatment is excellent in solubility and can be applied as a liquid product in addition to the formulation to increase the bioavailability and increase its usefulness, and furthermore can be utilized as a functional new material of cosmetics.

Figure 1 is a schematic diagram showing a preferred embodiment of the process for producing agaricus mushroom mycelium containing the organic selenium according to the present invention for each process.

Figure 2 is the MBRT time of the degree of organicization according to the amount of selenium added in the production method of agaricus mushroom mycelium containing the organic selenium according to the present invention.

3 is a growth curve of the selenium-containing Agaricus mycelium obtained according to the method of Example 1 of the agaricus mushroom mycelium containing the organic selenium according to the present invention.

4 is a growth curve of the selenium-containing Agaricus mycelium obtained according to the method of Example 2 of the agaricus mushroom mycelium containing the organic selenium according to the present invention.

Claims (11)

Mycelium culture step of liquid culture of Agaricus mushroom mycelium, introducing the inorganic selenium into the medium during the culture; And Method for producing an organic selenium-containing Agaricus mushroom mycelium, characterized in that it comprises a mycelium recovery step of removing the inorganic selenium remaining after the mycelium culture step and recovering the organic selenium-containing mycelium. The method of claim 1, After the mycelia recovery step, The method of producing an organic selenium-containing agaricus mushroom mycelium further comprises the step of preparing an aqueous extract of the mycelium to add an enzyme to the recovered mycelium to react with the mycelium. The method of claim 1, The mycelium culture step, An organic selenium-containing Agaricus mushroom mycelium, characterized in that the inorganic selenium is added to the beginning or the end of the culture. The method of claim 3, Method for producing an organic selenium-containing agaricus mushroom mycelium, characterized in that the inorganic selenium is added at a time point 48 to 72 hours from the start of the culture or 20 to 30 hours before the end of the culture. The method of claim 1, The inorganic selenium, A method of producing an organic selenium-containing Agaricus mushroom mycelium, which is sodium salt, calcium salt or selenium oxide of selenium. The method of claim 5, The inorganic selenium is Na ₂ SeO ₃ , Na ₂ SeO ₄ , CaSeO ₃ and SeO ₂ The production method of the organic selenium-containing agaricus mushroom mycelium, characterized in that at least one selected from the group consisting of. The method of claim 6, The inorganic selenium is produced in the organic selenium-containing Agaricus mushroom mycelium, characterized in that added to the medium in the amount of 5 to 100mg / L by the weight of selenium. The method of claim 2, The water-soluble extract manufacturing step of the mycelium, An organic selenium-containing agaricus mushroom mycelium characterized in that at least one mycelium cell wall degrading enzyme selected from the group consisting of cellulase, protease, glucanase, chitinase and lysozyme is treated with purified water and then sterilized and extracted simultaneously with hot water. Production method. A water-soluble extract of organoselenium-containing Agaricus mushroom mycelium or mycelium, characterized in that produced by the production method according to any one of claims 1 to 8. A health food composition comprising an organic selenium-containing Agaricus mushroom mycelium or a water-soluble extract of the mycelium according to claim 9 as an active ingredient.   A cosmetic composition comprising an organic selenium-containing Agaricus mushroom mycelium or a water-soluble extract of the mycelium according to claim 9 as an active ingredient.