KR100470734B1 - Method for preparation of exo-polymers from submerged mycelial culture of Ganoderma lucidum TG(KCTC 10241BP) and the use thereof - Google Patents

Abstract

The present invention relates to a method for producing a polymer (Exo-polymer) from Ganoderma lucidum TG (KCTC 10241BP) mycelium culture medium and its use, and to synthesize Ganoderma lucidum TG (KCTC 10241BP) mycelium Inoculate the medium and stir at 50 to 150 rpm while supplying air at a rate of 1vvm at a pH of 4 to 7 and a temperature of 25 to 35 ° C to incubate the liquid for 10 to 30 days, and then centrifuge the culture solution at 5,000 to 15,000 x g for 10 to 30 minutes. After separation, the supernatant was treated with ethanol to obtain a precipitate, lyophilized, dissolved in distilled water, centrifuged and dialyzed, and the separated polymeric material was composed of 17.2% protein and 82.8% carbohydrate, and contained a glycoprotein having a molecular weight of 780kDa. When administered to mice, it has a very good effect of increasing glycogen concentration in muscle and liver, reducing lactate accumulation in serum, and enhancing exercise power. .

Description

(Method for preparation of exo-polymers from submerged mycelial culture of Ganoderma lucidum TG (KCTC 10241BP) and the use approximate)

The present invention relates to a method for producing a polymer material from the culture solution of Ganoderma lucidum mycelium, and more particularly, to the use of the present invention, Ganoderma lucidum TG (KCTC 10241BP) mycelium in liquid culture of the mycelium The present invention relates to a method of producing a polymer material and a polymer material having an effect of increasing glycogen concentration in the muscle and liver, reducing serum lactate accumulation, and enhancing exercise power when the polymer material is administered to a mouse.

Recently, many studies have been conducted to find natural substances that can enhance athletic and endurance, but only a few studies have been reported. In 1972, Cureton proved that the fermentation effect of wheat fermented milk and its active ingredient is octacosanol (Cureton, TK 1972. Charles C Thomas, Springfield.), Costill et al. The effect on behavior was reported (Costill, DL, GP Dalsky, and WJ Fink. Med. Sci. Sports. 10: 155-158., 1978). In addition, many studies have been conducted on the potentiation effect of capsaicin, a hot ingredient of red pepper, and Kim investigated the effects of capsaicin on the improvement of swimming endurance (Kawada, T., S. Sakabe, T. Watanabe). , M. Yamamoto, and K. Iwai. Proc. Soc.Exp. Biol.Med. 188: 229-33. 1988; Kawada, T., T. Watanabe, T. Takaishi, T. Tanaka, and K. Iwai. ..... Proc Soc Exp Biol Med 183:..... 250-256 1986; Kim, KM, T. Kawada, K. Ishihara, K. Inoue, and T. Fushiki Biosci Biotech Biochem 61: 1718-1723 1997.).

 However, there are many pharmacologically active ingredients isolated from Ganoderma lucidum mycelium. Although this research is being conducted, there is no research on the polymer material with the enhancing effect produced by Ganoderma lucidum mycelium.

The present inventors focus on the above, while searching for a new substance having a potency enhancing effect, extracting a polymer material from Ganoderma lucidum TG (KCTC 10241BP), the polymer extract is effective in enhancing swimming endurance in mice By confirming that the present invention was completed.

Accordingly, an object of the present invention is to provide a method for producing a polymer material from a liquid culture of Ganoderma lucidum TG (KCTC 10241BP) mycelium.

 It is another object of the present invention to provide a polymer material having a motion enhancing effect produced by the method.

The above object of the present invention is achieved by culturing Ganoderma lucidum TG (KCTC 10241BP) mycelium, extracting a polymer material, confirming the effect of enhancing the exercise power by administering the polymer extract to a mouse, and analyzing the components of the extract It was.

The present invention is cultured spawn of Ganoderma lucidum TG (KCTC 10241BP) mycelium and then homogenized to prepare the inoculum, inoculated in 200mL Ganoderma lucidum synthetic medium and incubated at 120 ℃ air supply speed to 1rpm at 120rpm 18 days of liquid incubation with stirring to obtain a supernatant by centrifugation of the culture and to obtain a precipitate by ethanol treatment to prepare a Ganoderma lucidum polymer extract; Measuring swimming endurance enhancing effect by administering the polymer extract prepared in the step to the mouse; Administering the polymer extract to a mouse to investigate the effect of glycogen concentration in the liver and muscle and the concentration of lactic acid in serum and analyzing the sugar and protein content of the polymer extract by phenol sulfate, Lawley method and gas chromatography It consists of.

Optimum culture conditions of Ganoderma lucidum TG (KCTC 10241BP) mycelium of the present invention was incubated for 10-30 days with stirring at 50-150 rpm at a temperature of 20-35 ℃, pH 4.0-7.0 and preferably 25-30 ℃ , pH 4.0-5.0 was adjusted.

In this step, Ganoderma lucidum TG (KCTC 10241BP) mycelium liquid culture solution was obtained by centrifugation at 5,000 to 15,000 × g for 10 to 30 minutes, and the supernatant was preferably centrifuged at 10,000 to 11,000 × g for 15 to 25 minutes. Separated.

 In the step, the supernatant obtained from the culture solution was treated with ethanol 3 to 5 times the volume of the supernatant to obtain a precipitate. The precipitate was lyophilized and dissolved in distilled water to prepare a polymer material.

The results of animal experiments were expressed as mean ± standard deviation, and the mean of the group was verified by p <0.05 level compared with Duncan's multiplex test.

 Ganoderma lucidum mycelium used in the present invention was separated by the following procedure using Ganoderma lucidum TG (KCTC 10241BP) deposited in the Biotechnology Research Institute and subcultured every three months.

 After harvesting from Juwangsan, Cheongsong-gun, Gyeongbuk, the spores of fruiting bodies were cut in a clean room and spores were collected on a plate containing antibiotics (Plate; potato dextrose agar medium, pH 4.5). Subsequently, oak sawdust medium (water content of 60% by weight) containing 10% by weight of rice bran was placed in a seed bottle and sealed, followed by sterilization at 121 ° C. for 60 minutes. Then, after cooling to room temperature in a sterile chamber was inoculated with purely isolated bacteria and incubated at 30 ℃. After the bacteria were completely grown in the spawn bottle, they were transferred to a growth chamber, and the caps were removed, and cultured under conditions of a humidity of 90% by weight and a temperature of 30 ° C. When the fungi of the mushrooms were grown, the humidity was reduced to 75% and incubated. After the spat was produced, it was cultured for 65 days to obtain an fruiting body, and formed mycelium from the spores and deposited it on the May 3, 2002, the Biotechnology Research Institute Gene Bank, and received the accession number KCTC 10241BP as of May 10, 2002.

 The experimental animal used in the present invention was used as a 5 week old mouse (Japan SLC, Hamamatsu, Japan). The temperature of the animal laboratory was maintained at 22 ± 0.5 ℃, humidity 50 ± 5% and the contrast was adjusted at 12 hour intervals. Moisture was freely supplied during the experiment, followed by general diet.

Glycogen concentration in this step was measured using a method such as Fushiki, T (Fushiki, T., K. et al., J. Nutr. 125: 531-539), and the concentration of L-lactic acid in serum was Kyowa Medes. It was measured by enzymatic analysis using a commercial kit (Determiner LA, Taoism, Japan).

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: Ganoderma lucidum mushroom Ganoderma lucidum ) Culture of TG (KCTC 10241BP) Mycelia and Extraction of High Polymers

Ganoderma lucidum TG (KCTC 10241BP) mycelium was mixed with 200ml of Ganoderma lucidum TG (KCTC 10241BP) mycelium (galactose 0.1%, sucrose 0.9%, xylose 0.1%, glucose 0.9%, yeast extract 0.05%, peptone 0.2%). , potato dextrose broth 0.2%, NH ₄ H ₂ PO ₄ 0.05%, DL-serine 0.05%, KH ₂ PO ₄ 0.1%, CaCl ₂ 0.06%, MgSO ₄ · 7H ₂ O 0.2%, FeSO ₄ · 7H ₂ O 0.002 %, ZnSO ₄ · 7H ₂ O 0.002%, MnSO ₄ · H ₂ O 0.002%, tiamine-HCl 0.0001%) and incubated for 18 days while stirring at 120rpm at 30 ℃. The cultured mycelium culture solution was extracted by the process step shown in Figure 1, first centrifuged at 10,447 × g for 20 minutes, then obtained a supernatant and treated with ethanol four times the volume of the supernatant to obtain a precipitate after freezing Dried. The lyophilized powder was dissolved in distilled water to a concentration of 10 mg / mL and centrifuged at 10,447 × g for 20 minutes to obtain a supernatant, followed by dialysis and lyophilization to separate a polymer (exo-polymer).

Experimental Example 1: Investigation of the exercise endurance effect of the feed intake and body weight of the mouse when the exo-polymer of the present invention is administered to the mouse

The polymeric material extracted from Ganoderma lucidum TG (KCTC 10241BP) mycelium of Example 1 was orally administered to mice, and the effects of oral administration on feed intake and body weight of mice were investigated. Table 3 shows the changes in feed intake and body weight over the three weeks. The administration of exo-polymer had no significant effect on body weight and feed intake. Furthermore, mice given oral administration of exo-polymer had no change in overall behavior and no animals were killed. From the above results, it was found that oral administration of Ganoderma lucidum TG (KCTC 10241BP) mycelium-derived polymer material is harmless.

Changes in Body Weight and Feed Intake According to Administration of Polymer Extract of Ganoderma lucidum TG (KCTC 10241BP) Mycelium Days Body weight (g / 3wk) Feed Intake (g / day) Control ^* GL Dosage ^** Control ^* GL Dosage ^** 0 34.50 ± 0.49 34.49 ± 0.47 - - 2 35.23 ± 0.63 35.63 ± 0.63 4.64 ± 0.06 5.31 ± 0.06 4 35.80 ± 0.59 36.23 ± 0.57 4.45 ± 0.05 4.98 ± 0.05 6 35.49 ± 0.61 35.96 ± 0.69 4.56 ± 0.06 4.79 ± 0.06 8 35.15 ± 0.59 35.74 ± 0.54 3.91 ± 0.05 4.37 ± 0.05 10 35.13 ± 0.61 35.71 ± 0.49 4.12 ± 0.06 4.69 ± 0.04 12 35.53 ± 0.57 35.85 ± 0.53 4.09 ± 0.05 4.48 ± 0.05 14 36.18 ± 0.59 36.92 ± 0.48 4.22 ± 0.05 4.48 ± 0.04 16 36.37 ± 0.53 36.96 ± 0.47 4.01 ± 0.05 4.22 ± 0.04 18 36.64 ± 0.59 37.07 ± 0.42 3.81 ± 0.05 4.09 ± 0.04 20 36.78 ± 0.53 37.15 ± 0.56 3.65 ± 0.05 4.06 ± 0.05 The experimental results were expressed as mean ± standard deviation. Control group ^* was salt ingested mouse, GL treatment ^** was ingested macromolecules extracted from Ganoderma lucidum TG (KCTC 10241BP) mycelia culture. Mouse.

Experimental Example 2: Ganoderma lucidum mushroom Ganoderma lucidum ) Measurement of swimming endurance ability of mice treated with polymer extract of TG (KCTC 10241BP) mycelium

Matsymoto (Matsymoto et al., J. Appl. Physiol., Physiol. 81: 1843-1849) was used to measure the effect of swimming ability. After a week of preliminary testing, the animals were able to swim at a rate of 81 iter / min for 30 minutes. The mice were divided into two groups, a control group (12 animals) and a GL administration group (12 animals), and the GL administration group was 100 mg / kg body weight of the polymer extracted from the Ganoderma lucidum TG (KCTC 10241BP) mycelium. Daily oral administration was performed using the stomach tube, and the control group consumed saline.

 A swimming endurance test of the orally administered mice was conducted. After swimming one round, the mice had a 48 hour recovery time. The cycle was continued for 3 weeks, and the maximum swimming time of the mouse was measured every cycle, and when the experimental animal failed to come out of the water surface to breathe within 7 seconds, it was judged to be tired and measured. (Matsymoto et al., J. Appl. Physiol., Physiol. 81: 1843-1849). In order to avoid deviation due to physical activity according to the biological cycle, each cycle experiment was conducted at 10: 00-18: 00.

 The experimental results are shown in FIG. 2. The effect of the polymers of the present invention on the swimming ability of the GL and control mice did not appear to be superior until 10 days, but after 12 days, the swimming ability of the GL treatment was significantly improved and was longer than 10 minutes compared to the control. I swam.

Experimental Example 3 Measurement of Glycogen Concentration and Serum Lactic Acid Concentration in Liver and Muscle

 To determine the reason why the endurance is enhanced, the glycogen, liver glycogen and serum lactate content of the mouse muscles were examined before and after swimming in the control and GL treatments.

48 h after the end of the three-week experimental period in Experiment 2, a high-molecular substance extracted from Ganoderma lucidum TG (KCTC 10241BP) mycelium (100 mg / kg) in half of the mice in the control and GL administration mice (6 mice each). Body weight) and salinity, respectively, followed by a 2 hour rest time and swimming for 20 minutes. Muscle and liver glycogen levels and serum levels of lactic acid were measured immediately after 20 minutes of swimming, and the values were compared with the other half of mice treated for 3 weeks.

 As shown in FIG. 3, after 3 weeks of stabilized group, the control group showed lower levels of intramuscular glycogen and hepatic glycogen compared to the GL-treated group, and the serum lactate level was not high, but was relatively high. appear. Twenty minutes after the swimming test, the glycogen levels in muscle and liver were significantly decreased in the control and GL treatments, but the glycogen levels in the GL treatments were higher than the control. The control group had an intramuscular glycogen of 34.42%, whereas the GL group had 43.11%, and the liver glycogen levels were 85.29% in the control group and 95.30% in the GL group. Twenty minutes after the swimming test, lactate accumulation increased to 18.61% in the control of mice. In contrast, lactate accumulation in the GL administration did not change significantly.

In conclusion, Ganoderma lucidum TG (KCTC 10241BP) mycelium-derived polymer material of the present invention has an endurance enhancing effect, and the polymer material had no effect on swimming time for the first 10 days, but after 12 days Because of the ability to enhance swimming ability, a certain amount of time ('period of habituation') was required for the reaction effect of the administration of the polymer substance. In addition, glycogen levels in the liver and muscle groups of the stabilized group, which were measured 48 hours after the swimming endurance test of 3 weeks, did not increase, but were again administered with the polymer of the post-swimming experimental group, which had been swimming for another 20 minutes. Mice showed higher endurance due to higher glycogen levels in the liver and lower accumulation of serum lactate in the liver and muscle than the control.

Experimental Example 4 Analysis of Sugar and Protein Contents of the Polymeric Material of the Present Invention

The sugar and protein content of the extracellular polymer material of the present invention prepared in Example 3 was analyzed. Total protein is Lowry, OH, Rosebrough, NJ, Farr, L., and Rindall, RJ, J. Biol. Chem ., 193, 256 (1951)] and bovine serum albumin was used as a standard. Total carbohydrate content was analyzed by phenol sulfate method using glucose and galactose (1: 1, w / w) as a standard, and the composition of amino acids and carbohydrates was determined by hydrolysis and acetylation methods. By HPLC and gas chromatography based on (Song, CH, BK Yang, KS Ra, DH Shon, EJ Park, GI Go, and YH Kim. J. Microbiol. Biotechnol. 8: 277-279. 1998). Measured.

The exo-polymer obtained from the culture of Ganoderma lucidum TG (KCTC 10241BP) mycelium is dissolved in 0.2 M NaCl, equilibrated with 0.2 M NaCl, and flow rate of 5 ml / tube volume. The gel-filtration by Sepharose CL-6B column (2.6 * 99cm) gave the fraction of molecular weight 780kDa.

In chemical analysis, the polymer material of the present invention contained 82.8% carbohydrate and 17.2% protein, and no acidic sugar was detected. Six sugars constituted the carbohydrate moiety, and histidine, serine, alanine and valine were identified as major amino acids of the protein, Ganoderma lucidum TG (KCTC 10241BP) mycelium. -polymer) was found to be a glycoprotein.

As described above, the present invention has the effect of providing a polymer material and its extraction method from Ganoderma lucidum TG (KCTC 10241BP) mycelium culture medium, and the polymer material has a potency enhancing effect It is a very useful invention in the pharmaceutical industry and functional food industry.

1 is a schematic diagram illustrating a method of obtaining a polymer material from a liquid culture solution of Ganoderma lucidum TG (KCTC 10241BP) mycelium of the present invention.

 Figure 2 is a graph showing the endurance measurement time is a result of measuring the time that the mouse submerged in the water no longer rise above the water surface.

 3 is a graph measuring changes in the concentrations of intramuscular glycogen, liver glycogen, and serum lactate in the experimental group stabilized for 48 hours after swimming for 3 weeks and the experimental group for 20 minutes.

Claims (2)

(a) Ganoderma lucidum TG (KCTC 10241BP) mycelium 0.1% by weight galactose, 0.9% by weight sucrose, 0.1% by weight xylose, 0.9% by weight glucose, 0.05% by weight yeast extract, 0.2% by weight peptone 0.2% by weight dextrose broth, 0.05% by weight NH ₄ H ₂ PO _4, 0.05% by weight DL-serine, 0.1% by weight KH ₂ PO ₄ , 0.06% by weight CaCl ₂ , 0.2% by weight MgSO ₄ .7H ₂ O, FeSO ₄ · 7H ₂ O 0.002 _{wt.%, ZnSO 4 · 7H 2 O} 0.002 _{wt.%, MnSO 4 · H 2 O} 0.002 wt.%, tiamine-HCl 0.0001 inoculated into a synthetic medium composition, in weight percent, which was stirred with 100~150rpm at 25 ℃ While spawning for 5 to 15 days;  (b) homogenizing the seed culture of step (a) on an ice water bath;  (c) 0.5 to 2.0% of the homogenized spawn in step (b) is inoculated into the fermentor containing the medium, and stirred at 50 to 150 rpm at an air feed rate of 1 vvm, pH 4 to 7, and temperature of 20 to 35 ° C. Culturing daily;  (d) separating the supernatant by centrifuging the culture solution of step (c) at 5,000 to 15,000 × g for 10 to 30 minutes;  (e) The supernatant of step (d) was treated with ethanol 3 to 5 times the volume of the supernatant to obtain a precipitate, dissolved in distilled water, and centrifuged at 5,000 to 15,000 x g for 10 to 30 minutes to obtain the supernatant. Method of producing a polymer material from the liquid culture of Ganoderma lucidum mycelium, characterized in that consisting of a step of dialysis.  A composition for exercise power enhancer, comprising ganoderma lucidum extract produced by the method of claim 1 as an active ingredient.