CH634102A5 - Process for the preparation of a novel monokaryotic mycelium of Coriolus versicolor - Google Patents

Description

The invention relates to a method for producing new monocaryotic mycelium from Coriolus versicolor (Fr.) Quél., A known basidiomycete of the genus Coriolus of the Polyporaceae.

The great advantages of using polysaccharides by extracting Coriolus versicolor (Fr.)

Quél, or cultures thereof, as a basic component for medical preparations, food and drink have only recently become known, and various proposals have been made for producing this basidiomycete in artificial crops with high yield. So far, however, no advantageous method for multiplying this basidiomycete in high yields is known.

In the course of our own investigations with the aim of developing methods for the proliferation of Coriolus versicolor (Fr.) Quél, the following was found: If

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If this basidiomycete is grown in submerged culture and mechanical treatment is carried out by grinding or Scherang in a liquid medium, the basidiomycete loses the staple bonds belonging to its characteristic morphological properties and is converted into a monocaryotic mycelium which is stable and the characteristic property has an extremely high multiplication rate compared to the known dikaryotic mycelium.

io The method according to the invention is characterized by the features mentioned in claim 1. Preferred embodiments of the method have the features of claims 2-7.

15 In the drawings:

1 is a graphical representation of the comparison of a reproductive curve of monokaryotic mycelium, which is obtained from Coriolus versicolor (Fr.) Quél, according to the method according to the invention, in an aerated stirring culture, with a corresponding reproductive curve of the known dikaryotic mycelium,

2 and 4 microphotographs of the dikaryotic mycelium from oblique cultures of Coriolus versicolor (Fr.) Quél, and

3 shows the microphotograph of a monokaryotic mycelium obtained according to the invention.

In the graphic representation of FIG. 1, the mycelium concentration (g / liter) in the medium is plotted as the ordinate, the culture duration (hours) on the abscissa. Furthermore, (I) 30 shows the reproductive curve of the dikaryotic mycelium and (II) the reproductive curve of the monokaryotic mycelium.

It is generally believed that the fungi that form a sponge produce basidia spores, that germination of these spores produces primary mycelium, which generally consists of monokaryons, and that this mycelium forms the secondary mycelium, which consists of dikaryons, through adhesions. It is said that the monokaryotic mycelium is unable to produce the fruiting bodies, while the dikaryotic mycelium has this maturity. There are no reports 40 on the formation of monokaryotic mycelium from the spores of Coriolus versicolor (Fr.) Quél. In addition, the white aerial mycelium obtained in our own experiments by growing the spores was dikaryotic, which is probably due to the fact that the 45 monokaryotic mycelium from the spores is rapidly converted into the dikaryotic mycelium.

The monokaryotic mycelium obtained from the dikaryotic mycelium by the method according to the invention can be kept stable, which is a striking difference compared to the known dikaryotic mycelium. The following table I summarizes the differences between the monocaryotic mycelium of Coriolus versicolor (Fr.) Quél obtained according to the invention and the dikaryotic mycelium.

Table I

Criterion dikaryotic mycelium monokaryotic mycelium (according to the invention)

Appearance

1. Submerged culture

2. Plate culture microscopic examination

3. Formation of brackets

4. Mysel form non-suspended state Air mycelium is formed long and thinly suspended state no air mycelium is formed no shorter and considerably thicker than di-cariotic mycelium

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Table I (continued)

Criterion dikaryotic mycelium monokaryotic mycelium (according to the invention)

Physiological and biochemical properties

5. Propagation rate

6. Cellulose assimilation

7. Potassium nitrate as the only source of nitrogen

8. Thiamine

9. Litmus milk medium low positive no growth required acidified high weak positive

Growth not required not acidified

Furthermore, in connection with the properties 15 of the respective mycelium shown in the table above, the following findings can be found: The usual dikaryotic mycelium obtained by cultivating Coriolus versicolor (Fr.) , in front. In contrast, when the monokaryotic mycelium is grown, no pellets are formed and the culture takes on a cloudy appearance, as if slurry was suspended in water, and this is an obvious difference from the usual dikaryotic mycelium. In the course of the invention, a new method for counting the Nuclei are developed in the cell, which is carried out as follows: the basidial mycelium is first mixed with fixing fluid (according to Helly), then usually left to stand for about 24 hours and finally washed with water until it decolorises. The mycelium obtained is immersed in hydrochloric acid solution and the solution is heated to 60.degree. After cooling to room temperature and washing with water, the mycelium is immersed in nitric acid solution diluted 20-60 times and then washed again with water. The immersion time in the hydrochloric acid solution is 10 to 20 minutes, that in the nitric acid solution a few minutes. The fibrous cells obtained are spread on a glass slide and left on until the moisture has evaporated. Then Giemsa solution is added dropwise. As soon as staining occurs (approximately 10 minutes later), the cells are washed gently with water and dried. After drying, the cells are viewed under a light microscope at a thousandfold magnification and the circular, red-colored spots (viewed as nuclei) are counted. The number of nuclei can thus be determined by counting the red-colored spots in a cell.

The Helly fixing fluid used here is a solution, the base of which is prepared by dissolving 2.5 g of potassium bichromate, 1 g of sodium sulfate and 5 g of mercury chloride in 100 ml of water and mixed with 5 ml of formalin per 100 ml of base solution immediately before use .

The Giemsa solution is a solution for staining cell nuclei and is left to stand by dissolving 3.0 g of Azur-II-Eosin ss and 0.8 g of Azur in 250 ml of glycerol while heating to 60 ° C, adding 250 ml of methanol of the mixed solution for 24 hours and then filtering the solution. For use, 3 parts by volume of the stock solution thus prepared are diluted with 100 parts by volume of 60 phosphoric acid buffer solution (pH 6.4 to 6.8).

By measurement according to this method, it was found that the number of nuclei in a cell of the usual mycelium formed in the form of pellets is two, while the number of nuclei in a cell of the suspension-like mycelium produced according to the invention is one.

The monokaryotic mycelium with the specific properties specified above can be produced according to the invention by subjecting the dikaryotic mycelium from Coriolus versicolor (Fr.) Quél to a mechanical (physical) treatment by grinding or shearing in a liquid medium and subsequently or by treating the mycelium treated in this way is bred at the same time. In practice, this method can e.g. B. can be carried out in the following way:

1. When treating or cultivating the dikaryotic mycelium from Coriolus versicolor (Fr.) Quél, in a shaking culture, the mycelium is ground by adding inactive solid granular material, such as glass beads.

2. When the dikaryotic mycelium is treated or cultivated in a continuous submerged culture, the cultivation is carried out under shear stress on the mycelium with a stirring element.

3. When the dikaryotic mycelium is treated or cultivated in submerged culture, the mycelium is subjected to shear or grinding forces with a homogenizing device to such an extent that no mycelium pellets can be seen when viewed from the outside.

In the formation of the monokaryotic mycelium under the conditions specified above, the following additional measures are preferably used:

(i) An enriched nutritional state is provided by using a medium at 1.5-3 times higher concentration than usual, e.g. a glucose-yeast extract medium containing 5% glucose and 0.75% yeast extract.

(ii) The oxygen partial pressure is reduced in the atmosphere of the submerged culture. The reduced oxygen partial pressure can be generated by keeping the fermentation tank airtight or by introducing inert gas, such as nitrogen or carbon dioxide, into the fermentation tank.

(iii) The submerged culture is carried out continuously with the additional feeding of liquid medium.

The dikaryotic mycelium from Coriolus versicolor (Fr.) Quél can easily be converted into the monokaryotic mycelium using the methods 1-3 mentioned above, either as such or in a suitable combination with measures (i) to (iii) . If a sufficient amount of monokaryotic mycelium is not obtained in a culture run, the above-mentioned operation can be continued after homogenizing the culture until the desired amount of monokaryotic mycelium is obtained. The culture or cultivation is usually carried out at a temperature of 25 + 5 ° C for a period of 3 to 15 days. It has been found that the monocaryotic mycelium of Coriolus versicolor (Fr.) Quél obtained according to the invention always retains the same state and the same properties if the cultivation or culture is continued under the above-mentioned conditions for the production of the monokaryotic mycelium. This means that according to Ver25

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obtained monokaryotic mycelium also appears in the next generation as the same monokaryotic mycelium and maintains the properties of the monokaryotic mycelium as shown in Table I.

In the case of plate cultures or surface cultures (stationary cultures) of the monokaryotic mycelium, the aerial mycelium forms only slowly, but is generated if the cultivation is continued for several months. This aerial mycelium is dikaryotic, and when used to form fruiting bodies by inoculating appropriate beddings, these fruiting bodies are those of Coriolus versicolor (Fr.) Quél, itself. This means that the mycelium produced is exactly the same as the original mycelium.

These facts show that the inventive. Coriolus versicolor (Fr.) Quél monocaryotic mycelium derived from the original Coriolus versicolor (Fr.) Quél dikaryotic mycelium. The monokaryotic mycelium obtained according to the invention is a new mycelium which was obtained for the first time by the method according to the invention. The new mycelium is called Coriolus versicolor (Fr.) Quél. GX-101-3 and was filed under FERM-P No. 3686 on August 25, 1976 in the Fermentation Research Institute, Agency of Industriai Science and Technology, Chi-bashi, Japan, a Japanese government agency.

The characteristic features of the monocaryotic mycelium from Coriolus versicolor (Fr.) Quél obtained according to the invention are given in Table I, but the greatest technical importance of this fungus is its high propagation or propagation rate. The rate of proliferation of this fungus is 1.5 times higher than that of the original dikaryotic mycelium, and such a high rate of proliferation is of course extremely advantageous for industrial production.

The monokaryotic mycelium can be used for the same purposes as the dikaryotic mycelium from Coriolus versicolor (Fr.) Quél. For example, a nitrogen-containing polysaccharide can be obtained by extraction with an aqueous medium, such as water, dilute alkaline solution or dilute acidic solution, and this substance can be used for the production of medicaments, such as antitumor agents, immunity activating agents, antivirally active agents, anifungal or fungicidal agents, anti-leprosy agents, appetite enhancers and the like can be used.

Various enzymes such as protease, amylase and the like can also be obtained by low-temperature extraction. Furthermore, the mycelium itself or its extracts or residues can be used for food, beverages, animal feed and fertilizers.

In addition, the mycelium obtained according to the invention can be used for all purposes, such as Coriolus versicolor (Fr.) Quél.

The invention is explained in more detail below with the aid of examples of preferred embodiments. Percentages are based on weight, unless otherwise noted.

example 1

Production of monokaryotic mycelium

100 ml of liquid medium with 5% glucose (manufactured by Showa Sangyo Co., Ltd.) and 0.75% yeast extract (manufactured by Kyokuto Seiyaku Kogyo Co., Ltd.) was pipetted into a 500 ml Erlenmeyer flask. After 20 minutes steam sterilization at 120 ° C. in an autoclave, the medium was inoculated with 1 ml of a mycelium suspension formed by dispersing the mycelium from Coriolus versicolor (Fr.) Quél. The mycelium originated from a 20-day stationary culture at 25 ° C. using 50 ml of liquid medium containing 3% glucose and 0.5% yeast extract in 60 ml of physiological saline and breaking up the mycelium mass with the aid of a mixer for 3 min a speed of 6000 rpm.

After inoculation, the shaking culture was started at a speed of 200 rpm at 25 ° C. Three days after the start of the culture, the culture material was aseptically transferred to a 200 ml mixing beaker (manufactured by Sakuma Seisakujo). After the material was ground with a homogeneous mixer (manufactured by Sakuma Seisakujo) at a speed of 10,000 rpm for 10 minutes, the shaking culture was immediately resumed and continued for a total of 7 days. The mycelium grown in this way had no staple bindings and showed only a weak formation of aerial mycelium on the usual agar plate medium. The result of the microscopic examination after the staining according to the above information showed that the mycelium obtained was consistently monokaryotic.

Staining

1 ml of the broth containing the mycelium, which had been obtained in the above-mentioned manner, was mixed with 10 ml of water and then centrifuged at 2000 to 5000 G for 5 min. The supernatant liquid was separated off, the mycelium was transferred to a test tube and fixing liquid according to Helly was added. After standing for 24 hours, the separated cells were washed with 10 ml of water until decolorization. The cells were then placed in 10 ml of hydrochloric acid and heated to 60 ° C. for 15 minutes. The mixture was then cooled to room temperature, washed with 10 ml of water, immersed in 10 ml of a 20-50 times diluted nitric acid solution and washed 2-3 times with 10 ml of water each.

The fibrous cells obtained were spread on a glass slide and the liquid was allowed to evaporate. Then a few drops of Giemsa solution were added to the cells. After standing for 15 minutes, the cells were washed lightly with water and then dried.

When the nucleus-colored cells obtained in this way are examined under a microscope with a magnification of a thousand times, each nucleus shows up as a circular red-colored spot. The number of nuclei can therefore be determined simply by counting the circular, red-colored spots in a cell of the mycelium. The mycelium obtained could not acidify litmus milk and showed no liquidity for gelatin.

Multiplication of the monokaryotic mycelium

The monokaryotic mycelium obtained in the above-described manner was fed into a 20 liter fermentation flask (manufactured by Kyoritsu Riko Co., Ltd.) with 12 liters of a liquid medium containing 5% glucose and 0.75% yeast extract. whereupon 2 kg / cm2 of steam were blown directly into the fermentation flask. After steam sterilization for 20 min at 120 ° C. and subsequent cooling, 1 liter of a suspension containing the monokaryotic mycelium was inoculated at a rate of 0.5 g / liter, and then immediately the cultivation at an aeration rate of 0.5 volume Air per volume of medium per minute and a shaking speed of 550 rpm. Dikaryotic mycelium was grown under identical conditions for comparison purposes. Comparing the rates of proliferation of these monokaryotic and dikaryotic mycelia with respect to the time required to reach a mycelium concentration of 8 g / liter, it was found that the monokaryotic mycelium required only a quarter of the culture period of the dikaryotic mycelium (see FIG. 1). It was also be4

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confirms that the growth yield of monokaryotic mycelium was approximately 20% higher than that of dikaryotic mycelium.

Example 2

The kernel number was calculated as in Example 1 on mycelium obtained from a slant culture from Coriolus versicolor (Fr.) Quél. As a result, it was found that, according to the photographic representation of Fig. 2, all the cells were dikaryotic and that no monokaryotic mycelium was recognizable.

This mushroom, which serves as the starting material, is called Coriolus versicolor (Fr.) Quél. Designated CM-101 and deposited under FERM-P No. 2412 on December 25, 1973 in the above-mentioned institute.

100 ml of liquid medium containing 5% glucose and 0.75% yeast extract was placed in a 500 ml Erlenmeyer flask and distilled by heating. This medium was then inoculated with the above-mentioned dikaryotic mycelium using a platinum wire eyelet and subjected to a 3-day shaking culture (preculture) in a room kept at 25 ± 2 ° C. This created a mycelium in the form of pellets. The culture broth containing this mycelium in the form of pellets was homogenized for 5 minutes with a homogeneous mixer (manufactured by S. Seisakujo) and then subjected to a shear treatment, as a result of which the pellets were practically completely disappeared. The cultivation was continued under the above conditions. Four days later (main culture), the mycelium obtained in the form of pellets was homogenized again for 5 minutes and then subjected to a shear treatment. The concentration of the fungal cells at this stage was 11 g / liter.

1 ml of culture broth containing the homogenized mycelium was added to the same liquid medium as that used for the first culture run and then subjected to a second culture run under the same conditions as in the first run. However, the breeding period has been changed somewhat, i.e. the preculture was carried out for 3 days and the main culture also for 3 days. The fungal cell concentration was practically the same as when the shaking culture was run for the first time.

The third culture run was continued in a similar manner, the fungal cell concentration reaching practically the same value as in the first run of the shaking culture through 2 days of pre-culture and 3 days of main culture.

The fourth culture run was carried out in the same manner, giving a culture broth with a fungal cell concentration of 12 g / liter, i.e. higher than in the first culture run, namely through a 2-day pre-culture and a 2-day main culture. The mycelium obtained was not in the form of pellets and remained dispersed as a slurry, so that no homogenization treatment was required.

Microscopic examination of the fungal cells showed that there was no bracket formation, as can be seen in the usual dikaryotic mycelium, and that the width was almost twice that of the dikaryotic mycelium.

The measurement of the number of nuclei according to the method described in Example 1 showed that these cells are all those of monokaryotic mycelium as shown in the microphotograph of FIG. 3.

When the mycelium is grown under the same conditions as in the previous culture runs, the propagated mycelium consists of monokaryotic mycelium and has all of the properties of the monokaryotic mycelium shown in Table I.

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The monokaryotic mycelium was also found to have a rate of growth twice that of the dikaryotic mycelium.

10 g of the dried product of the monokaryotic mycelium obtained in the above-described manner was extracted with 300 ml of hot water at 95-100 ° C for 3 days. The extract solution was concentrated under reduced pressure at 30 ° C and then diluted to 90% with pure ethanol. The precipitate formed was dried to give 0.2 g of gray powder. Chemical analysis of the powder showed that the substance is a high molecular weight nitrogenous polysaccharide. When this substance is administered to mice that have had solid Sarcoma 180 cells transplanted, the substance shows high antitumor activity.

Example 3

100 ml of liquid medium containing 5% glucose and 0.75% yeast extract were fed into a 500 ml ErIenmeyer flask, in which 8 g of glass beads with diameters of 2 to 5 mm were placed. After heat sterilization, this mixed medium was inoculated with the dikaryotic mycelium from Coriolus versicolor (Fr.) Quél, of the type used in Example 1, using a platinum wire loop and then subjected to a shaking culture at 25 + 2 ° C. for 7 days.

1 ml of broth of the mycelium thus obtained was added to the same medium as that used with the glass beads, and further subjected to the second shaking culture for 6 days. The resulting product was also subjected to a third shake culture run lasting 5 days. The mycelium formed in this way showed no binding and was about 1.5 times larger than the original dikaryotic mycelium. The core count according to the method given in Example 1 showed that it was monocaryotic mycelium.

1 ml of this broth was used to inoculate 100 ml of a liquid medium containing 5% glucose and 0.75% yeast extract but no glass beads and grown at 25 + 2 ° C. The mycelium concentration obtained was 10.5 g / liter three days after the start of the culture, while a similar culture of the dikaryotic mycelium showed a concentration of 7 g / liter 4 days after the start of the culture.

Example 4

100 ml of liquid medium containing 5% glucose and 0.75% yeast extract was placed in a 500 ml Erlenmeyer flask. After heat sterilization, this medium was inoculated with the dikaryotic mycelium from Coriolus versicolor (Fr.) Quél, of the type given in Example 1, using a platinum wire loop and then subjected to a shaking culture lasting 3 days. After a homogenization treatment, the fermenter was completely wrapped in a polyethylene bag, sealed against the ambient air and subjected to a 4-day culture.

At the end of the cultivation, the fermenter atmosphere contained 5% CO 2. One ml of culture broth with the mycelium thus generated was used to inoculate medium as in the first culture run and then subjected to the second run of shaking culture in the same manner as in the first run. This breeding was repeated twice. The measurements carried out as in Example 1 confirm that the mycelium produced in this culture method is monocaryotic throughout.

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Example 5

Mycelium samples from a slant culture of Coriolus versicolor (Fr.) Quél were subjected to the core determination described in Example 1, which showed that the mycelium was consistently dikaryotic and that, according to the photograph from FIG. 4, there was no monocaryotic mycelium. Accordingly, the mycelium obtained from this culture is dikaryotic. The mushroom is Coriolus versicolor (Fr.) Quél. CM-103 and was deposited under FERM-P No. 2414 on December 25, 1973 at the aforementioned depository.

100 ml of liquid medium containing 5% glucose and 0.75% yeast extract were placed in a 500 ml Erlenmeyer flask, after heat sterilization with the mycelium from Coriolus versicolor (Fr.) Quél. CM-103 was inoculated with a platinum wire loop and then subjected to a 7 day shaking culture at 25 ± 2 ° C. The mycelium from this culture was dikaryotic and had a concentration of 10.8 g / liter.

The dikaryotic mycelium thus obtained was used in a proportion of 0.01% to inoculate 20 liters of liquid medium which contained 10% glucose and 1.5% yeast extract in solution, and then the submerged culture with agitation 5 in a fermenter at 25 + 2 ° C using a paddle stirrer at a speed of 500 rpm. After a culture period of 7 days, the mycelium concentration was 10.2 g / liter broth. 20 ml of this broth was used to inoculate the same medium and the second culture run was carried out under the same conditions for 6 days. The third culture run was carried out under similar conditions for 5 days, the fourth run for 4 days.

At the end of the fourth culture run, the i5 broth was in the form of an even slurry and contained the resulting mycelium in a concentration of 11.5 g / liter. The mycelium showed no staple binding and was consistently monokaryotic.

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Claims (7)

634102 PATENT CLAIMS 1. A method for producing a new monocaryotic otic mycelium from Coriolus versicolor (Fr.) Quél., Characterized in that a dikaryotic mycelium from Coriolus versicolor (Fr.) Quél is subjected to a shear or grinding treatment in a liquid medium and that the mycelium treated in this way subsequently or simultaneously cultivated in submerged culture. 2. The method according to claim 1, characterized in that the shearing or grinding treatment with a homogeneous mixer, a stirrer or by using inactive solid granular material is achieved. 3. The method according to claim 1, characterized in that the submerged culture is carried out in a culture medium which contains 5 to 10 wt .-% glucose and 0.75 to 1.5 wt .-% yeast extract. 4. The method according to claim 1, characterized in that the submerged culture is carried out in an airtight or in a fermentation tank supplied with inert gas. 5. The method according to claim 1, characterized in that the submerged culture is carried out continuously with additional feed of liquid culture medium. 6. The method according to claim 1, characterized in that the submerged culture is carried out with repeated inoculation of fresh culture medium with culture medium already used for the production of monocaryotic mycelium. 7. The method according to claim 2, characterized in that glass beads are used as the inactive solid granular material.