KR100391181B1 - The manufacturing method of exopolysaccharide by liquid culturing of Paecilomyces tenuipes - Google Patents

Abstract

The present invention produces a protein polysaccharide having the effect of improving the liver function, enhance the anti-cancer and immune system activity through the snow fungus mycelia mycelia liquid culture, the optimum liquid culture conditions ie organic nitrogen source, inorganic nitrogen source, temperature, pH, culture temperature and Snowflower Cordyceps strains that maximize the usefulness of protein polysaccharides by activating the development of protein polysaccharides into new functional foods, health foods, and medicines by allowing mass production of protein polysaccharides by varying the conditions such as culture time. It relates to a method of producing a protein polysaccharide by liquid culture.

KCTC 18063P of Snow Cordyceps sinensis strain was implanted in subculture, incubated for 3 to 5 days, inoculated into preculture liquid medium, and then shaken at 120 rpm for shaking culture at 24 ° C. for 7 days to be used as preculture. step; After the first step, inoculated at 5% concentration to the protein medium polysaccharide production liquid medium pre-cultivated snow Cordyceps sinensis mycelium in the conditions of organic nitrogen source, inorganic nitrogen source, pH index, culture temperature, culture time A second step of shaking culture at 120 rpm for shaking incubator for one day; After the second step, the culture solution was filtered using a filter cloth and 100% ethanol was added to twice the amount of the culture filtrate, and left for 24 hours at -20 ° C, and then the supernatant was removed. Separating a third step; The protein polysaccharide is hot-air dried to obtain a powder, and then dissolved in water to freeze-dried the dialysate dialyzed for 12 hours to produce a protein polysaccharide by the snow flower Cordyceps sinensis, characterized in that the liquid culture of the protein How to produce polysaccharides

Description

Method for producing protein polysaccharides by liquid culture of the Snow Cord Cordyceps strains {The manufacturing method of exopolysaccharide by liquid culturing of Paecilomyces tenuipes}

The present invention relates to a method for producing protein polysaccharide (EPS; Exopolysaccharide) by liquid culture of the snow Cordyceps sinensis strain under optimal conditions, and more specifically, a protein polysaccharide having an effect of improving liver function, enhancing anticancer and immune system activity. Is produced through the liquid culture of snow fungus Mycelium mycelium, and it is possible to mass-produce protein polysaccharides other than bacteria by adjusting various conditions such as optimum liquid culture conditions, that is, organic nitrogen source, inorganic nitrogen source, temperature, pH, culture temperature and incubation time. The present invention relates to a method for producing protein polysaccharide by liquid culture of a snow Cordyceps sinensis strain that can activate the development of protein polysaccharides into new functional foods, health foods and medicines, thereby maximizing the usefulness of protein polysaccharides.

In general, the name Cordyceps sinensis is derived from the insect's body in winter and grass-like in summer, and it is a fungus that enters the insect body by invading and killing insects and then forms fruiting bodies using the nutrients of the host. It belongs to the genus Cordyceps sinensis of the ergot, which is widely distributed in the world in various forms such as pupa cordyceps, chafer beetle, stink bug cordyceps, bee cordyceps, bubble insect cordyceps, snowflake cordyceps.

In particular, the Snow Cordyceps is a representative imperfect generation of Cordyceps that usually invade the pupa to form a bunch of mushrooms. It is composed of orange yellow sack and twig-shaped head, and the head is covered with white spores such as flour. Spores are easily dissipated by stimulation and are scattered, and animal polysaccharides have been shown to have anticancer activity and immune boosting effect. Currently, Snow Cordyceps sinensis can be used as a food, and is widely known in China, Japan, and Korea, and is a kind of Cordyceps sinensis grown in many places.

It has been proved through various experiments that the protein polysaccharide has antitumor activity, cancer metastasis inhibitory effect and activity of the immune system. The protein polysaccharide is β-glucoside containing small size monosaccharides such as glucose and fructose. It is a general feature to form a basic skeleton by a bond, i.e., β- (1 → 3), and to have a molecular weight of about 100,000 to 500,000 having a β- (1 → 6) branched chain. Protein polysaccharides do not directly attack cancer cells, but have the property of exhibiting physiological activity by enhancing immune activity.

Protein polysaccharides produced to date are typically extracted from plants such as barley, ginseng, wormwood, ginkgo, chlorella, and some seaweeds, and most of the protein polysaccharides currently used are produced from edible or medicinal mushrooms. The extraction method is obtained by hot water extraction of fruiting bodies or mycelium. This technical method has a problem in that the supply and demand of raw materials due to the limitation of the production of fruiting bodies and a large amount cannot be obtained when extracting the protein polysaccharide from the mycelium. In addition, some mushrooms that are not artificial culture is almost impossible to mass production of protein polysaccharides from the fruiting body.

The conventional method for extracting protein polysaccharide is 'application method for extracting and purifying immunoactive substance from cultured Ganoderma lucidum mycelium and patented immunoactive substance extracted therefrom', patent application No. 1990-019877, antitumor immune enhancement Ganodermarushidum, which produces an effective protein polysaccharide (G 009 '), strain Schizophilim comb IY 818, which produces a protein polysaccharide (S 818) having an anti-tumor immune enhancing effect, patent application No. 1990-019879. Patent Application No. 1991-010157, 'Cloud mushroom extract protein polysaccharide and a method for producing the same', there is a method such as US Patent 4225672.

In addition, compared to the above mushrooms, Cordyceps sinensis has a variety of physiological activities, there are advantages such as being available for food and medicinal use. Due to these advantages, Japanese Patent JP-0093092 and European Patent EP-0067000, which are methods for extracting and purifying protein polysaccharides produced from Cordyceps genus, have been previously filed. In the case of extraction and purification, the recovery rate of protein polysaccharide is low, making it difficult to commercialize, which makes it difficult to mass produce.

In order to activate the development of new functional foods, health foods and medicines using protein polysaccharides that exhibit excellent effects of increasing the activity of the anti-cancer and immune system, a new protein that enables mass production of protein polysaccharides by culturing certain strains There is a need for a method of producing polysaccharides.

The present invention has been made in order to solve the above-mentioned conventional problems, by incubating the optimal conditions of the organic nitrogen source, inorganic nitrogen source, temperature and pH in the culture of the snow Cordyceps sinensis strain, the protein polysaccharide from the Snow Cordyceps sinus strain The purpose of the present invention is to maximize the usefulness of the protein polysaccharide by enabling the production of large amounts of the protein polysaccharide to enable the development of new functional foods, health foods and medicines.

In order to achieve the above object, the plant was inoculated in the subculture medium of Snowflower Cordyceps sinensis strain KCTC 18063P, incubated for 3 to 5 days, inoculated in a liquid culture medium for preculture, and shaken at 120 rpm for shaking for 7 days at 24 ° C. A first step of using the pre-culture solution; After the first step, inoculated at 5% concentration to the protein medium polysaccharide production liquid medium pre-cultivated snow Cordyceps sinensis mycelium in the conditions of organic nitrogen source, inorganic nitrogen source, pH index, culture temperature, culture time A second step of shaking culture at 120 rpm for shaking incubator for one day; After the second step, the culture solution was filtered using a filter cloth and 100% ethanol was added to twice the amount of the culture filtrate, and left for 24 hours at -20 ° C, and then the supernatant was removed. Separating a third step; The protein polysaccharide is hot-air dried to obtain a powder, and then dissolved in water to freeze-dried the dialysate dialyzed for 12 hours to produce a protein polysaccharide by the liquid culture of the snowflake Cordyceps sinensis strain comprising a fourth step to produce a protein polysaccharide. We wanted to implement the method.

1 is a bar graph showing the effect of the organic nitrogen source on the protein polysaccharide produced by one embodiment of the present invention

Figure 2 is a line graph showing the effect of the concentration of organic nitrogen source on the protein polysaccharide produced by one embodiment of the present invention

Figure 3 is a bar graph showing the effect of inorganic nitrogen source on the protein polysaccharide produced by one embodiment of the present invention

Figure 4 is a line graph showing the effect of pH on the protein polysaccharide produced by one embodiment of the present invention

Figure 5 is a line graph showing the effect of temperature on the protein polysaccharide produced by one embodiment of the present invention

Figure 6 is a line graph showing the production process according to the incubation time of the protein polysaccharide produced by one embodiment of the present invention

Snow strain Cordyceps sinensis applied to the present invention is to try to isolate the protein polysaccharide produced in the strain by collecting the fruiting body wild in Yeongsil region of Jeju Island, if the strain is cultured under the appropriate conditions in a predetermined medium anticancer and immune system It was found that it is possible to produce a useful protein polysaccharide that has the effect of enhancing the activity, but since the strain is not known in Korea, it was deposited with KCTC 18063P on December 19, 2000, to the Genetic Resource Center Gene Bank of Biotechnology. .

The present invention relates to a method for producing a protein polysaccharide having an effect of enhancing the anticancer effect and the activity of the immune system from the snow Cordyceps Sinensis strain, by collecting the fruiting bodies of wild in the Yeongsil region of Jeju Island to isolate the Snow Cordyceps Sinensis strain After culturing the liquid in the liquid, the cultured mycelium is adjusted to an organic nitrogen source, an inorganic nitrogen source, temperature, pH, and incubation time so that optimal conditions are established, thereby establishing an optimal production condition of the protein polysaccharide and thereby mass production of the protein polysaccharide. Make it possible.

Intraperitoneal injection of the protein polysaccharide produced by the present invention at doses of 25 and 100 mg / kg showed suppression of 82.8% and 85.1% of proliferation in sarcoma 180 ascites cancers transplanted into the abdominal cavity of ICR mice, respectively. Increasing the expression of IL-2 receptor in CD8 + T cells in the spleen responsible for sexual immunity increased the cell diameter (FSC value) by 6.6% and showed the activation effect of CD8 + T cells. In addition, in vitro , T lymphocytes were activated to generate T lymphocytes.

As described above, it has been demonstrated through the experiment that the snowflake Cordyceps sinensis polysaccharide produced by the present invention not only inhibits the proliferation of cancer cells but also has an effect of enhancing immunity.

Hereinafter, with reference to the drawings will be described in detail centering on the embodiment and the experimental example according to an embodiment of the present invention.

EXAMPLE

(1) strain

Snowflake Cordyceps sinensis strain used in the present invention, after collecting wild fruiting bodies in Yeongsil area of Jeju Island, the fruiting bodies at least 1000 medium (Czapek) used for the culture of fungi containing antibiotics to inhibit the growth of bacteria and other contaminating bacteria -Use mycelium obtained by tissue culture in Dok agar medium (CDA).

(2) Strain passage medium

24 g of potato glucose agar medium (Potato dextrose agar medium; PDA) is dissolved in 1 liter of distilled water, and sterilized by autoclaving at 121 ° C. for 20 minutes to pour 30 ml in a petri-dish to make a flat medium.

(3) Liquid medium for preculture

1 g of distilled water is added to 4 g of glucose, 4 g of yeast extract, and 10 g of malt extract, and then dissolved. The solution is adjusted to pH 7.0 and sterilized by autoclaving at 121 ° C. for 20 minutes to be used as a liquid medium for preculture.

(4) Liquid culture for whole culture and production of protein polysaccharide

After cultivating the stored strain in strain passage culture medium and cultured for 3 to 5 days, the grown strain was isolated aseptically using a cork borer having a diameter of 8 mm in 500 ml of the preculture medium. After inoculation, shaking culture at 120rpm in a shaker at 24 ℃ for 7 days to use as a pre-culture solution, the cork borer used in this case uses a saw-shaped cylindrical tube used for drilling of wood or rubber stopper. The precultivated snowflake Cordyceps sinensis is inoculated at 5% concentration in a liquid medium for the production of protein polysaccharide using a pipette and shaken at 120 rpm for shaking for 7 days at 24 ° C. under the same conditions.

(5) Extraction and Separation of Protein Polysaccharide from Culture Media

After the above-described process of filtering the snowflower Cordyceps sinensis culture cultured for 7 days using a filter cloth, to obtain a predetermined culture filtrate, 100% ethanol in twice the amount of the culture filtrate to precipitate the protein polysaccharide in the culture filtrate After addition and left at -20 ° C. for 24 hours, the supernatant is removed and the precipitated protein polysaccharide is separated. The separated protein polysaccharide was hot-air dried to obtain a powder, dissolved in water to remove monosaccharides and salts, and dialyzed for 12 hours (cut-off MW 12,000, Superance BUS-200), followed by freeze drying of the dialysate. Measure the weight.

Experimental Example 1 (Organic Nitrogen Source Determination on Protein Polysaccharide Production)

The medium and components used in the present invention are weighed and dissolved in water, and then pH is adjusted using a pH meter, and the temperature is set to 121 ° C and sterilized for 20 minutes.

Organics such as yeast extract, soytone, peptone, tryptone, malt extract, and skim milk in Czapek Dox minimal medium After further adding 2.0% of the concentration of the nitrogen source, it is incubated for 7 days to determine the conditions for the optimum production of the extracellular protein polysaccharide organic nitrogen source. At this time, the composition of the CD minimum medium was composed of 3% sucrose, 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4 · 7H2O, 0.05% KCI, and 0.001% FeSO4 · 7H2O. do.

As a result of measuring the type of organic nitrogen source affecting the production of protein polysaccharide, a bar graph as shown in FIG. 1 is formed. In other words, Dryweight of EPS (g / ℓ), which indicates the amount of lyophilized protein polysaccharide to be extracted per liter of culture filtrate, is 3.2g for yeast extract, 1.8g for tryptone, 1.7g for soyton, and degreasing. 1.2g for milk powder, 1.1g for peptone and 0.5g for malt extract.

Therefore, as shown in Experimental Example 1, the organic nitrogen source that is most excellent in protein polysaccharide production is represented by yeast extract and tryptone, and skimmed milk powder and malt extract have relatively low yields.

Experimental Example 2 (Determination of Organic Nitrogen Sources on Protein Polysaccharide Production)

In Experimental Example 1, the mixing ratio of yeast extract and tryptone was 0.5: 1, 1: 1, 2 to determine the yield of protein polysaccharide according to the mixing ratio of yeast extract and tryptone, which are excellent sources of protein polysaccharide. After culturing for 7 days with: 1, 3: 1, 4: 1, the conditions for optimal concentration of organic nitrogen source for extracellular protein polysaccharide are determined.

As a result of measuring the mixing ratio of yeast extract, which is an organic nitrogen source affecting protein polysaccharide production, and tryptone, a broken line graph is formed as shown in FIG. In other words, dry weight of EPS (g / L) is 2.4g when yeast extract and tryptone are 1: 1, 0.8g when 2: 1, 0.2g and 4: 1 for 0.5: 1 and 3: 1. 1 indicates that it can produce 0.1 g.

Therefore, as shown in Experiment 2, the mixing ratio of yeast extract and tryptone which is the best for the production of protein polysaccharide shows the highest yield when mixed in an amount of 1: 1.

Experimental Example 3 (Determination of Mineral Water Sources on Protein Polysaccharide Production)

Inorganic nitrogen sources such as NaNO2, (NH4) 2SO4, NH4Cl, (NH4) 2PO4, and NaNO3 were added to a medium in which organic nitrogen sources were determined in a modified Czapek Dox medium in which yeast extract and tryptone were added to CD medium by 1%. By adding 0.5% and incubating for 7 days, the conditions for optimal production of extracellular protein polysaccharide inorganic nitrogen source are determined.

As a result of measuring the major components of the inorganic nitrogen source affecting the production of protein polysaccharide, a bar graph as shown in FIG. 3 is formed. That is, the dryweight of EPS (g / ℓ) is 1.8g for the initial medium without adding inorganic nitrogen sources, 2.4g for the addition of NaNO2, 2.3g for the addition of NaNO3, and 2.2g for the addition of (NH4) 2SO4. It appears that it can produce 2.0 g when (NH 4) 2 PO 4 is added and 1.8 g when NH 4 Cl is added.

Therefore, as shown in Experiment 3, the production of protein polysaccharide was highest when NaNO2, an inorganic nitrogen source, was added to the modified medium in which yeast extract and tryptone were added in 1%, and NH4Cl was added. In this case, it shows low production of protein polysaccharide as in the case of the initial medium without addition of inorganic nitrogen source.

Experimental Example 4 (Measurement of Initial pH of Medium on Protein Polysaccharide Production)

In general, the optimum mycelial growth pH of basidiomycete is known to be weakly acidic. In the experimental example according to the present invention, the yeast extract and tryptone were added to the CD medium to determine the effect of pH on the production of protein polysaccharide. The initial pH of the medium is gradually changed within the pH range of 3 to 10, and cultured for 7 days to determine the conditions for the optimal production pH of the extracellular protein polysaccharide.

As a result of measuring the pH index affecting the protein polysaccharide production, a line graph as shown in FIG. 4 is formed. In other words, the dryweight of EPS (g / ℓ) is 3.3g when pH index is 7, 2.7g when pH index is 6, 2.4g when pH index is 8, 2.0g when pH index is 9, pH index is It is shown that it can produce 0.4 g at 5 and 0.3 g at pH 3, 4 and 10.

Therefore, as shown in Experimental Example 4, when the pH index is 7, it shows the highest production of protein polysaccharide, and relatively low production in acidic or alkaline.

Experimental Example 5 (Determination of Culture Temperature on Protein Polysaccharide Production)

The culture temperature is gradually changed within the range of 10 to 40 ° C., and the pH index is set to 7 to be cultured for 7 days to determine the conditions for the optimal production culture temperature of the extracellular protein polysaccharide.

As a result of measuring the incubation temperature affecting the protein polysaccharide production, a line graph as shown in FIG. 5 is formed. That is, the dryweight of EPS (g / ℓ) is 2.6g when the culture temperature is 30 ℃, 2.4g at 24 ℃, 2.0g at 20 ℃, it can be produced 0.2g at 37 ℃.

Therefore, as shown in Experimental Example 5, the culture temperature shows the highest production of protein polysaccharide at 30 ° C, and shows that almost no protein polysaccharide is produced at 37 ° C or higher.

Experimental Example 6 (Protein Polysaccharide Production Decision with Culture Time)

Optimal medium conditions for the production of protein polysaccharides from Snow Cordyceps sinensis strains were set to conditions of optimum organic nitrogen source, inorganic nitrogen source, initial pH and culture temperature determined in Experimental Example 5 for 9 days Make a decision.

As a result of the incubation time affecting the production of protein polysaccharide, a line graph as shown in FIG. 6 is formed. That is, the dryweight of EPS (g / ℓ) is 3.0g on the 9th day, 2.4g on the 8th day, 1.8g on the 7th day, 1.4g on the 6th day, 1.4g, the 5th day 0.8g, 0.4g on the 4th day and 0.2g on the 3rd day.

Therefore, as shown in Experimental Example 6, under the optimum culture conditions determined in Experimental Example 1 to Experimental Example 5, the highest protein polysaccharide production was shown on the day 9 of the culture time, and the protein from the day of the 3rd culture time. It can be seen that polysaccharides begin to produce and continue to increase steadily.

As described above, the optimum conditions for increasing the production of protein polysaccharides through various experiments, such as organic nitrogen source, inorganic nitrogen source, pH index, incubation temperature and incubation time, in a predetermined medium by liquid culture of S. aureus By doing so, it is a useful invention to enable the development of new functional foods, health foods and medicines using the protein polysaccharide.

The present invention described above is capable of various substitutions, modifications, and changes without departing from the technical spirit of the present invention for those skilled in the art to which the present invention pertains. It is not limited to the drawings.

The present invention has been limited to the production of the protein polysaccharide of the fruiting body and mycelium itself in the present invention by the liquid culture of the snow Cordyceps sinensis strain, so that the mass production of protein polysaccharide, the conditions for producing protein polysaccharide on a predetermined medium, that is, organic Development of protein polysaccharides into new functional foods, health foods and medicines by setting nitrogen sources, inorganic nitrogen sources, pH and incubation temperature to the optimum conditions through various experiments to produce large amounts of protein polysaccharides from the Snow Cordyceps sinensis strains. It can be activated to maximize the usefulness of the protein polysaccharide.

Claims (7)

In the method of producing a protein polysaccharide by liquid culture of the snow Cordyceps Sinensis strain, KCTC 18063P of Snow Cordyceps sinensis strain was implanted in subculture, incubated for 3 to 5 days, inoculated into preculture liquid medium, and then shaken at 120 rpm for shaking culture at 24 ° C. for 7 days to be used as preculture. step; After the first step, inoculated at 5% concentration to the protein medium polysaccharide production liquid medium pre-cultivated snow Cordyceps sinensis mycelium in the conditions of organic nitrogen source, inorganic nitrogen source, pH index, culture temperature, culture time A second step of shaking culture at 120 rpm for shaking incubator for one day; After the second step, the culture solution was filtered using a filter cloth and 100% ethanol was added to twice the amount of the culture filtrate, and left for 24 hours at -20 ° C, and then the supernatant was removed. Separating a third step; The protein polysaccharide is hot-air dried to obtain a powder, and then dissolved in water to freeze-dried the dialysate dialyzed for 12 hours to produce a protein polysaccharide by the snow flower Cordyceps sinensis, characterized in that the liquid culture of the protein How to produce polysaccharides The method of claim 1, After adding 2% of the concentration of yeast extract, tryptone, soybean, skim milk powder, peptone, malt extract, which are organic nitrogen sources, to the protein medium for producing the protein polysaccharide, it is incubated for 7 days to produce protein polysaccharide. Method of producing protein polysaccharide by liquid culture of Snow Cordyceps Sinensis strain The method of claim 1, After mixing the yeast extract, which is an organic nitrogen source, and tryptone in a ratio of 0.5 to 4: 1 in the liquid medium for the production of protein polysaccharide, and cultured for 7 days to produce a protein polysaccharide, the snow flower Cordyceps spp. How to Produce Protein Polysaccharides The method of claim 1, After the yeast extract and tryptone were added to the medium for the production of protein polysaccharide by 1%, the concentration of inorganic nitrogen sources NaNO2, NaNO3, (NH4) 2SO4, (NH4) 2PO4 and NH4Cl was added for 7 days, Method of producing a protein polysaccharide by liquid culture of the snow Cordyceps Sinensis strain, characterized by culturing to produce a protein polysaccharide The method of claim 1, In the medium to which yeast extract and tryptone were added 1% to the medium for producing the protein polysaccharide, the pH index of the medium was gradually changed within the range of 3 to 10 to incubate for 7 days to produce the protein polysaccharide. Method for producing a protein polysaccharide by liquid culture of the snow Cordyceps sinensis strain characterized in that The method of claim 1, It is characterized in that to produce a protein polysaccharide by culturing the culture temperature in the range of 10 ~ 40 ℃ for 7 days with a pH index of 7 in the medium to which yeast extract and tryptone are added 1% to the liquid medium for protein polysaccharide production. Method of producing protein polysaccharide by liquid culture of snowflake Cordyceps sinensis strain The method of claim 1, Yeast extract and tryptone are added to the liquid medium for protein polysaccharide production by 1%, and concentrations of inorganic nitrogen sources NaNO2, NaNO3, (NH4) 2SO4, (NH4) 2PO4 and NH4Cl are added, and then the pH index is added. Method of producing a protein polysaccharide by liquid culture of the snow Cordyceps Sinensis strain characterized in that the culture temperature is set to within the range of 10 to 40 ℃ and incubated for 9 days to produce protein polysaccharides.