GB1566625A - Method of producing monokaryotic mycelia of coriolus versicolor - Google Patents

Abstract

If a dikaryotic mycelium of Coriolus versicolor (Fr.) Quél., a fungal species of the genus Coriolus of the Polyporaceae, is subjected to mechanical treatment by grinding or shearing in a liquid medium, and the mycelium treated in this way is subsequently or simultaneously cultured in submerged culture, a monokaryotic mycelium is produced which differs from the dikaryotic mycelium in morphological and physiological respects. The monokaryotic mycelium obtained in this way is a novel product and characterised by its extremely high growth rate in comparison to the known dikaryotic mycelium. The material obtained by extraction from cultures of the noval mycelium moreover has an outstanding physiological activity.

Description

(54) A METHOD OF PRODUCING NOVEL MONOKARYOTIC MYCELIA OF CORIOLUS VERSICOLOR (71) We, KUREHA KAGAKU KOGYO KABUSHIKI KAISHA, a Japanese body corporate, of No. 8, Horidome-cho l-chome, Nihonbashi, Chuo-ku, Tokyo, Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a method of producing monokaryotic mycelia from Conogus versicojor (Fr.) Qu.

Polysaccharides obtained from the extraction of Comics versicoter (Fr.) Fuel, or the culture medium thereof are known as useful base components for the preparation of drugs, foods and drinks. Various attempts for pro- ducing such Basidiomycete fungi in high yields by artificial cultivation have been proposed.

The present invention is predicated on our observation that when the Basidiomycete fungus Conotus versicolor (Fr.) Quel is subjected to submerged culture and a mechanical treatment such as grinding or shearing, the clamp connections (which is an intrinsic morphological characteristic of this fungus) tend to be broken and the mycelia transformed into monokaryotic mycella; and that the thus formed monokaryotic mycelia are reasonably stable; and have a high propogation rate com- pared with the dikaryotic mycella.

Accordingly, the present invention provides a method of producing monokaryotic mycelia of Coridus versicolor (Fr.) Quel, characterized in that dikaryotic mycelia of a strain of said fungal species are subjected to mechanical treatment and, either simultaneously or subsequently, to submerged culture, in a culture medium containing 510% by weight of glucose and 0.751.5% by weight of yeast extract in an atmosphere in which the partial pressure of oxygen is below the partial pressure of oxygen in the air.

In the practice of the invention the dikaryotic mycelia may be subjected simultaneously to submerged culture and the mechanical treatment, or if desired submerged culture may be carried out after the mechanical treatment of the dikaryotic mycelia.

It is generally believed that the fungi fruiting bodies produce the basidiospores and that such spores germinate to form the primary mycelia consisting, usually, of monokaryons, and that such mycelia fuse together to form secondary mycelia consisting of the dikaryons.

It is said that the monokoryotic mycelia are incapable of forming the fruiting bodies and that such formation is confined to the dikaryotic mycelia. We are not aware of any report concerning the generation of monokaryotic mycelia from the spores of Coriotus versicotor (Fr.) Qua1. Moreover, the white aerial mycelia obtained in our experiments by cultivating such spores were dikaryotic: this appears to be due to the fact that the monokaryotic mycelia from the spores are rapidly converted into the dikaryotic ones.

Monokaryotic mycelia obtained from the dikaryotic mycelia according to the practice of this invention have been found to be more stable than the known dikaryotic mycelia.

Table 1 below lists the differences between the monokaryotic mycelia of Carious verso color (Fr.) Quail. and the dikaryotic mycelia TABLE 1 Dikaryotic Items mycelia Monokaryotic mycelia Appearance 1. Submerged culture Non-suspended Suspended condition.

condition.

2. Plate culture Aerial mycelia are Not formed formed.

Microscopic observation 3. Formation of clamp Observed. None.

connections 4. Shape of mycelium Long and fine. Shorter and far thicker than dikaryotic mycelium.

Physiological and biochemical properties 5. Propagating rate Low. High.

6. Cellulose assimilation Positive. Slightly positive 7. Potassium nitrate as a sole nitrogen source No growth. Growth.

8. Thiamine Required. Not required.

9. Litmus milk medium Acidified. Not Acidified.

The following facts are to be further noted in connection with the properties of the respective mycelia shown in the above Table.

The ordinary dikaryotic mycelia obtained by cultivation of Coriolus versicolor (Fr.) Fuel.

by a conventional method are usually in the form of pellets. On the other hand, the monokaryotic mycelia, when cultivated, are not formed as pellets and the culture assumes a turbid condition similar in appearance to a suspension of pulp in water.

We have also devised a method for counting nuclei in the cells, and this method is as follows: Helly's fixing fluid is first added to the Basidiomycete mycelia, and then those mycelia are allowed to stand for usually about 24 hours and then washed with water until they are decolored. The thus obtained mycelia are immersed in 1 - N hydrochloric acid solution and the solution is heated to a temperature of 600 C. After cooling to room temperature and washing with water, they are further immersed in 20 to 50 times diluted nitric acid solution, followed by additional washing with water. The period of immersion is from ten to twenty minutes in the hydrochloric acid solution and a few minutes in the nitric acid solution. The thus obtained fibrous cells are spread on a slide glass and left thereon until moisture evaporates away, and then Giemsa's solution is added dropwise thereto. At the point when staining has been accomplished with the solution (approximately 10 minutes later), the cells are washed lightly with water and then dried. After drying, the cells are examined under a light microscope of 1,000 magnifications and the circular red-stained spots (considered as nuclei) are counted. Thus, the number of nuclei can be determined by counting the red-stained spots in one cell.

"Helly's fixing fluid" used herein is a solution of which the base is prepared by dissoling 2.5 gr of potassium bichromate, 1 gr of sodium sulfate and 5 gr of mercuric chloride in 100 ml of water, and immediately before use, such base solution is further added with formalin in an amount of 5 ml per 100 ml of the solution.

"Giemsa's solution" is a nucleus staining solution prepared by dissolving 3.0 gr of azur II eosine and 0.8 gr of azur in 250 ml of glycerin by heating them to 6()0C, further adding thereto 250 ml of methyl alcohol, allowing the mixed solution to stand for 24 hours and then filtering the solution. In use, the thus prepared stock solution is diluted by adding a phosphoric acid buffer solution (pH 6.4- 6.8) in an amount of 100 ml for 3 ml of the stock solution.

As a result of the measurements by the abovedescribed method, it was found that the number of nuclei in one cell of the conventional pellet-shaped mycelia is 2 whereas that of the mycelia obtained according to this invention is 1.

Suitable methods for use in the practice of the invention are as follows: (1) Shaking culture of the dikaryotic mycelia of Contolus versicolor (Fr.) Fuel: the mycelia being ground by adding inactive solid granular materials, for example glass beads.

(2) Continuous submerged culture of the dikaryotic mycelia, such culture being performed while shearing the mycelia with an agitating element.

(3) Submerged culture of the dikaryotic mycelia, the mycelia being sheared or ground by a homogenizer to such an extent that no pellet-shaped mycelia are observed upon external observation.

In producing monokaryotic mycelia under the abovementioned conditions according to the present invention, the following techniques are also applied: (i) the culture medium employed contains 510% by weight of glucose and 0.75- 1.5% by weight of yeast extract, which concentrations are higher than those employed conventionally in culturing such fungi; and (ii) the atmosphere above the culture medium has a partial pressure of oxygen below the partial pressure of oxygen in the air, which condition may be achieved by keep ing the fermenter airtight, or by passing an inert gas e.g. nitrogen or carbon dioxide, into the fermenter.

An additional technique which is adopted in preferred embodiments of the invention is to carry out the submerged culture continuously while additionally supplying nutrients to the culture medium.

The dikaryotic mycelia of Coriolus versicolor (Fr.) Quél can be reasonably easily converted into the monokaryotic mycelia by employing the abovedescribed procedures. If a desired amount of monokaryotic mycelia can not be obtained in one cultivation run, the above-mentioned operation may be repeated after homogenizing the culture until the desired amount of monokaryotic mycelia is obtained. Cultivation is usually carried out at a temperature of 25+ 50C for a period of 3 to 5 days.

We have found that the monokaryotic mycelia of Coriolus versicolor (Fr.) Qua1.

obtained by the practice of this invention are reproduced in the same state and with the same properties if cultivation is repeated under the above-mentioned conditions. This means that monokaryotic mycelia produced by the practice of this invention are obtained as the same monokaryotic mycelia in a subsequent generation, thus maintaining the properties of monokaryotic mycelia shown in Table 1.

In plate culture or surface culture (station ary culture) of the monokaryotic mycellia, we have observed that they are reluctant to form aerial mycelia, although aerial mycelia are eventually produced if cultivation is continued for several months. Those aerial mycelia are dikaryotic, and when they are inoculated into bed log to form fruiting bodies, they prove to be Coriolus versicolor (Fr.) Quel. This re vealed that the mycelia produced are just the same as the original ones.

These facts indicate that the monokaryotic mycelia of Coriolus versicolor (Fr.) Fuel. are derived from the original dikaryotic mycelia of Coriolus versicolor (Fr.) Quel.

The monokaryotic mycelia of this invention are novel; they have been named Coriolus versicotor (Fr.) Fuel. GX-101-3 and deposited under FERM-P No. 3689 on August 25, 1976 in the Fermentation Research Institute, Agency of Industrial Science and Technology (Chiba Shi, Japan), a Japanese governmental organ.

The characteristic features of the monokaryotic mycelia of Co rio lus versicolor (Fr.) Fuel. according to this invention are as shown in Table 1, but the greatest industrial significance of this fungus is its high rate of propagation. The propagation rate of this fungus is 1.5 to 10 times as high as that of the known dikaryotic mycelia; such a high propagation rate is beneficial for industrial production.

The present monokaryotic mycelia can be used for all the same purposes as the dikaryotic mycelia of Coriolus versicolor (Fr.) Qutl.

For instance, it is possible to obtain nitrogen-containing polysaccharides by extracting the mycelia with an aqueous medium (e.g.

water, dilute alkaline solution or dilute acid solution): such nitrogen-containing polysaccharide substances have been found to be useful in the preparation of drugs e.g. antitumor agents, immunity activating agents, antiviral drugs, antifugal agents, anti-leprous drugs, appetite promoting drugs.

It is also possible to isolate various kinds of enzymes e.g. protease and amylase, by lowtemperature extraction. Further, the mycelia of extracts or residues thereof can be used for food and drink, feed for animals and as fertilizer for plants.

The invention will now be further des cribed with reference to the following Examples and the accompanying drawings referred to therein.

In the drawings: Fig. 1 is a graph showing a propagation curve (II) in aerated and agitated culture of monokaryotic mycelia derived from Coriolus versicolor (Fr.) Fuel. and a similar propagation curve (I) of known dikaryotic mycelia, the mycelia concentration (g/l) in the medium is plotted as ordinate and the cultivation time (hrs) as abscissa; Figs. 2 and 4 are microphotographs of the dikaryotic mycelia obtained from slant culture of Coriolus versico;or (Fr.) Fuel.; and Fig. 3 is a microphotograph of the monokaryotic mycelia.

In the Examples percentages are given by weight unless indicated otherwise.

EXAMPLE 1: Production of monokaryotic mycelia: 100 ml of a liquid medium containing 5% of glucose (produced by Showa Sangyo Co., Ltd.) and 0.75% of yeast extract (produced by Kyokuto Seiyaku Kogyo Co., Ltd.) was pipetted into a 500-ml conical flask, and after 20-minutes steam sterilization at 1200C in an autoclave, the medium was inoculated with 1 ml of a suspension of mycelium prepared by dispersing the mycelium of Coriolus verso cotor (Fr.) Fuel. (obtained from 20-day stationary culture at 250C by using 50 ml liquid medium containing 3% of glucose and 0.5% of yeast extract in 60 ml of physiological saline solution, by breaking up the mycelia mat with a blender at speed of 6,000 r.p.m. for 3 minutes), and then shaking culture was started at the speed of 200 r.p.m.

at 250C. 3 days after the start of cultivation, the cultivated material was transferred aseptically into a 200-ml blender cup (mfd. by Sakamu Seisakujo), and after grinding the material by a homomixer (mfd. by Sakuma Seisokujo) at the speed of 10,000 r.p.m. for 10 minutes, shaking culture was immediately resumed to practice shaking culture for the total period of 7 days. The thus cultivated mycelia had no clamp connections and were poor in generation of aerial mycelia in a standard agar plate medium. The result of microscopic examination after staining described below showed that the obtained mycelia were all monokaryotic.

Staining: 1 ml of the broth containing the mycelia obtained in the manner described above is added with 10 ml of water and then subjected to centrifugal separation at 2,000 to 5,000 G for 5 minutes. The supernatant liquid is eliminated and the mycelia are transferred into a test tube, to which Helly's fixing fluid is added.

After 24-hour standing, the separated cells are washed with 10 ml of water until they are decolored. Then the cells are put into 10 ml of 1N hydrochloric acid and heated at 600C for 15 minutes, followed by cooling to room temperature, washing with 10 ml of water, 2minute immersion in 10 ml of 20 to 50 times diluted nitric acid solution and 2 to 3 times washing with 10 ml of water.

The obtained fibrous cells are spread on a slide glass to let moisture evaporate away and then a few drops of Giemsa's solution are added onto the cells, and after 15-minute standing, they are washed lightly with water and then dried.

When the thus nucleus-stained cell is examined under a microscope of 1,000 magnifications, each nucleus is observed as a circular red-stained spot. Therefore, the number of nuclei can be easily determined by counting the circuit red-stained spots in one cell of the mycelium. The obtained mycelia could not acidify litmus milk and had no gelatinliquefying ability.

Propagation of monokaryotic mycelia: The monokaryotic mycelia obtained in the way described above were added with 12 litres of a liquid medium containing 5% of glucose and 0.75% of yeast extract into a 20-litre jar fermenter (mfd. by Kyoritsu Riko Co., Ltd.), followed by blowing of 2 kg/cm2 of steam directly into the jar fermenter. After steam sterilization at 1200C for 20 minutes and cooling, 1 litre of suspension containing the monokaryotic mycelia was inoculated (at the rate of 0.5 g/l), immediately followed by cultivation at aeration rate of 0.5 v.v.m. and agitating speed of 550 r.p.m. For the sake of comparison, cultivation of the dikaryotic mycelia was carried out under the completely same conditions as those used for cultivation of the monokaryotic mycelia. When the propagation rates of these mono- and dikaryotic mycelia are compared by way of the time required for attaining the mycelia con centration of 8 g/l, it is to be noted that the monokaryotic mycelia require only 1/4 of the cultivation time needed for the dikaryotic mycelia (see Fig. 1). It was also confirmed that the propagation yield of the monokaryotic mycelia increased about 20% over that of the dikaryotic mycelia.

EXAMPLE 2: Calculation of the number of nuclei was made, after the manner of Example 1, on the mycelia obtained from slant culture of Coriolus versicolor (Fr.) Quel. As a result, it was found that, as shown in the photograph of Fig. 2, all of these cells are dikaryotic and no monokaryotic myceliia were detected.

This original fungus was named Coriolus versicotor (Fr.) Quél. CM-101 and deposited under FERM-P No. 2412 on December 25, 1973 in the afore-mentioned governmental organ.

100 ml of a liquid medium containing 5% of glucose and 0.75% of yeast extract was put into a 500 ml conical flask and sterilized therein under heating. This medium was then inoculated with the above-stated dikaryotic mycelia by using a platinum loop and subjected to 3 day shaking culture (preculture) in a room adjusted to a temperature of 25 + 20C. There were consequently produced pellet-shaped mycelia. The broth containing these pellet-shaped mycelia was homogenized by a homo-blender (mfd. by Sakuma Seisakujo) for 5 minutes and then subjected to a shearing treatment, whereby the pellet form had substantially disappeared. Cultivation was continued under the above-mentioned conditions, and 4 days later (main culture), the produced pellet-shaped mycelia was again homogenized for 5 minutes and then subjected to a shearing treatment. The concentration of cells of the fungus at this stage was 11 g/l.

1 ml of broth containing the homogenized mycelia was added to the same liquid medium as used in the first run of cultivation and then subjected to a second run of cultivation under the same conditions as in the first run. However, the cultivation period was slightly changed, that is, the preculture was performed for 3 days and the main culture also for 3 days. The fungal cells concentration was of substantially the same level as in the first run of shaking culture.

The third run of cultivation was further continued in the similar way, with the fungal cells concentration reaching substantially the same level as that of the first run of shaking culture by 2 days of preculture and 3 days of main culture.

Likewise, the fourth run of cultivation was carried out, obtaining a broth with fungal cells concentration of 12 g/l, higher than that of the first run of cultivation, by 2-day preculture and 2-day main culture. The obtained mycelia were not in the form of pellets and stayed dispersed in the form of pulp, requiring no homogenization treatment.

The fungal cell, as observed by a microscope, had no clamp connections which are found in the ordinary dikaryotic mycelia, and was about twice as wide as that of the dikaryotic mycelium.

From measurement of the number of nuclei by the method described in Example 1, it was ascertained that these cells are ail monokaryotic as shown in the microphotograph of Fig. 3.

When the mycelia was further cultivated under the same conditions as in the previous run of cultivation, the propagated mycelia were constituted from the monokaryotic mycelia and had all of the properties possessed by the monokaryotic mycelia shown in Table 1.

It was also found that the monokaryotic mycelia were more than twice as high in propagation rate as the dikaryotic mycelia.

10 gr of the dried product of the monokaryotic mycelia obtained in the above-described method were extracted with 300 ml of hot water at 95 to 1000C for 3 hours. The extract solution was concentrated under reduced pressure to 300C, pure ethanol was added to the concentrate to form an ethanolic solution containing 90% ethanol, and the produced precipitate was dried, yielding 0.2 gr of gray powder.

A chemical analysis of this gray powder revealed that this substance was a nitrogencontaining high-molecular weight polysaccharide. When this substance was administered to mice to which cells of solid type Sarcoma-180 had been transplanted, it de monstrated a high anti-tumour activity.

EXAMPLE 3: 100 ml of a liquid medium containing 5% of glucose and 0.75% of yeast extract were fed into a 500-ml-capacity conical flask to which was already added 8 gr of glass beads having diameters of 2 to 5 mm, and this mixed medium, after heat sterilization, was inoculated with the dikaryotic mycelia of Coriolus versicotor (Fr.) Quail., as employed in Example 1, by using a platinum loop and then subjected to 7-day shaking culture at 25+ 20C.

1 ml of broth containing the thus obtained mycelia was supplied to the medium with added glass beads as mentioned above and subjected to a further 6day shaking culture.

The resultant product was further subjected to a third run of shaking culture for a period of 5 days.

The thereby produced mycelia had no clamp connections and were about 1.5 times as wide as the original dikaryotic mycelia, and the result of counting nuclei by the manner of Example 1 revealed that they were mono karyotic mycelia.

1 ml of this broth was inoculated into 100 ml of a liquid medium containing 5 of glucose and 0.75% of yeast extract and not containing any glass beads and cultivated at 25+ 20C. The produced mycelia con centration 3 days after the start of cultivation was 10.5 g/l, while the result of a similar culture of the dikaryotic mycelia showed 7 g/l concentration 4 days after start of cultiva tion.

EXAMPLE 4: 100 ml of a liquid medium containing 5% of glucose and 0.75% of yeast extract were put into a 500-ml conical flask, and this medium, after heat sterilization, was inocu lated with the dikaryotic mycelia of Coriotus uersicolar (Fr.) Fuel. as employed in Example 1 by using a platinum loop and then suS jected to shaking culture for 3 days. After a homogenization treatment, the entirety of the fermenter was covered by a polyethylene bag and sealed against the external air followed by a 4-day cultivation.

At the point of completion of the cultivation, 5.0% of CO2 gas was contained in the fermenter atmosphere.

1 ml of broth containing the thus produced mycelia was inoculated into the medium after the manner of the first run of cultivation and then subjected to a second run of shaking culture in the same way as the first run. Such cultivation was further repeated twice. As the result of a nucleus determination conducted as in Example 1, it was ascertained that the mycelia produced in this process of cultivation were all monokaryotic.

EXAMPLE 5 The mycelia obtained from slant culture of Coriolus versicolor (Fr.) Fuel. were sampled out and subjected to the nucleus determination following the procedure described in Example 1, whereby it was ascertained that all of the mycelia were dikaryotic and no monokaryotic mycelia were present as seen in the microphotograph of Fig. 4. This indicates that the mycelia obtained from this run of cultivation were dikaryotic mycelia.

This fungus is Coriolus versicolor (Fr.) Fuel. CM-103 and deposited under FERM-P No. 2414 on December 25, 1973 in the aforementioned governmental organ.

100 ml of liquid medium containing 5% of glucose and 0.75% of yeast extract was put into a 500 ml capacity conical flask and, after heat sterilization, inoculated with the mycelia of the Coriolus versicolor (Fr.) Quel.

CM-103 by using a platinum loop, followed by a 7-day shaking culture at 25+ 20C. The mycelia in this culture were dikaryotic and reached the concentration of 10.8 g/l.

The thus obtained dikaryotic mycelia were inoculated by 0.01 ,/ into 20 litres of a liquid medium having dissolved therein 10% of glucose and 1.5% of yeast extract and subjected to submerged culture at 25 + 20C under agitation in a fermenter using a paddle type agitator rotating at the speed of 500 r.p.m.

After 7-day cultivation, the mycelia reached a concentration of 10.2 g/l in the broth. 20 ml of this broth was inoculated into the same medium and a second run of cultivation was performed under the same conditions for 6 days. Similarly cultivation was further repeated for 5 days in a third run and for 4 days in a fourth run.

After completion of the fourth run of cultivation, the broth was in the form of uniform pulp and contained the produced mycelia at the concentration of 11.5 g/l. The mycelia had no clamp connections and were all monokaryotic mycelia.

WHAT WE CLAIM IS: 1. A method of producing monokaryotic mycelia of Coriolus versicolor (Fr.) Quél.

characterized in that dikaryotic mycelia of a strain of said fungal species are subjected to mechanical treatment and, either simultaneously or subsequently, to submerged culture, in a culture medium containing 510% by weight of glucose and 0.75-1.5% by weight of yeast extract in an atmosphere in which the partial pressure of oxygen is below the partial pressure of oxygen in the air.

2. A method according to claim 1, in which the submerged culture is carried out for 3 to 15 days and at least one additional quantity of nutrient is supplied to the culture medium.

3. A method according to Claim 1 or Claim 2, wherein the submerged culture is carried out by successively culturing the mycelia in two or more batches of said culture medium.

4. A method according to any one of the preceding claims, in which the mechanical treatment is shearing or grinding with a homogenizer or an agitator or is grinding by use of inactive solid granular materials.

5. A method according to Claim 4, in which the inactive solid granular materials are glass beads.

6. A method according to Claim 1, and substantially as described with reference to any one of the Examples herein.

7. Monokaryotic mycelia of Coriolus versi- color (Fr.) Quel, whenever produced by a method according to any one of the preced

Claims (1)

1. ing claims.

   8. A method of producing a therapeutically active nitrogen containing polysaccharide, which comprises extracting monokaryotic mycelia according to Claim 7 with an aqueous solvent and isolating the polysaccharide from the extract solution.

   9. A polysaccharide obtained by the method of Claim 8.