KR0156986B1 - Preparation process of polysaccharide by cultivating ganoderma lucidum - Google Patents

Abstract

The present invention relates to a method for producing β-1,3-glucan by liquid culture of Ganoderma lucidum under optimum conditions, which method has a C / N ratio of 7-9.5: 1 in the mycelium of Ganoderma lucidum, culture the liquid under conditions of pH 4.0-6.0, temperature 20-40 ° C., agitation speed 80-140 rpm to accumulate β-1,3-glucan in the medium, and harvest the β-1,3-glucan from the culture medium. Characterized by including.

Β-1,3-glucan produced from Ganoderma lucidum not only has excellent anticancer activity, but also has excellent gelling ability, and thus can be usefully used for preparing gelated foods or pharmaceuticals having anticancer activity in addition to food or pharmaceuticals.

Description

Production method of polysaccharides by liquid culture of Ganoderma lucidum

1 is a bar graph showing the effect of the stirring speed in the liquid culture of Ganoderma lucidum mycelium.

2 is a bar graph showing the effect of pH on the liquid culture of Ganoderma lucidum mycelium.

3 is a bar graph showing the effect of temperature on the liquid culture of Ganoderma lucidum mycelium.

4 is a bar graph showing the cell production and biopolymer production according to the nitrogen source in the liquid culture of Ganoderma lucidum mycelium,

5 is a bar graph showing cell production and biopolymer production according to the type of inorganic salt in liquid culture of Ganoderma lucidum mycelium.

FIG. 6 is a graph showing cell production and biopolymer production according to the concentration of KH ₂ PO ₄ , which is an inorganic salt, in liquid culture of Ganoderma lucidum mycelium.

7 is a graph showing gel permeation chromatography analysis of polysaccharides obtained from mycelium and culture medium after liquid culture of Ganoderma lucidum mycelium.

8 is a graph showing gel permeation chromatography analysis of polysaccharides obtained from culture medium after liquid culture of Ganoderma lucidum mycelium.

Figure 9 is a photograph showing the antimicrobial activity of the polysaccharide obtained from the mycelium (top) and culture medium (bottom) after the liquid culture of Ganoderma lucidum mycelium.

10 is a graph showing the anti-complement activity of polysaccharides obtained from culture medium after liquid culture of Ganoderma lucidum mycelium.

11 is a graph showing the anti-complement activity of polysaccharides obtained from the mycelia after liquid culture of Ganoderma lucidum mycelium.

12 is a gel filtration chromatography result for determining the molecular weight of the polysaccharide obtained from the culture medium after the liquid culture of Ganoderma lucidum.

Figure 13 is a photograph showing the gel-forming ability of the polysaccharide obtained from the culture medium after liquid culture of Ganoderma lucidum.

The present invention relates to a method for producing an anticancer polysaccharide from Ganoderma lucidum mushroom, and more particularly, liquid mycelium of Ganoderma lucidum mushrooms to accumulate extracellular polysaccharides in the medium, and harvesting the polysaccharides from the culture medium. The present invention relates to a method for producing polysaccharides from Ganoderma lucidum mushroom and new uses of the polysaccharides.

Ganoderma lucidum is known to be effective in hypertension, diuresis, interpolation, tonic, cardiac, gastric ulcer, fever, anemia, tuberculosis, bronchitis, and sarcasm. Ganoderma lucidum also has effects on cancers such as gastric cancer, lung cancer, and esophageal cancer, and β- (1,3) -D-glucan (hereinafter referred to as polysaccharide) having a branch of β- (1-6) -glucosyl has anticancer activity. It is revealed. This polysaccharide has an average molecular weight of 50-20 million Daltons and a specific optical intensity [α] _D of + 80-20 °.

Ganoderma lucidum polysaccharides are used as health foods in Japan (JHFA Food Handbook, pages 24 and 102, Japan Health Food Association, 1990) .Acute toxicity at oral administration is 1 g / kg in rats and 3.3 g / kg in rats. It is 1.78g / kg in Mole Mot (Mushroom Cultivation Education, p. 458, Nonghyup College, 1987).

As a method of obtaining such anti-cancer active polysaccharides from Ganoderma lucidum mushroom, a method of extracting it from the Ganoderma lucidum produced by solid culture using an organic solvent (Hyunjin, et al. , p. 58-68 (1990)) and two methods (homology) of extracting and separating useful substances from mycelia or culture filtrates cultured by liquid culture are known.

In the case of the former solid culture, it is difficult to supply a mushroom fruit body of uniform quality, a process of extracting and separating useful materials from the fruit body is difficult and requires a lot of labor. On the other hand, in the case of liquid culture, it is possible to always culture under constant conditions, to obtain a uniform mycelium and a culture solution, and there is an advantage that the acquisition of desired useful substances is simple.

However, there have been reports of trace polysaccharides present in culture filtrates obtained by liquid culture of ganoderma lucidum (Sone et al., Agric. Biol. Chem., 49, 2642, 1985). Since polysaccharides were not intended to produce polysaccharides, but were limited to the growth conditions for mycelial growth or to the components of mycelium extracts, the amount of polysaccharides obtained in these studies was very low, 0.39-0.85 g / l, but the production of extracellular polysaccharides from Ganoderma lucidum. It was only to show the possibility of. In addition, in the case of liquid culture of the hyphae, the recovery rate of the polysaccharides is lower than that obtained by extracting from the fruiting body.

Under these circumstances, the present inventors studied extensively to find the optimal fermentation conditions capable of accumulating extracellular polysaccharides at an increased yield in order to solve the problem while taking advantage of the liquid culture described above. The invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing polysaccharides of Ganoderma lucidum by accumulating extracellular polysaccharides by liquid culture of mycelium of Ganoderma lucidum and harvesting the polysaccharides from the culture medium.

In order to achieve the above object, the present invention is liquid mycelium cultured under the conditions of C / N ratio of 7 to 9.5: 1, pH 4.0-6.0, temperature 20-40 ℃, stirring speed 80-140 rpm The method for producing β-1,3-glucan by liquid culture of Ganoderma lucidum mycelium characterized by accumulating β-1,3-glucan in the medium and harvesting the β-1,3-glucan from the culture medium. to provide.

The present invention also provides glucose 3-10% (w / v), yeast extract 0.1-1.0% (w / v), (NH ₄ ) ₂ HPO ₄ 0.05-0.3% (w / v) as a medium for the liquid culture. And a medium containing KH ₂ OP ₄ 0.01-0.2% (w / v).

Furthermore, the present inventors extensively examined the activity of the polysaccharides of Ganoderma lucidum mushroom obtained by the above-mentioned method, and found that it showed not only the anticancer activity already revealed but also the outstanding gelation ability. Accordingly, the present invention also provides a novel use of using the polysaccharide of Ganoderma lucidum as a gelling agent.

Other objects and applications of the present invention will become apparent to those skilled in the art from the following detailed description.

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

Ganoderma lucidum strain used in the present invention is ganoderma lucidum No. 1 state that was sold by the Rural Development Administration, which can always be sold by the Rural Development Administration.

Fermentation conditions of the present invention has a C / N ratio in the range of 7-9.5: 1, pH 4.0-6.0, temperature 20-40 ℃, stirring speed 80-140 rpm. After culturing for about 7 days under the above conditions, the size of the pellet can be maintained at 2-3.5 mm. If the pellets were larger than this, it was difficult to transfer oxygen or absorb nutrients into the cells, resulting in a decrease in the amount of cells and the production of polysaccharides.

In addition to glucose, water-soluble starch, mannose, lactose, maltose can be used as the carbon source, and mal extract, ammonium sulfate, ammonium chloride as the nitrogen source, dipotassium hydrogen phosphate and magnesium sulfate as the inorganic salt can be used. It doesn't happen.

Liquid culture can be performed by all methods of flask culture, batch type, fed-batch, continuous culture.

A method for measuring the cell mass and the amount of polysaccharide produced in the present invention is introduced.

(1) Cell weight: The culture solution was centrifuged at 2000 g for 30 minutes to obtain a precipitate, and washed three times with distilled water. After drying for 24 hours at 60 ℃ weight was measured.

(2) Polysaccharides: Polysaccharides include polysaccharides (MSW) obtained from cells and polysaccharides (BSW) from culture filtrates.

Cellulose polysaccharides (MSW: water-soluble biopolymers obtained from mycelium) were suspended in distilled water with 5% of the dried cells, and refluxed for 24 hours in a 100 ° C water bath. The precipitate was obtained by centrifugation at 2500 g for 30 minutes, the supernatant was concentrated, dialyzed for 5 days with a dialysis membrane (250-7U Dialysis sack manufactured by Sigma), and freeze-dried at 0.05 Torr for 24 hours.

The polysaccharide (BSW: water-soluble biopolymer obtained from the culture medium) in the culture filtrate was obtained by ultrafiltration for 20 hours using a molecular weight 100,000 ultrafilter (product of Sartorius, model name Sartocon mini SM 17521), and then doubled acetone. Was added to obtain a precipitate. It was dissolved in distilled water with stirring for 24 hours, dialyzed for 5 days, and lyophilized at 0.05 lot for 24 hours. The supernatant obtained by suspending in distilled water and centrifuged at 2500g for 30 minutes was concentrated and freeze-dried.

The anticancer effect of polysaccharides was measured according to the method of Scdieero et al. (Scudieero et al., Cancer Res., 48, 4827, 1988). Rat leukemia cell line L1210 received from ATCC was suspended in RPMI 1640 medium with 10% calf serum and inoculated in a 96 well plate. Dissolve the polysaccharides in phosphate buffer and add 20 ul to each well. The inoculation amount is diluted so that the absorbance at 540 nm is about 0.6-0.7 after the experiment. Incubate at 37 ° C. for 4 days in an incubator supplied with 5% carbonic acid gas, and 0.1 mg of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazollum bromide manufactured by Slgma) Add to the wells and incubate for another 4 hours. Subsequently, the plate is centrifuged at 100 g for 5 minutes to remove the medium, and 150 μl of dimethyl sulfoxide is dissolved to dissolve formazan, and the absorbance at 540 nm is measured.

The present inventors not only measured the anticancer activity of the polysaccharides obtained by the present invention but also investigated various other functions, and for the first time, found that β-glucan, the polysaccharide of Ganoderma lucidum mushroom, had gelation ability. Thus, the present invention also provides novel uses of β-glucans as gelling agents.

The present invention will be described in detail by way of examples, but the inventive concept is not limited to the embodiments.

First, various experimental methods performed in order to find the strain and the optimum medium composition used in the present invention.

(1) Strains The strain used in the present invention was Ganoderma lucidum (Zone 1), and was sold from Suwon Rural Development Administration. After incubating at 30 ° C. for 7 days in P. D. A. (potato dextrose agar) medium of Table 1, it was preserved at 4 ° C. and used for experiments with subculture every 8 weeks.

(2) Medium composition and preparation

The spawn medium and the biopolymer production medium used in this experiment are the shaking culture medium (Table 2) of Ganoderma applanatum. After 15 minutes autoclaving at 121 ℃ was used, pH was adjusted to 0.1N HCl or 0.1N NaOH.

(3) Cultivation method For preculture of inoculated bacteria, the mycelium grown on the PDA plate medium was made using a 5 mm diameter stainless steel pipe to make a mycelium disk. Was inoculated into a 100 ml Erlenmeyer flask. After incubation at 30 ° C. for 7 days, 5% (v / v) of the whole culture solution was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of the above medium, followed by rotation shaking culture at 100 ° C. at 30 ° C. for 4 days.

Example 1

Influence of the stirring speed

The culture was carried out under the conditions described above, but was incubated at 30 ° C. for 7 days while changing the stirring speed to 0 to 250 rpm, and the growth of the cells and the production of the biopolymers were investigated according to the stirring speed.

The results are shown in FIG.

As shown in FIG. 1, the productivity of the microbial cells and the biopolymer was the best when the stirring speed was 100 rpm, and was slightly decreased. This was thought to be because normal cell growth was difficult due to the increase of shear force at agitation speed of 100rpm or higher, whereas oxygen transfer into the cell and absorption or utilization of medium nutrients were increased as the size of mycelium increased under agitation speed below 100rpm. This was considered to be because of the difficulty and the growth of mycelia.

Example 2

Effect of pH and Temperature

The influence of pH was investigated by rotating shaking culture at 30 ° C. for 7 days by adjusting the medium of Table 2 to have a pH range of 4 to 8, and the influence of temperature was rotated for 7 days at pH 5 at a temperature range of 20 to 40 ° C. After shaking culture, the mycelial growth and dry weight of biopolymer were measured and investigated. Other culture conditions were the same as described above.

The results are shown in FIGS. 2 and 3, respectively.

As shown in FIG. 2, the production of cell and biopolymers was the best at pH 5, and the growth was relatively good at pH 4-7, but the production of cell growth and biopolymers was rapidly decreased at pH 8.

On the other hand, as shown in Figure 3, the cell growth and biopolymer productivity was good in the temperature range of 25 ℃ ~ 30 ℃, the growth of the cells was the best at 25 ℃, the production of biological polymer was somewhat high at 30 ℃. However, above 30 ℃, the growth of the cells and the production of biopolymers showed a sharp decrease. Therefore, the growth of the cells was the best at 25 ℃, considering the productivity of the biological polymer, the culture temperature is preferably in the range of 25 ~ 30 ℃.

Example 3

Impact of Carbon Sources

Influence of carbon source was incubated at 30 ° C for 7 days by adding various carbon sources such as fructose, xylan, mannose, sucrose, soluble starch at 5% (w / v) concentration in place of glucose, which is the carbon source, in the basic medium of Table 2 The dry weight of the mycelium and the biological polymer was measured and investigated. As a result, cell growth was best in soluble starch and relatively good in mannose, lactose and maltose.

On the other hand, the productivity of the biopolymer was the best in the monosaccharide glucose, relatively good also in fructose, xylose, maltose and soluble starch.

Therefore, glucose is the optimal carbon source for the production of biopolymers in cells. As a result of experiments by changing the concentration of glucose, the growth of cells increased rapidly up to 3% (w / v), but slowly increased at higher concentrations. Production peaked at 5% (w / v) and slightly decreased at higher concentrations. This is expected to be due to the significant decrease in conversion efficiency to polysaccharides with the addition of high concentrations of glucose.

Therefore, the concentration of glucose was determined to be 5% (w / v) representing the maximum production of biological polymers, and also predicted the catabolite repression due to the high concentration of glucose (fed batch), fed batch We can predict that culture will be effective.

Example 4

Effect of Nitrogen Sources

The effect of the nitrogen source is the same as that of the carbon source by adding various nitrogen sources shown in FIG. 4 at a concentration of 0.5% (w / v) instead of the nitrogen source of the base medium under the optimum carbon source concentration obtained in Example 3 of the basic medium composition shown in Table 2. It was investigated. When two or more nitrogen sources were used, the total nitrogen source concentration was adjusted to 0.5% (w / v).

The results are shown in FIG.

As can be seen from the results of FIG. 4, the productivity of the microbial cells and the biopolymer was good even when the yeast extract and the malto juice were mixed with the organic nitrogen source as in the control group, but the yeast extract and the inorganic nitrogen source (NH) HPO were mixed. When added, the productivity slightly increased. The highest biopolymer productivity was obtained when yeast extract 0.4% (w / v) and (NH) HPO 0.1% (w / v) were added.

On the other hand, the same experiments with varying concentrations of these nitrogen sources showed that the yield of biopolymer was the best at the yeast extract concentration of 0.5% (w / v), and the cell growth was the best at 1.5% (w / v). In addition, when 0.1% (w / v) of inorganic nitrogen (NH) HPO was added, there was no significant effect on the growth of the cells, but there was some synergistic effect on the production of the biopolymer.

Example 5

Influence of Inorganic Salts

Cell growth and living organisms of various inorganic salts using an inorganic salt of the kind shown in FIG. The effect on polymer production was investigated. In the case of using two or more kinds of inorganic salts, the concentration of the total inorganic salts was adjusted to 0.15% (w / v).

The results are shown in FIG.

As can be seen in FIG. 5, cell growth was best when the control group KHPO, KHPO and MgSO7HO were mixed at 0.05% (w / v) concentration and 0.15% (w / v) concentration was added. The production of the polymer showed almost the same yield as the mixed addition even when KHPO was added alone at a concentration of 0.15% (w / v).

Therefore, KHPO is most appropriate for inorganic salts, and the result of examining the effect of its concentration is shown in FIG. As shown in FIG. 6, even when inorganic salts were not added, the production of biopolymers was about 7.6 g / 1, showing the highest productivity at 0.05% (w / v) concentration. Production no longer increased.

Therefore, the concentration of KHPO was 0.05% (w / v), and the biopolymer production at this time showed the maximum value of 15.4 g / 1.

From the results of the optimum medium composition obtained from the above, glucose 5% (w / v), yeast extract 0.15% (w / v), divalent ammonium phosphate 0.1% (w / v) and monovalent potassium phosphate salt 0.05% (w / v).

Example 6

Production and separation of biopolymers

Ganoderma lucidum was incubated for 7 days under the optimum conditions established in Examples 1 to 5 above, and the biopolymer was isolated as follows.

1) Separation from Mycelium

The culture solution was centrifuged at 2500 × g for 30 minutes to obtain mycelium from the precipitate, which was washed with distilled water several times, and freeze-dried to obtain a yellow mycelium powder (crude mycelium powder: CMP).

The mycelium powder thus obtained was refluxed with distilled water with stirring for 24 hours in an oil bath at 100 ° C., and centrifuged at 2500 × g for 30 minutes. The supernatant was concentrated and dialyzed, and freeze-dried at 0.05torr for 24 hours to obtain MWS (water soluble biopolymer obtained from mycelium) which is yellow biopolymer fraction from mycelium.

2) Isolation from Culture Filtrate

Separation of extracellular biopolymers from the culture filtrate was carried out in a concentrated filtrate obtained by centrifugation of the filtrate for 15-20 hours using an ultrafiltration system (Sartocon Mini SM 17521, West Germany) with a membrane having a molecular weight limit of 100,000. Two times acetone was added to obtain a precipitate.

A small amount of water was added to the precipitate, suspended, concentrated on a rotary evaporator, and dialyzed for 4-5 days. After lyophilization for 24 hours at 0.05torr to give an extracellular crude biopolymer (crude exo-biopolymer, CBP), and the sample was dissolved by stirring for 24 hours in distilled water at room temperature.

The supernatant was obtained by centrifugation at 2500 × g for 30 minutes, and the supernatant was freeze-dried at 0.05torr for 24 hours to obtain a water-soluble extracellular biopolymer fraction, BWS (water-soluble biopolymer obtained from the culture solution).

As a result, the cell weight was 18.8 g / 1, the NWS polysaccharide was 8.27 g / 1, and the BWS polysaccharide was 15.4 g / 1.

3) Purification of Biological Polymers

① DEAE-cellulose (C1) ion exchange chromatography

The biological polymer samples MWS and BWS isolated from the mycelium and the culture filtrate were subjected to DEAE cellulose (C1) ion exchange chromatography. As a result, first, MWS, a mycelium extract, obtained the heavy component fraction (MWS-DN) from the non-adsorption sphere and the acid component fraction (MWS-DA) from the adsorption sphere, respectively.

In general, acidic polymers having polyvalent anionic properties form insoluble complexes with tetravalent ammonium salts, especially CPC (cethyl pyridinium chloride) or cetavlon (cethyl trimethyl ammonium bromide). MWS-DA is treated with CPC, resulting in CPC complexes. Therefore, it was confirmed that the polymer is an acidic polymer having polyvalent anionic properties. In addition, both sugar and protein components were detected in the heavy and acid components, which were considered to be protein-deficient polysaccharides.

On the other hand, in the case of BWS isolated from the culture filtrate, the heavy component fraction (BWS-DN) was obtained at the non-adsorption port, and the acid component (BWS-DA) was obtained at the adsorption port.

Acid peaks were thought to be composed of various acidic polymers with different ionic strengths due to the detection of several peaks according to the concentration gradient (0.-2.0M NaCl). Could.

BWSs were further identified by the methylation analysis as having a β-1,3 binding backbone.

② Gel Chromatography

Gel permeation chromatography (GPC) by Sepharose CL-48 was performed on various neutral and acidic polymer samples MWS-DN, MWS-DA, BWS-DN and BWS-DA obtained by DEAE-cellulose (C1) ion exchange chromatography. The results for BWS-DN and BWS-DA are shown in FIGS. 7 and 8, respectively.

First, MWS-DN, the heavy component fraction of the mycelium extract, represented two sugar fractions containing protein, and was considered to be a mixture of two kinds (MWS-DN-GI and MWS-DN-GII) having different molecular weights. In addition, MWS-DA, which is a mycelium extract acid component extract, was considered to be a mixture of two kinds (MWS-DA-GI, MWS-DA-GII) with different molecular weights. High. However, BWS-DN, an extracellular biopolymer heavy chain, consisted of only two sugars with different molecular weights (BWS-DN-GI, BWS-DN-GII) as shown in FIG. As shown in FIG. 8, it consisted of two kinds of sugar mixtures.

Fractions of relatively high molecular weight (MWS-DN-GI, MWS-DN-GI, BWS-DN-GI, BWS-DA-GI) were collected by the above GPC, and lyophilization was performed for the following experiments, respectively.

Example 7

Biological Activity of Biological Polymers

(1) antimicrobial activity test

The antimicrobial activity of the biopolymer was measured by a paper disk diffusion method. A paper plate was placed on the agar medium inoculated with the test bacteria, and 15 µl of each prepared 1 mg / ml concentration sample was inoculated, incubated at the proper growth temperature of each test bacteria, and growth inhibition of the test bacteria was observed.

As for the test bacteria used for the measurement of antimicrobial activity, Bacillus cereus was used as gram positive bacteria and E. coli was used as gram negative bacteria. The results of the antibacterial activity test using the culture filtrate and the biological polymer samples (10 species) are shown in FIG.

MWS-DA-GI, an acidic biological polymer extracted from the mycelium, is a gram-negative bacterium. The Collie showed a clear zone of 19 mm. In addition, BWS-DN, an extracellular neutral biopolymer, showed a growth inhibition rate of about 13 mm against Gram-positive bacillus Bacillus cereus, but other biopolymers did not show specific antimicrobial activity.

(2) anticoplementary activity test

Yellow complement activity was measured using the method of Yamada et al. (Yamada, H. et al., Studies on Polysaccaride from Angelica acutiloba, plants Medics p.164 (1984)).

Various biological polymer samples were prepared at various concentrations (100-1000 µg / ml), 30 µl of this sample, 30 µl of guinea pig complement (27.5 units / ml), and GVB. 30 μl (Gelatin Veronal Buffered Saline, pH 7.4) was added to a small test tube. After reacting at 37 ° C for 30 minutes, 100 µl of antibody sensitized sheep erythrocytes were added as an indicator and allowed to react for 60 minutes in a 37 ° C water bath. After that GVB 900 μl of buffer was added and 20 μl of 0.5 M EDTA was added to stop the reaction, and the absorbance of the supernatant obtained by centrifugation at 400 × g for 5 minutes was measured at 541 nm.

Anti-complement activity was calculated as the inhibition rate (TCH,%) on the total complement hemolytic activity of the control group as follows.

% Complement complement activity = [(TCH of control TCH-treated) / TCH of control] X 100

In general, in the case of polysaccharides, anticancer activity is indirectly predicted by measuring anticomplement activity since anticancer activity and anticomplement activity are proportional to each other.

Therefore, BWS and MWS and 8 kinds of biological polymer samples fractionated and purified from them were prepared in different concentrations and anti-complementary experiments were performed in order to examine the anticancer activity. The results are shown in FIGS. 11 and 12. .

As shown in FIG. 11, BWS-DA and BWS-DA-GI showed 30% anti-complement activity in the extracellular biopolymer samples, and other samples showed 15-20% anti-complement activity. Activity was shown.

On the other hand, as shown in FIG. 12, MWS and MWS-DA-GI, which are biological polymers derived from mycelium, exhibited relatively high anti-complement activity of about 60-70%. In this case, the TCH of MWS and MWS-DA-GI is 420 µg / ml and 600 µg / ml, respectively. This was somewhat lower than 320 μg / ml of Krestin reported.

From the above results, it was determined that MWS and MWS-DA-GI, which have excellent anti-complement activity, as well as extracellular biological polymers including BWS, are likely to have strong anticancer activity.

Example 8

Molecular Weight Measurement

The molecular weight was determined by Cooper's method by gel chromatography [Cooper, T. G., The Tools of Biochemistry, New York, John wiley and sons, p. 171-172 (1991)] was measured as follows.

Sepharose CL-4B equilibrated with 0.4 M NaCl solution was charged to the column (2.5 × 95 cm) and the sample and standard sugar (two kinds other than Blue dextran), respectively, and fractionated by 4.0 ml at a flow rate of 40 ml / hr.

Sugars were quantified by the phenol-sulfuric acid method, and elution volume (Ve), void volume (Vo), and gel bead volume (Vx) were obtained, respectively, and then the distribution coefficient (15) was used. Kav) was calculated. The molecular weights of the biological polymer samples were calculated from the standard curve obtained by plotting the distribution coefficient Kav and the logarithm of the molecular weight.

Kav: Partition coefficient Vx: Gel bead volume

Ve: Elution volume Vt: Total volume

Vo: void volume:

After gel chromatography on standard dextran and purified samples, the partition coefficient (Kav), which is the partition coefficient, was obtained, respectively, and plotted with the logarithm of the molecular weight, showing BWS-DA-GI, MWS-DA-GI, and BWS-DN-GI. The molecular weight of was shown in Figure 13.

From FIG. 13, the molecular weights of BWS-DA-GI and MWS-DA-GI were about 1,200,000 and 1,000,000 Daltons, and BWS-DN-GI, which is a fraction of BWS-DN, was 600,000 Daltons. The molecular weight of was varied.

Example 9

Absorption Rate and Gelation Capacity of Ganoderma lucidum Polysaccharides

Absorption capacity was modified by the tea bag method to weigh a certain amount of dry sample (Wo), put it in a dialysis membrane and swell it with excess distilled water, and then filter the site of the gel and measure the weight (W ₁ ). The water absorption was calculated from the following equation.

Meanwhile, gelling ability was prepared by dissolving various polysaccharide samples including biological polymer samples and xanthan at a concentration of 2.5% (w / v), and then placing them in a dialysis membrane and leaving them for 24 hours. Then, the dialysis membrane was carefully removed to form a gel. Was taken.

As a result, the absorption rate of BWS was similar to that of guar gum among commercially available polysaccharides, but was significantly lower than that of sodium alginate or gellan gum and somewhat higher than xanthan, carrageenan and pullulan. In addition, as a result of examining the water absorption at different concentrations, the water absorption rate of BWS showed a tendency to increase exponentially as the sample concentration was increased, but the MWS had almost no water absorption capacity.

On the other hand, the result of examining the gelation ability of BWS is the same as FIG.

MWS had no gelation ability but BWS formed a gel in the dialysis membrane as can be seen in FIG. In general, gel is a crosslinked polymer having a three-dimensional retinal structure, and swelling occurs due to interaction with a solvent. The swelling degree at this time depends on the crosslinking density, and the higher the crosslinking density, the lower the swelling degree.

The gel ability of such biopolymers has the potential to be used in medicine, pharmacy, and the like, which are widely studied in recent years, such as artificial muscles and sustained release control of medicines.

Claims (4)

In the method for producing a polysaccharide having anticancer activity by liquid culture of Ganoderma lucidum mushroom, the method of the mycelium of Ganoderma lucidum mushroom has a C / N ratio in the range of 7-9.5: 1, pH 4.0-6.0, temperature 20-40 ℃ Ganoderma lucidum mycelium, characterized in that the liquid culture under the conditions of agitation speed 80-140 rpm to accumulate β-1,3-glucan in the medium, and harvesting the β-1,3-glucan from the culture medium Method for producing β-1,3-glucan by liquid culture of The method of claim 1 wherein the C / N ratio is about 8.3: 1. The medium for liquid culture according to claim 1, wherein the medium for liquid culture is glucose 3-10% (w / v), yeast extract 0.1-1.0% (w / v), (NH ₄ ) ₂ HPO ₄ 0.05-0.3% (w / v). ) And KH ₂ PO ₄ 0.01-0.2% (w / v). A gelling agent composition comprising β-1,3-glucan obtained by the method of claim 1 as an active ingredient.