WO2008062983A1

WO2008062983A1 - Hericium erinaceus mycelium comprising rice bran and ginseng steamed red and cultivation method thereof

Info

Publication number: WO2008062983A1
Application number: PCT/KR2007/005823
Authority: WO
Inventors: Hyun-Jun Park; Young-Deok Kim; Shin-Young Lee
Original assignee: Cj Cheiljedang Corporation
Priority date: 2006-11-22
Filing date: 2007-11-20
Publication date: 2008-05-29
Also published as: JP2010510305A; KR100826446B1

Abstract

The present invention relates to a production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate and Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red prepared by the same, aiming at the increase of production of mycelium by using rice bran or red ginseng extract based on liquid culture. More precisely, the invention relates to a production method of Hericium erinaceus mycelium comprising the steps of first cultivation of Hericium erinaceus and second cultivation of the primary culture solution with the addition of rice bran or red ginseng extract and Hericium erinaceus mycelium prepared by the method. According to the method of the present invention, the problems of the conventional solid culture method such as high costs, low productivity, ir¬ regularity of the product quality and deterioration of the product quality during shipping/ handling can be overcome and thus high quality Hericium erinaceus mycelium can be mass- produced with low costs. And the produced mycelium can be effectively used for the treatment of senile disease, cancer and immune disorder and for the activation of brain functions owing to the rice bran or red ginseng extract used as a substrate.

Description

HERICIUM ERINACEUS MYCELIUM COMPRISING RICE BRAN AND GINSENG STEAMED RED AND CULTIVATION

METHOD THEREOF

Technical Field

[1] The present invention relates to a production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate and Hericium erinaceus mycelium comprising the active ingredients of rice bran or ginseng steamed red prepared by the same, more precisely, a method for the mass-production of Hericium erinaceus mycelium comprising the active ingredients of rice bran or ginseng steamed red by using the rice bran or ginseng steamed red as a substrate with low costs, and Hericium erinaceus mycelium prepared by the above method, which has a treatment effect on senile disease, cancer and immune disorder and activates brain functions. Background Art

[2] Hericium erinaceus belongs to Basidiomycota, Aphyllophorales, Hydnaecae,

Hericium. Hericium erinaceus is one of important functional mushrooms having excellent effects and great potential for industrial applicability.

[3] Hericium erinaceus has pharmacologically active constituents such as NGF stimulator, HeIa cell growth inhibitor, immune regulator, anticancer component and dietary fiber (β-Glucan), etc. In particular, hericenone originated from the fruit body is a natural substance which was first identified and has been known to have the activity equal to adrenalin. Erinacine has been known to be the one who has the most powerful activity.

[4] It is well known that the sports drink Ηoutou' consumed by Chinese athletes in

1990 Asian games was made to contain Hericium erinaceus.

[5] Hericium erinaceus is one of edible mushrooms, which is distributed widely in the northern temperate regions of the northern hemisphere including Korea, China and Japan. This mushroom is a kind of white rot fungi which is parasitic on a broad leaf tree such as oak and chestnut, and the population grows mostly in the fall. Owing to the development of new cultivation technique by Liu, Hericium erinaceus is now able to be artificially cultivated (Liu. CY. 1981. Technique of cultivation of monkey head mushroom. Edible Fungi, No. 4, 33). The artificial cultivation such as hypae cultivation using PE (polyethylene) bottle with solid media has been already established in Japan, so that the mushroom is now sold at the market throughout the year [Ko. H.K. 1998. Cultural characteristics of Hericium erinaceus. M.S. thesis, Korea Univ.]. The artificial cultivation of Hericium erinaceus has recently been started in Korea.

[6] The major chemical constituents of Hericium erinaceus are carbohydrate and protein, and some of minerals or fat is included (Eisenhut, R., Fritz, D. and Tiefel, P. 1995. Investigations on nutritionally valuable constituents of Hericium erinaceus (Bull.:Fr) Pers. Gartenbauwissenschaft, 60(5), 212-218). The amounts of mineral as included are larger in the order of Na, P and Mg. The ash content might be varied with the log for cultivation or germ cart.

[7] The taste components of the mushroom are free amino acids, free sugars (trehalose, manitol, arabinitol, glucose, etc.) and organic acids (malic acid, fumaric acid, citric acid, α-ketoglutaric acid, etc.). In addition, nucleic acid component such as adenosine, guanine and guanoadenylic acid is included as a taste component, which is known to interact with amino acids to retain the taste longer as the synergic effect.

[8] Vitamin Bl, B 2 and niacin are detected but neither Vitamin A nor C is detected in this mushroom. Hericium erinaceus generally includes ergosterol which changes into vitamin D2 in the presence of light and heat and helps Ca absorption to play a certain role in preventing osteoporosis.

[9] In addition to the nutritional components, Hericium erinaceus contains pharmacologically active constituents such as HeIa cell growth inhibitor, NGF stimulator, immune regulator (BRM effect), anticancer polysaccharides, lectin, dietary fiber such as β-glucan, chitins and hetero-polysaccharides, and physiological activities corresponding to each component have been reported by recent studies [Mizuno, T. 1995. Yamabushitake, Hericium erinaceum: Bioactive substances and medicinal utilization. Food Reviews International, 11(1), 173-175].

[10] Hericium erinaceus is a popular herb medicine in China. Hetero-polysaccharides of

Hericium erinaceus have been known to have anticancer activity (particularly against stomach cancer, esophageal cancer, liver cancer and skin cancer) based on immune regulation. It has been also reported that Hericium erinaceus has therapeutic effect on stomach ulcer, duodenal ulcer, stomach cancer and esophageal cancer by strengthening immune system [Yang, Q.Y., and Jong, S. C. 1989. Medical mushrooms in China. Mush. ScL, 12(Part 1), 631-643] and another report says this mushroom promotes the anticancer activity and immune function [Ahn, D. K. 1992. Medicinal fungi in Korea. Kor. J. Mycol., 20,154-165 ].

[11] The effect of phenol-related compounds originated from fruit body having NGF stimulating activity (hericenone C, D, E, Y-A-8-C) or erinacine A - I originated from mycelium on the central nerve regeneration and the improvement of Alzheimer type dementia has also been reported.

[12] Since the new cultivation technique of Hericium erinaceus having the above effect was developed in 1981 by Liu, artificial cultivation of the mushroom based on solid culture has been tried. However, to produce fruit body by solid culture, it is important to regulate light, temperature and humidity properly. Extraction yield of the active ingredients by solid culture is very low, suggesting that the artificial cultivation of Hericium erinaceus by solid culture has disadvantages of high costs, low productivity, irregular quality and deterioration of quality of the product during shipping/handling and thus industrial mass-production seems to be very difficult.

[13] To overcome the above problem, liquid culture of Hericium erinaceus has been tried by Grigansky and Lomberth based on the conventional liquid culture which has been applied to the culture of approximately 60 kinds of edible mushrooms since Humfeld firstly tried the liquid culture for agaricus in 1948. However, the culture was performed in flask level, so that the yield of the mycelium was as low as 2.7 - 9.2 g/L and the culture time was as long as 10 days, requiring optimization of the culture condition.

Disclosure of Invention Technical Problem

[14] It is an object of the present invention, in order to to overcome the above mentioned problems, to provide a production method of Hericium erinaceus mycelium in which rice bran or red ginsing extract is added to the primary culture solution of Hericium erinaceus and which then is secondly cultivated to obtain Hericium erinaceus mycelium since the inventors found out that the rice bran or red ginseng extract was very effective in the mass-production of Hericium erinaceus mycelium. Precisely, according to the method of the present invention, the problems of the conventional solid culture method such as high costs, low productivity, irregularity of the product quality and deterioration of the product quality during shipping/handling can be overcome and thus high quality of Hericium erinaceus mycelium can be mass- produced with low costs.

[15] It is also an object of the present invention to provide Hericium erinaceus mycelium prepared by the method. Hericium erinaceus mycelium containing the rice bran and red ginseng as a substrate has a treatment effect on senile disease, cancer and immune disorder and activates brain functions.

[16] The above object of the present invention can be achieved by the following embodiments of the present invention. Technical Solution

[17] The present invention is described in detail hereinafter.

[18] To achieve the above object, the present invention provides a production method of

Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate comprising the steps of preparing a first cultivation solution of Hericium erinaceus mycelium and adding rice bran or red ginsing extract to the first cultivation solution and secondly cultivating the cultivation solution. [19] The present invention also provides Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red, which was produced by the method of the present invention. [20] In a preferred embodiment of the present invention, the first cultivation is performed by the following steps; preparing a medium comprising 5% of glucose,

0.2% of the mixture of yeast extract and peptone (1:1) and 0.2%of the mixture of KH

2

PO and MgSO (1:1); performing seed-culture in the conditions of pH 4 - 6, 20 - 3O⁰C and stirring speed of 100 - 200 rpm, with 50 mL of working volume and inoculation rate of the mycelium of 3 - 10% in the prepared medium; and performing main culture with the seed cultured above in a bio-reactor in the conditions of pH 5 - 6, 20 - 3O⁰C, stirring speed of 100 - 200 rpm and aeration rate of 1 - 5 vvm for 4 - 8 days.

[21] In another preferred embodiment of the present invention, the second cultivation is performed by the following steps; adding rice bran or red ginseng extract extracted with hot water to the main culture solution in the first cultivation to be at the concentration of 1 - 7% (v/v); and culturing the culture solution at 20 - 3O⁰C with 100 - 200 rpm for 2 - 3 days.

[22] In another preferred embodiment of the present invention, Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red is freeze- dried and pulverized to be applied in the pharmaceutical composition for the treatment of senile disease, cancer and immune disorder.

[23] The objects and characteristics and advantages of the present invention are better understood with the attached figures and the following descriptions.

[24] Figure 1 is a flow chart illustrating the production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate comprising the steps of first cultivation of Hericium erinaceus and second cultivation of the primary culture solution with the addition of rice bran or red ginseng extract.

[25] The first cultivation is performed by the following steps; preparing a medium; performing seed-culture; and performing main culture.

[26] To prepare an optimum medium for the culture of functional mycelium, the medium is composed of 5% of glucose, 0.2% of the mixture of yeast extract and peptone (1:1) and 0.2% of the mixture of KH₂PO₄ and MgSO₄ (1:1).

[27] The seed culture is performed in the medium prepared above under pH 4 - 6, at 20 -

3O⁰C and at stirring speed of 100 - 200 rpm, with 50 mL of working volume and inoculation rate of the mycelium of 3 - 7%. The main culture of the seed cultured above is performed to produce functional mycelium in a bio-reactor under pH 5 - 6, at 25 - 30⁰C with stirring speed of 100 - 200 rpm and aeration rate of 1 - 5 vvm for 4 - 8 days.

[28] The second cultivation is performed after adding rice bran or red ginseng extract to the primary culture solution. Precisely, 1 - 7% (v/v) of rice bran or red ginseng extract extracted with hot water is added to the primary culture solution, followed by further culture at 20 - 30⁰C with 100 - 200 rpm for 2 - 3 days. Centrifugation is performed to produce the mycelium. Brief Description of the Drawings

[29] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

[30] Figure 1 is a flow chart illustrating the production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate according to a preferred embodiment of the present invention. Best Mode for Carrying Out the Invention

[31] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

[32] The optimum medium composition and mycelium productivities according to the different time points of rice bran addition are described hereinafter and the optimum conditions for the seed culture for the comparison are also illustrated in experimental example 1.

[33]

[34] Experimental Example 1

[35] Mixture experiment was performed as described in Table 1 based on simplex- centroid design using MA (mixture amount) of 4 factor mixture components (water, C- source, N-source and mineral) and 6-level amounts (0, 1, 1/2, 1/3, 1/4 and 1/6).

[36] Table 1

Mixture experimental conditions according to simplex-centroid design using MA {mixture amount)

[37] Culture was performed in a medium with optimum composition under the conditions of inoculation rate of 5% and stirring speed of 100 rpm at 10 - 3O⁰C for 9 days. From the result of the culture, mixing ratio among components for the optimum composition was determined. As a result, optimum mixture composition was determined as follows; glucose 5%, the mixture of yeast extract and peptone (1:1) 0.2%, and the mixture of KH PO and MgSO (1:1) 0.2%.

2 4 ^σ 4

[38] The culture solution which had been cultured for 9 days to evaluate the optimum medium composition proceeded to the primary liquid culture at 25 - 35⁰C with aeration rate of 0.2 - 2.0 vvm and stirring speed of 100 - 300 rpm for approximately 8 days under the same conditions as described in table 2. After establishing the optimum culture condition, seed-culture was performed under pH 5 - 6, at 25 - 3O⁰C with stirring speed of 100 - 200 rpm for 4 - 8 days.

[39] Table 2

Variables for the determination of major factors for culture and their stages

[40] The primary liquid culture (main culture) was performed in the optimum medium determined above under the culture conditions provided above. 5 days later when the mycelium was fully grown, rice bran was added, followed by the secondary culture to increase the mycelium production.

[41] The amount of the rice bran added above was 1 - 7% (v/v) and more preferably 3 - 6% (v/v). The secondary culture was preferably performed at 20 - 5O⁰C (more preferably 20 - 3O⁰C) with standing or stirring. During the entire 8 day culture, the growth of mycelium was checked according to the time point of the rice bran addition. And the results are shown in Table 3.

[42] Table 3

Comparison of mycelium production

[43] As shown in Table 3, when rice bran was added on the 0 day of culture (which means from the beginning), the growth of mycelium was rather retarded which was consistent with the result of conventional mycelium culture with garlic, etc. When rice bran extract was added after one or more days from the culture start, the growth of mycelium seemed to be retarded at first. But, as the addition of rice bran extract was delayed longer, the production of mycelium increased rapidly. For example, when rice bran extract was added on 4 - 5 days later, the production of mycelium was far greater than that resulted from the conventional liquid culture, etc.

[44] More precisely, when rice bran or red ginseng extract was added in the early stage of culture, the mycelium production was approximately 1.5 g/L. When rice bran or red ginseng extract was added on the 4^th 5^th day of the primary culture, the production was 16.65 - 17.89 g/L in a 20, 75, and 300 L fermentor, which was at least 160 - 300% increased comparing with the production by the conventional liquid culture (4.25 - 10.77 g/L).

[45] The production method of Hericium erinaceus mycelium of the invention using rice bran or ginseng steamed red as a substrate and the Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red prepared by the above method can be modified. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims. Industrial Applicability

[46] As explained hereinbefore, the production method of Hericium erinaceus mycelium of the invention overcomes the problems of the conventional solid culture such as high costs, low productivity, irregularity of products, and deterioration of the production quality during shipping/handling and thereby favors mass-production of Hericium erinaceus mycelium. And the produced mycelium can be effectively used for the treatment of senile disease, cancer and immune disorder and for the activation of brain functions owing to the rice bran or red ginseng extract used as a substrate.

Claims

[1] A production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate comprising the steps of first cultivation of Hericium erinaceus to prepare primary culture solution and second cultivation of the primary culture solution with the addition of rice bran or red ginseng extract.

[2] The production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate according to claim 1, wherein the first cultivation is performed by the following steps; preparing a medium comprising 5% of glucose, 0.2% of the mixture of yeast extract and peptone (1:1) and 0.2% of the mixture of KH PO and MgSO (1:1); performing seed-culture using the

2 4 4 prepared medium under pH 4 - 6, at 20 - 3O⁰C and at stirring speed of 100 - 200 rpm, with 50 mL of working volume and inoculation rate of the mycelium of 3 - 7%; and performing main culture with the seed cultured above in a bio-reactor under pH 5 - 6, at 25 - 3O⁰C with stirring speed of 100 - 200 rpm and aeration rate of 1 - 3 vvm for 4 - 8 days.

[3] The production method of Hericium erinaceus mycelium using rice bran or ginseng steamed red as a substrate according to claim 1, wherein the second cultivation is performed by the following steps; adding rice bran or red ginseng extract extracted with hot water to the main culture solution of the first cultivation at the concentration of 1 - 7% (v/v); and culturing the culture solution at 20 - 3O⁰C with 100 - 200 rpm for 2 - 3 days.

[4] A Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red, which is produced by one of the methods of claim 1 - claim 3.

[5] The Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red according to claim 4, wherein the Hericium erinaceus mycelium containing active ingredients of rice bran or ginseng steamed red is freeze-dried and pulverized to be applied in a composition for the treatment of senile disease, cancer and immune disorder.