JP2003038165A - Culture method of Cordyceps mycelium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing Cordyceps sinensis mycelia enabling the mycelia to easily be supplied in large quantities. SOLUTION: This method for culturing Cordyceps sinensis mycelia involves using a medium containing 0.1-6.0 wt.% of sugar alcohol(s) such as glycerol and/or mannitol. By this method, the yield of the mycelia can be increased by a factor of 1.2 to 1.3 compared with conventional methods where saccharide(s) such as glucose or sucrose is added as the carbon source to media.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for artificially culturing mycelium of Cordyceps sinensis isolated from a fungus of Cordyceps sinensis that parasitizes larvae of bat moths.

[0002]

BACKGROUND OF THE INVENTION A group of fungi that parasitize insects and arachnids, form endophytic sclerotia in their bodies, and form stalks with a club-shaped to spherical shape, is called Cordyceps. These companion Cordyceps sinensis parasitize the larvae of the lepidopteran bat moths and are distributed in the Sichuan, Yunnan, Aomi, Gansu, Hubei and Oribe provinces of China and from Tibet to alpine mountains in Nepal and the Himalayas. Altitude 3 over the zone
It is said that it grows naturally in the rough grasslands of 000 to 4000 m.
The zodia of the Cordyceps have long been eaten in China for the purpose of nourishing and strengthening, and in recent years, cordycepin, which has a strong antibacterial action against cancer cell suppressive and tubercle bacillus, has been isolated as a physiologically active ingredient, and the demand has increased. ing. As a result, it has become over-captured at the place of origin and is difficult to obtain.

Therefore, in recent years, a method for artificially culturing mycelium of Cordyceps has been proposed. For example, a method for cultivating mycelium of Cordyceps by using a synthetic liquid medium containing a trace amount of amino acids and vitamins B in glucose while adding an extract of an insect of the same species as the parasitic insect of Cordyceps sinensis to the medium (Japanese Patent Laid-Open No. HEI 10-96). -11770) and a method for artificially cultivating Cordyceps sinensis using a medium whose main component is the composition component of silkworm pupa (Japanese Patent Laid-Open No. 10-42691).

[0004]

However, in the above-mentioned conventional culturing method, the handling is troublesome, for example, using an extract of an insect of the same species as the parasitic insect of Cordyceps sinensis and a constituent component of silkworm pupa as a medium. In addition to the slow growth of mycelia, there is also a problem that the yield of mycelia is low.

[0005] Therefore, an object of the present invention is to provide a culturing method capable of easily supplying a large amount of mycelium of Cordyceps sinensis.

[0006]

Means for Solving the Problems The present inventors have focused on accumulating sugar alcohol in the body to make an antifreeze liquid for the insects parasitic on Cordyceps sinensis, and by adding sugar alcohol to the medium. The inventors have found a culturing method that is simpler than the conventional culturing method and that can increase the yield.

That is, the method for cultivating mycorrhiza of the Cordyceps sinensis according to the present invention is characterized by culturing in a medium containing sugar alcohol. It was found that culturing the Cordyceps sinensis mycelia in a medium containing sugar alcohol promotes the growth of the mycelia and increases the yield. The sugar alcohol content is preferably 0.1 to 6.0% by weight. This is because if the sugar alcohol is contained in a small amount, the effect is exhibited, while if it is too large, the growth is inhibited by the alcohol. The sugar alcohol added is preferably trivalent glycerol, tetravalent erythritol, hexavalent sorbitol and mannitol for the growth of mycelium.

In addition to the sugar alcohol, the medium preferably contains a nitrogen source and inorganic salts necessary for the growth of microorganisms, and if necessary, a carbon source other than the sugar alcohol. Therefore, both glycerol and glucose may be included in the medium as carbon sources. The medium can be used as a liquid medium by dissolving nutrients in water, or can be used as a solid medium by solidifying with gelatin or agar. When the Cordyceps mycelium is cultured using such a medium, the Cordyceps mycelium can be completely spread in about 7 days.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. In the present invention, as the sugar alcohol added to the medium, those recognized as food additives can be used. For example, lactitol, maltitol,
Glycerol, erythritol, xylitol, sorbitol, mannitol and the like can be used,
Particularly, trivalent glycerol, tetravalent erythritol, hexavalent sorbitol and mannitol are preferable. Also,
In order to increase the yield of mycelium and prevent inhibition by alcohol, the amount of sugar alcohol added to the medium is 0.1 to 6% by weight, preferably 1 to 6% by weight.

The medium in the present invention contains, in addition to the sugar alcohol, nutrients such as a carbon source, a nitrogen source, and inorganic salts necessary for the growth of the mycelia during culture. In addition, vitamins and minerals are also appropriately contained. The initial pH of the medium is preferably 4.5 to 6.0.

Examples of the above carbon source include sugars such as glucose, fructose, maltose, sucrose and trehalose. The amount of saccharide added is preferably 0.1 to 10.0% by weight of the whole medium.

Further, examples of the nitrogen source include peptone, yeast extract, skim milk powder and the like. The amount of the nitrogen source added is preferably 0.1 to 5.0% by weight of the whole medium.

Examples of inorganic salts include phosphates such as potassium phosphate and sodium phosphate.

In the present invention, by using the liquid medium, more mycelia can be obtained in a shorter period than the conventional liquid culture. The liquid medium can be obtained by dispersing a predetermined amount of the above-mentioned sugar alcohol, saccharide, protein and inorganic salt in water and sterilizing by autoclaving.

The Cordyceps mycelium cultured in the present invention is mainly classified into the Cordyceps genus (Cordyceps), in which Cordycep sinensis (Cordycep) is contained.
s sinensis), Cordycep militaryis (Cordycep
s militaris), Cordycepse japonensis (Cor
dyceps japonensis), but especially at an altitude of 30
Sugar alcohol has a great effect on Cordyceps sinensis, which grows naturally in the high mountains of 0 to 4000 m.

[0016]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1: Effect test of sugar alcohol added to liquid medium First, inoculum is prepared. In a 300 ml Erlenmeyer flask without rim, peptone 3%, glucose 6%, potassium phosphate 0.1%
%, Sodium phosphate 0.1%, water 150 ml, pH
A liquid medium of 5.5 was prepared, and PDA medium (4.0 g of potato extract, 20.0 g of glucose, 1 agar of agar) was added to this liquid medium.
The mycelium of Cordyceps sinensis, which had been plane-cultured with 5.0 g of water and 1 L of water, was inoculated, and after reciprocal shaking culture at room temperature of 25 ° C. for 14 days under dark conditions, this was used as a seed culture.

Next, a liquid medium is prepared. Yeast extract 4
%, Glucose 4%, potassium phosphate 0.1%, sodium phosphate 0.1%, water 1L initial pH 5.0.
Make a liquid medium prepared as described above as a basic medium, and add 4% each of equal amounts of glycerol, erythritol, sorbitol, mannitol, glycerol and glucose instead of the above-mentioned glucose, and dispense each 100 ml into a 300 ml rimless Erlenmeyer flask. After that, it was sterilized by autoclaving.

After allowing the above liquid culture medium to cool to room temperature,
The inoculum is punched out with a cork borer having an inner diameter of 4 mm and the disc piece is inoculated. After inoculation, static culture is performed under dark conditions at room temperature of 25 ° C. and humidity of 60%.

Mycelia are completely infested on the surface of the liquid culture within 14 days from the start of the culture. The infested mycelium is collected and blow-dried at 60 to 80 ° C. to complete the production.

The yield of the collected mycelium was calculated by the ratio calculation when the yield of the dried product cultured in the basic medium containing glucose was 1.0. The result is shown in FIG.

As shown in FIG. 1, the yield of mycelium was higher when sugar alcohols such as mannitol and glycerol were used, as compared with glucose which has been conventionally used as a carbon source in a medium for Cordyceps sinensis hyphae. It was found to increase by 20 to 30%. In addition, the yield of mycelium was also increased in the case of an equal mixture of glycerol and glucose as compared with glucose alone.

Example 2 The amount of sugar alcohol added to the liquid medium is the same as in Example 1 above. Next, make a liquid medium. Yeast extract 4%, glucose 1%, potassium phosphate 0.1%, sodium phosphate 0.1%, water 1
A liquid medium adjusted to an initial pH of 5.0 with a medium composition of L is prepared as a basic medium. Also, instead of glucose in this basic medium, glycerol, erythritol, sorbitol,
Add mannitol 1% each, add 300m
100 ml of each was dispensed into a rimless Erlenmeyer flask and sterilized by autoclaving. Furthermore, the amount of glucose added to the basal medium was 2%, 3%, 4% in the same manner as above.
Make something increased by 5% and 6%. In addition, glycerol, erythritol, sorbitol, and mannitol were used instead of glucose for 2% and 3%, respectively.
%, 4%, 5% and 6% were added separately and this was added to 300m
100 ml of each was dispensed into a rimless Erlenmeyer flask and sterilized by autoclaving. A liquid medium prepared by removing glucose from the above basic medium is also prepared.

After the above liquid medium is allowed to cool to room temperature, the inoculum is punched out with a cork borer having an inner diameter of 4 mm and the disc piece is inoculated. After inoculation, static culture is performed under dark conditions at room temperature of 25 ° C. and humidity of 60%.

Mycelia are completely infested on the surface of the liquid culture within 14 days from the start of the culture. The infested mycelium is filtered, collected, washed well with water, and dried by blowing at 60 to 80 ° C., and the result is completed.

The yield of the collected mycelia was calculated by calculating the ratio of the yield obtained when 1% glucose was added to the basal medium to the culture, and the yield was 1.0. The result is shown in FIG.

As shown in FIG. 2, the addition amount is 1 to 6%.
At that time, all of the sugar alcohols used in this example had higher mycelial yields than glucose, especially very high yields with glycerol. In addition, the yield of all sugar alcohols is about 3%, which is the highest.

[0028]

Industrial Applicability As described above, according to the method for cultivating mycelium of Cordyceps sinensis according to the present invention, the mycelium of Cordyceps sinensis can be easily supplied in a large amount by culturing in a medium containing sugar alcohol. There is an effect such as.

[Brief description of drawings]

FIG. 1 is a graph showing the yield ratio of mycelium depending on the type of sugar alcohol.

FIG. 2 is a graph showing the yield ratio of mycelia depending on the added amount of sugar alcohol.

Claims (5)

[Claims] 1. A method for culturing mycelium of Cordyceps sinensis, which comprises culturing using a medium containing sugar alcohol. 2. The method for cultivating the Cordyceps mycelium according to claim 1, wherein a nitrogen source, an inorganic salt and, if necessary, a carbon source other than the sugar alcohol are contained in the medium in addition to the sugar alcohol. 3. The method for culturing Cordyceps mycelium according to claim 1 or 2, wherein the sugar alcohol is contained in an amount of 0.1 to 6.0% by weight. 4. The method for culturing the Cordyceps mycelium according to claim 1, wherein the sugar alcohol is one or more selected from glycerol, erythritol, sorbitol and mannitol. 5. The method for cultivating Cordyceps mycelium according to claim 1, wherein Cordyceps sinensis is used as the Cordyceps mycelium.