KR20010046778A - Polymer obtained from Phellinus linteus basidiocarp and mycelial and process for preparation thereof - Google Patents

Abstract

PURPOSE: Polysaccharide obtained from a fruit body and a mycelium of Phellinus linteus and a process for preparing the same are provided, which exhibit excellent immune activity similar to polysaccharide obtained from a mycelium of natural Phellinus linteus. CONSTITUTION: The polysaccharide is obtained by extracting a mycelium of Phellinus linteus with hot water. Phellinus linteus is cultured by a process consisting of: culturing a mycelium at 21 to 22deg.C, disassembling a polypropylene bag for the growth of a mycelium when a color of the mycelium starts to change from yellowish brown to black and planting in a culture medium. The liquid cultivation of a mycelium of Phellinus linteus comprises the steps of culturing in a plate culture medium containing a potato dextrose agar medium at 25deg.C for 10 days and culturing in a medium for the production of polysaccharide at pH 4.5 at 25deg.C.

Description

Polysaccharide obtained from Phellinus linteus basidiocarp and mycelial and process for preparation

The present invention relates to a polysaccharide obtained from Phellinus linteus fruiting body and mycelium, and a method for producing the same. More specifically, the present invention relates to a polysaccharide excellent in immunological activity extracted and isolated from Prunus mushroom fruiting bodies and mycelium, and a method for producing the same.

Research on immunopotentiators that stimulate the immune system and affect anticancer effects or host resistance has been investigated from higher plants, higher fungi, bacteria, algae, and others (Franz). , 1989). In particular, many studies are being conducted on polysaccharides produced from mushrooms (basidiomycetes). Ikekawa et al. (1968) show that the extract obtained by hydrothermal extraction of fruiting bodies of edible fungi, including polyporaceae, has significant anti-tumor activity against host transplantation against animal transplant cancers such as sarcoma 180. Found. Polysaccharides such as Lentinan (Chihara et al., 1970) from shiitake mushrooms, Schizophyllan (Tabata et al., 1981) from skirt mushrooms, Krestin (Tsukagoshi and Ohashi, 1974) from fingering, and Meshima (Han et al., 1995) from situation mushrooms are currently It is widely used as an anticancer and immunotherapy agent.

Fruiting hot water extracts of Phellinus linteus ( Phellinus linteus) have been shown to have an anti-immune effect due to the combination of chemotherapy after surgery for liver cancer and patient resection with Ikekawa (1968). Immune activity (Lee et al., 1996) and anticancer activity (Jeong et al., 1993) have also been demonstrated from mycelial culture extracts. The mechanism of anti-tumor activity of polysaccharides extracted from situational mushrooms is not clear, but it is reported that they exhibit anti-tumor effects mainly by activating the immune system such as macrophage or complement system. Suzuki et al., 1989). In particular, Okuda et al. (1973) demonstrated a close relationship between antitumor activity and loss of serum hemolytic activity, and observed that activation of complement system components was involved in antitumor activity.

As a study on the physiological activity of natural mushrooms from nature, Ikekawa (1968) reported that fruiting hot water extracts were superior to the cancers of the digestive system, promoting cellular mediated immune responses (Maeda and Chihara, 1971). Or anti-cancer effects by restoring the antibody-producing ability of cancer patients with reduced immunity (Nomoto et al., 1975). However, since natural mushrooms are not easy to be harvested and quality uniformity has not been secured, the necessity of artificial cultivation of these mushrooms has emerged. ). In addition, studies on the anticancer activity of endo-polymers (Chung et al., 1993) and immunological activities (Kim et al., 1996) obtained by hydrothermal extraction of mycelium from situational mushrooms have been recently conducted. come. However, there have been no studies comparing the immunological activity of the fruiting body hydrothermal extract polysaccharides of artificial cultivated mushrooms and natural mushrooms, and endo-polymer and extracellular polysaccharide (exo-polymer) by liquid culture. No comparative studies have been reported on the immunological activity of).

The present inventors investigated the availability of artificially grown situational mushrooms as a substitute for natural situational mushrooms by measuring and comparing the immunological activity of polysaccharides obtained from natural state fruiting bodies and those obtained from artificially grown situational fruiting bodies. The fungal mycelium obtained by cultivation was measured by measuring the immune activity of the mycelial polysaccharide and the extracellular polysaccharide.

Accordingly, an object of the present invention is to provide a polysaccharide extracted and separated from the situation mushroom fruiting body and mycelium. Another object of the present invention to provide a method for producing the polysaccharide.

The object of the present invention is to recover the polysaccharides from the artificial mushroom cultured fruiting body and the situation mushroom fruiting body collected in nature and to obtain the in-cell polysaccharide and the extracellular polysaccharide from the liquid mushroom cultured mycelium in the same manner, and then This was achieved by measuring complement activity.

Hereinafter, the configuration and operation of the present invention.

Figure 1 shows the process of extracting and separating the polysaccharides from the artificial cultivated situation mushroom fruiting body.

2 shows a process of extracting and separating the intracellular polysaccharides and the extracellular polysaccharides from the liquid cultured mycelium mycelium.

Cultivating the situation mushroom mycelium, when the color is changed from yellowish brown to black, moving to fruiting body cultivator to induce mushrooms to artificially culture the situation mushroom; Cultivating the situation mushroom hyphae in PDA medium to obtain liquid mycelium by liquid culture using a synthetic medium; Hot water was extracted from the artificial mushroom cultured fruiting bodies, centrifuged, and then lyophilized, DIW dissolved, and centrifuged again to obtain polysaccharides from the artificial cultured fruiting mushrooms. After dividing the supernatant, the mycelium part was extracted with ethanol, centrifugation, lyophilization, DIW dissolution and centrifugation to obtain polysaccharides in the cells, and the supernatant part was subjected to ethanol precipitation, centrifugation, lyophilization, DIW dissolution and centrifugation. Obtaining an extracorporeal polysaccharide; It consists of measuring the anti-complement activity of the fruiting body polysaccharide, intracellular microsaccharides, extracellular polysaccharide obtained in the above step by the Mayer method.

Phellinus linteus ( Phellinus linteus) used in the artificial culture of fruiting bodies in the present invention was collected from Hongcheon, Gangwon-do, and isolated and identified (Song et al., 1997).

PDA (potato dextrose agar; Difco Co., Ltd.) was used as a medium for seed storage in the present invention, and the composition of the polysaccharide production medium was galactose 1g / L, sucrose 9g / L, xylose (Xyrose) 1g / L, Glucose 9g / L, Yeast extract 0.5g / L, Peptone 2g / L, Potato dextrose broth 2g / L, NH ₄ H ₂ PO ₄ 0.5g / L, DL-Serine 0.5g / L, KH ₂ PO ₄ 1g / L, CaCl ₂ 0.6g / L, MgSO ₄ ㆍ 7H ₂ O 2g / L, FeSO ₄ 0.02g / L, ZnSO ₄ 0.02 g / L, MnSO ₄ 0.02 g / L, thiamine-HCl 1 × 10 ⁻⁴ / L and the pH was adjusted to 4.5.

In the present invention, the spawn medium for fruiting body culture was sterilized by adding 10% of rice bran to oak sawdust, and wood for artificial cultivation used mulberry as a substrate.

IgM hemolysin sensitized sheep erythrocyte (EA cell) in the reagents used in the present invention was purchased from Japan Freeze-Drying Research Institute, and 5,5'-diethylbarbituric acid sodium salt was manufactured by Merk Co., Ltd. Normal hunman serum (NHS) was prepared and used directly by the inventors in a fresh state, and other reagents were commercially available first or express reagents.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: Fruiting body production by artificial culture

The optimum temperature for mycelial growth of Phellinus linteus was 25 ℃ ~ 30 ℃, but initially it was incubated at 21 ~ 22 ℃ to minimize the occurrence of various bacteria, and after 25 ~ 30 days when mycelium adhered to the whole surface of the logs From 25 ℃ was maintained at the optimum temperature. There are some differences depending on the thickness of the logs, but when the mycelium color starts to yellowish brown and turns to black (about 2 to 3 months), the polypropylene bag is dismantled and transferred to the grower for fruiting. Work was done. After the mycelial culture is completed in the incubation room, the logs are completely watered after removing the spawn seed used as inoculum on the upper surface, and when they are transferred to the soil, they protrude about 5-7cm to the ground and bury them. Induction of fats and oils and flanks.

Example 2: Mycelial Culture by Liquid Culture

Situation mushroomsPhellinus linteus) The mycelia were incubated on a plate medium containing PDA medium at 25 ° C. for 10 days, and then punched with 8 mm cork bore in diameter and used as an inoculum. The cells were incubated at 25 ° C. and 120 rpm for 30 days.

Example 3: Polysaccharide recovery from fruiting bodies and mycelium

The polysaccharide from the mycelium liquid culture in Example 1 was recovered by hydrothermal extraction of the situation mushroom fruiting body grown in Example 1 and the situation mushroom obtained in nature, as shown in FIG. 2. The polysaccharide and extracellular polysaccharide were separated and recovered. That is, the mycelium liquid culture was centrifuged at 10447 × g / 20 min, and precipitated at 4 ° C. for 24 hours to recover the precipitate by centrifugation. After freeze-drying, 10 mg / 1mL (DIW) was dissolved to separate the water-soluble polysaccharide, and the supernatant was taken and lyophilized again to use as a sample.

Experimental Example 1 Anti-complement Activity Measurement

Anti-complement activity was measured by Mayer method (Kabat and Mayer, 1961). Intracellular cultured polysaccharide (Fraction I) obtained in Example 3, in vitro polysaccharide (endo-polymer; Fraction III), microbial polysaccharide (Fraction II) obtained from nature, fruit mushroom polysaccharide (Fraction II), liquid cultured 50 μl of this sample was mixed with NHS (normal human serum) and GVB ⁺⁺ (gelatine veronal buffered saline, pH 7.2) using each of exo-polymer (Fraction IV) as a sample. After that, 350 μl of GVB ⁺⁺ was added to the reaction solution, which was continuously diluted from 10 to 160 times, and 750 μl of GVB ⁺⁺ and 250 μl of positive sensitized blood cells (10 ⁸ cells / ml) were added for 1 hour. After the reaction, PBS (phophate buffered saline, pH 7.4) was added in 2.5 mL portions, and the absorbance of the supernatant after centrifugation was measured at 412 nm. On the other hand, the control group was measured without the sample under the same conditions, and the anti-complement activity was expressed as the inhibition rate of TCH ₅₀ or ITCH ₅₀ compared with the control complement hemolysis (50% of total complement hemolysis, TCH ₅₀ ). In order to assay the correct activity of the sample, anti-complement polysaccharide CAP-0 (ITCH ₅₀ = 61.50%) of known titer was used as the standard.

As a result, as shown in Table 1, the anti-complement activity of the natural fruit mushroom fruit water extract polysaccharide was 65.77%, and in the case of artificial cultivation, 63.94% showed similar activity. Therefore, there was a possibility of artificial situation mushroom in terms of immune activity. In addition, as shown in Table 2, as a result of comparing the immune activity of the liquid cultured polysaccharides and the liquid cultured polysaccharides, the anti-complement activity of the extracted polysaccharides was 41.95% and the anti-complex polysaccharides. The activity was higher with 21.87%.

Anti-complement Activity of Sacred Mushrooms and Sacral Mushroom Polysaccharides from Nature source Anti-complement activity (ITCH ₅₀ %) ± SD ^a Artificial Culture Situary Mushroom Fruit Polysaccharide (Fr I) 63.94 ± 6.48 Natural situation mushroom fruiting body (Fr Ⅱ) 65.77 ± 2.54

Anti-complement Activity of Polysaccharides and Extracellular Polysaccharides in Strains of Liquid Culture source Anti-complement activity ((ITCH ₅₀ %) ± SD ^a Liquid Culture Situation Mushroom Polysaccharides (Fr Ⅲ) 41.95 ± 6.48 Liquid Culture Situation Mushroom Extracellular Polysaccharides (Fr IV) 21.87 ± 7.45

As described above, the artificial mushroom cultured fruiting body has an excellent effect of showing similar immunological activity as that of the polysaccharide obtained from the natural fruiting fruiting body, and also has the effect of exhibiting significant immune activity even in liquid cultured polysaccharides. Therefore, it is a very useful invention in the biopharmaceutical industry.

Claims (4)

Immune activity polysaccharide, characterized in that obtained by hydrothermal extraction from the artificially grown situation ( Phellinus linteus ) fruiting body. According to claim 1, Situation mushroom artificial cultivation of the mycelium initially at 21 ~ 22 ℃, the mycelium sticks to the entire surface of the wood and maintained at 25 ℃ from the start of growth, the mycelium color is yellowish brown to black When it starts to change the polypropylene bag (polyprophylene bag) for fruiting body growth and transfer to the cultivator planting polysaccharide with an immunological activity, characterized in that to induce mushroom development. Polysaccharide in cells with immune activity, characterized in that obtained by hot water extraction from liquid culture mycelium mycelium. The method of claim 3, wherein the liquid culture of mushroom mushroom mycelium is a mushroom mushroom The mycelia were incubated on a plate medium containing PDA (potato dextrose agar) at 25 ° C. for 10 days, and then, as inoculum, galactose, sucrose, xylose, glucose, yeast extract, peptone, potato dextrose broth, NH₄H₂PO₄, DL-Serine, KH₂PO₄, CaCl₂, MgSO₄7H₂O, FeSO₄, ZnSO₄, MnSO₄, Polyamine in the cell with immunoactivity, characterized in that it is cultured at 25 ℃, 120rpm in a medium for polysaccharide production containing thiamine-HCl, pH 4.5.