DK147128B - PROCEDURE FOR THE PREPARATION OF A MONOCARYOTIC MYCELIUM OF A BASIDIOMYCETIC FUNGI OF THE ART CORIOLUS VERSICOLOR - Google Patents

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PATENT AND TRADEMARKET

(21) Patent Application No: 3851/77 (51) Lnt.CI.3: C12N 1/14 (22) Filing Date: 30 Aug 1977 (41) Alm. available: 01 Mar 1978 (44) Submitted: 16 Apr 1984 (86) International Application No :- (30) Priority: 30Aug1976JP51 / 103379 31 Aug 1976JP51 / 104186 (71) Applicant: 'KUREHA KAGAKU KOGYO KABUSHIKI KAISHA; Tokyo, JP.

(72) Inventor: Chlkao 'Yoshlkuml; JP, Yoslilo * Omura; JP, Toshihlko * Wada; JP, Hlromltsu “Makita; JP,

Takao * Ando; JP, Norlyuki * Toyoda; JP, Kenlchi 'Matsunaga; JP.

(74) Plenipotentiary: Lehmann & Ree Engineering (54) Process for the preparation of a monokaryotic mycelium of a basidiomycetes fungus of the species Coriolus versicolor

The invention relates to a method for producing a monokaryotic mycelium of Coriolus versicolor (Fr.) Quel., Which is a basidiomycet belonging to the genus Coriolus of the Polyporaceae family, the mycelium of which has hitherto been known only in the dikaryotic form.

More recently, the great utility of the polysaccharides obtained by extraction of Coriolus versicolor (Fr.) Quel has been recognized. or a culture thereof as a basic ingredient in the manufacture of drugs or foods and beverages, and JU various attempts to produce this basidiomycet in high N yield on cultivation have been proposed. Nevertheless, Γ "so far there has been no advantageous method for propagating the r-basidiomycet in high yield.

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During studies to obtain high performance propagation of Coriolus versicolor (Fr.) Quel. it was found that when this basidiomycet is subjected to submergence by performing a mechanical treatment, namely grinding or shear in a liquid medium, the basidiomycetine loses the islet cell shape, which is its natural morphological property, and is transformed into a monokaryotic mycelium, thus forming monokaryotic mycelium is stable and also has the special property of having a very high rate of propagation compared to the known dikaryotic mycelium.

The method of the invention for the preparation of a monokaryotic mycelium of Coriolus versicolor (Fr.) Quel. is accordingly characterized in that a dikaryotic mycelium of the fungal species is subjected to a shear or grinding treatment with a homogenizer or stirrer during or without the use of a chemically inert solid granular material in an aqueous liquid culture medium containing substantially 5 10% by weight of glucose and 0.75-1.5% by weight of yeast extract, after which or simultaneously the shared or formal mycelium is grown in a submers culture, and the thus produced monokaryotic mycelium having a higher propagation rate than the dicharyotic mycelium is recovered from the culture medium.

The invention will now be described in more detail with reference to the drawing, in which: FIG. Figure 1 is a graph depicting comparatively a growth curve in an aerated and stirred culture of the Coriolus versicolor (Fr.) Quel monolaryotic mycelium obtained by the method of the invention and a corresponding growth curve of the known dicharyotic mycelium; . 2 and 4 are photomicrographs of the dikaryotic mycelium derived from an oblique culture of Coriolus versicolor (Fr.) Quel. 3 is a photomicrograph of the monokaryotic mycelium provided by the present invention.

In the graphical representation of FIG. 1, the mycelium concentration (g / l) in the substrate is plotted as ordinate and the culture time (hours) as abscissa. Furthermore, (I) denotes the growth curve of the dikaryotic mycelium and (II) denotes the growth curve of the monokaryotic mycelium.

It is a general assumption that fungi forming a toad hatch form basidia spores and that these spores germinate to form the primary mycelium usually consisting of monokaryotic hyphae and that this mycelium is fused to form secondary mycelium consisting of dicaryotic hyphae. It is claimed that the monokaryotic mycelium has no ability to form fruit bodies, but that the dikaryotic mycelium possesses this ability. There is no literature on the formation of the monokaryotic mycelium from the spores of Coriolus versicolor (Fr.) Quel. Furthermore, the white air mycelium formed in the experiments by culturing the spores was dikaryotic, and this appears to be due to the fact that the monokaryotic mycelium from the spores was rapidly converted to dikaryotic mycelium.

The monokaryotic mycelium formed from the dikaryotic mycelium of the present invention can be kept stable, which is a striking difference from the existing dikaryotic mycelium. The following Table 1 shows the differences between the monokaryotic mycelium of Coriolus versicolor (Fr.) Quel. Obtained by the method of the present invention and the dikaryotic mycelium.

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Table 1

Dikaryotic mycelium Monokaryotic mycelium (according to the invention)

Occurrence 1. Submer's cultivation Unsuspended Suspended state.

condition.

2. Plate culture Aerial mycelium formed. Not formed.

Microscopic examination 3. Formation of islet cell Observed. None.

4. Mycelium form Long and fine. Shorter and far thicker than dikaryotic mycelium.

Physiological and biochemical properties 5. Rate of propagation Low. High.

6. Cellulose Assimilation Positive. Slightly positive.

7. Potassium nitrate as the sole source of nitrogen No growth. Vaskst.

8. Thiamin Required. Not required.

9. Lactate Milk Sub S Treats Acid. Not leavened.

It should be further noted in connection with the properties set forth in the above table that the usual dikaryotic mycelium is obtained from the cultivation of Coriolus versicolor (Fr.) Quel. according to a conventional method takes the form of small balls. On the other hand, the monokaryotic mycelium that emerges after cultivation does not take the form of beads, and the culture exhibits a turbidity state such as pulp suspended in water, which is a distinct difference from the usual dikaryotic mycelium. In addition, counting of nuclei in the cell was carried out for the first time by the inventors in the present application, and for this count the following equipment and technique are used.

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Helly's fixative fluid is first added to the basidiomycet mycelium, and then this mycelium is allowed to stand for usually approx. 24 hours and then washed with water until decolorized. The mycelium thus obtained is immersed in 1N hydrochloric acid solution and the solution is heated to a temperature of 60 ° C. After cooling to room temperature and washing with water, it is further immersed in 20-50 times dilute nitric acid solution, followed by further washing with water. The immersion period is from 10 to 20 minutes in the hydrochloric acid solution and a few minutes in the nitric acid solution. The fibrous cells thus obtained are spread on a slide and thereafter remain until the moisture has evaporated and then the solution of Giem-sa is dropped. At the time of staining with the solution (about 10 minutes later), the cells are lightly washed with water and then dried. After drying, the cells are observed under a light microscope with a magnification of 100x and the circular red-colored spots (considered to be nuclei) are counted. Thus, the number of cores can be determined by counting the red-colored spots in one cell.

"Helly's fixative fluid" used in this connection is a solution of which a base is prepared by dissolving 2.5 g of potassium dichromate, 1 g of sodium sulphate and 5 g of mercuric chloride in 100 ml of water, and immediately before use, this basic solution is added to further formalin. in an amount of 5 ml per ml. 100 ml of the solution.

"Giemsa's Solution" is a nuclear staining solution prepared by dissolving 3.0 g of azure II eosin (a mixture of equal amounts of azure I, methylene blue and eosin) and 0.8 g of azure II (a mixture of equal amounts of azure I and methylene blue) in 250 ml of glycerine by heating to 60 ° C, further adding 250 ml of methyl alcohol, curing the mixed solution for 24 hours and then filtering the solution. In use, the stock solution thus prepared is diluted by adding a phosphoric acid buffer solution (pH 6.4-6.8) in an amount of 100 ml to 3 ml of the stock solution.

As a result of the measurements by the method described above, it was found that the number of nuclei in one cell of the usual spherical mycelium is two, whereas the number in the suspension type mycelium according to the invention is one.

In the method of the invention, the dikaryotic mycelium of Coriolus versicolor (Fr.) Quel can be used. is ground by the addition of inactive solid granular materials, such as glass beads, or the mycelium can be subjected to shear stresses by means of a stirring element.

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The comminution is performed to such an extent that no spherical mycelium is seen when viewed from the outside.

The atmosphere of the submerse culture is preferably maintained at a reduced oxygen partial pressure. The reduced oxygen partial pressure can be provided by keeping the fermenter airtight or by letting an inert gas such as nitrogen gas or carbon dioxide gas into the fermenter.

Preferably, the submerse culture is carried out continuously with further application of the liquid substrate.

In one embodiment of the method according to the invention, the submerse culture is carried out by successively grafting one of a plurality of culture media.

Cultivation is usually carried out at a temperature of 25 -5 ° C over a period of 3-15 days.

It was found that the monokaryotic mycelium of Coriolus versicolor (Fr.) Quel. obtained by the method according to the invention, the same condition and the same properties are always retained if cultivation to produce monokaryotic mycelium is continued under the above conditions. This means that the monokaryotic mycelium obtained by the method of the invention emerges as the same next-generation monokaryotic mycelium, thereby maintaining the monokaryotic mycelium properties shown in Table 1.

In plate cultivation or surface cultivation (stationary cultivation) of the monokaryotic mycelium, it is incapable of forming aerial mycelium, but aerial mycelium will be formed if cultivation is continued for several months. This aerial mycelium is dikaryotic, and when inoculated into growth tree (bed log) to form fruiting bodies, these were found to be Coriolus versicolor (Fr.) Quel. self. This showed that the mycelium formed is exactly the same as the original.

These results indicate that the monokaryotic mycelium of Coriolus versicolor (Fr.) Quel. prepared by the method of the invention is derived from the original dikaryotic mycelium of Coriolus versicolor (Fr.) Quel.

The monokaryotic mycelium produced by the method of the invention is a new mycelium that was first developed by the method of the invention and this new mycelium was named Coriolus versicolor (Fr.) Quel. GX-101-3 and deposited under the number FERM-P No. 3686 on August 25, 1976 in The Fermentation Research Institute, Agency of Industrial Science 7 147128 and Technology (Chiba-shi, Japan), which is a Japanese government agency.

The characteristic features of the monokaryotic mycelium of Coriolus versicolor (Fr.) Quel. produced by the method of the invention is as shown in Table 1, but the major industrial significance of the fungus lies in its high propagation rate. The propagation rate of the fungus is from 1.5 to 10 times as high as the propagation rate of the original dikaryotic mycelium, and it is clear that this high propagation rate is extremely advantageous in industrial production.

The monokaryotic mycelium can be used for the same purpose as the dicharyotic mycelium of Coriolus versicolor (Fr.) Quel.

Eg. it is possible to provide nitrogen-containing polysaccharide by extracting the mycelium with an aqueous medium (such as water, dilute alkaline solution or dilute acidic solution), and this nitrogen-containing polysaccharide material can be used to prepare drugs such as antitumorigenic agents, immunity stimulants, antiviral agents, antiviral agents, agents, anti-leprosy agents, appetite stimulants, etc.

It is also possible to recover various types of enzymes, such as protease, amylase, etc. by low temperature extraction. Furthermore, the mycelium itself or its extracts or residues can be used for food and beverages, animal feed and plant fertilizers.

The invention will now be described in more detail by means of some preferred embodiments. In the following description of embodiments, percentages are by weight unless otherwise indicated.

Example 1

Preparation of Monokaryotic Mycelium: 100 ml of a liquid substrate containing 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 conical flask and after 20 minutes of steam sterilization at 120 ° C in an autoclave, 1 ml of a suspension of mycelium obtained by dispersing the mycelium of Coriolus versicolor (Fr.) was inoculated into the medium.

Quel. (obtained from 20 days of stationary culture at 25 ° C using 50 ml of liquid substrate containing 3% glucose and 0.5% yeast extract in 60 ml of physiological saline solution by breaking the mycelia mat with a blender at a speed of 6000 rpm per minute for 3 minutes), after which shaking was started using the speed of 200 rpm. minute at 25 ° C. Three days after the start of cultivation, the cultured material was aseptically transferred to a 200 ml blender beaker (manufactured by Sakuma Seisakujo) and after grinding the material with a homogenizer mixer (manufactured by Sakuma Seisakujo) at the rate of 10,000 rpm. Minute for 10 minutes, rumination was resumed immediately, so that rumination was practiced for a total period of 7 days. The mycelium thus grown had no cartilage cell formation and the formation of air mycelium in a conventional agar plate substrate was modest. The results of microscopic examination following the staining described below showed that the resulting mycelium was completely monokaryotic.

dyeing; 1 ml of the culture liquid containing mycelium obtained in the manner described above is added to 10 ml of water and then subjected to centrifugal separation at 2000-5000 G for 5 minutes. The supernatant liquid is removed and the mycelium is transferred to a test tube to which Helly's fixative fluid is added. After 20 minutes of standing, the separated cells are washed with 10 ml of water until decolorized. The cells are then placed in 10 ml 1N hydrochloric acid and heated to 60 ° C for 15 minutes followed by cooling to room temperature, washing with 10 ml water, 2 minutes immersion in 10 ml 20-50 times diluted nitric acid solution and 2-3 times washing with 10 ml. water.

The resulting fibrous cells are spread on a slide to allow moisture to evaporate and then a few drops of Giem-sa's solution is added to the cells, and after 15 minutes standing they are lightly washed with water and then dried.

When the thus-stained cell is observed under a microscope at the magnification 100x, each nucleus is seen as a circular red-stained spot. Therefore, the number of cores can easily be determined by counting the circular red stains in one cell of the mycelium. The resulting mycelium could not acidify litmus milk and had no gelatin-depleting ability.

Propagation of monokaryotic mycelium:

The monokaryotic mycelium obtained in the manner described above was charged with 12 liters of a liquid substrate containing 5% glucose and 0.75% yeast extract in a 20 liter glass fermenter (manufactured by Kyoritsu Riko Co., Ltd.) which was previously sterilized. 2 right by supplying 2 kg / cm of steam directly into the glass fermenter.

After steam sterilization at 120 ° C for 20 minutes and cooling, 1 liter of suspension containing the monokaryotic mycelium (at a rate of 0.5 g / l) was inoculated, after which cultivation at an aeration rate of 0.5 min. ^ and a stirring speed of 550 rpm. minute followed immediately. For comparative purposes, culture of the dikaryotic mycelium was performed under completely the same conditions as those used in culture of the monokaryotic mycelium. When comparing the rates of propagation of these mono- and dikaryotic mycelia by the time required to achieve a mycelial concentration of 8 g / l, it is noted that the monokaryotic mycelium requires only 1/4 of the culture time of the dikaryotic mycelium (see Figs. 1). It was also confirmed that the propagation yield of the monokaryotic mycelium increased by approx. 20% relative to the yield of the dikaryotic mycelium.

Example 2

Calculation of the number of cores was performed in the manner described in Example 1 on mycelium obtained by oblique cultivation of Coriolus versicolor (Fr.) Quel. As a result, it was found that all of these cells are dikaryotic, as seen in the photograph of FIG. 2, and no monokaryotic mycelium was detected.

This known fungus is called Coriolus versicolor (Fr.)

Quel. CM-101 and is deposited under the number FERM-P No. 2412 on December 25, 1973, in the aforesaid Japanese government agency.

100 ml of a liquid substrate containing 5% glucose and 0.75% yeast extract were placed in a 500 ml conical flask and sterilized therein under heating. This substrate was then inoculated with the aforementioned dikaryotic mycelium using a platinum graft cartridge and subjected to 3 days of shaking (culture) in a room whose temperature was controlled to 25 -2 ° C. As a result, mycelium was formed in the form of beads. The culture liquid containing this mycelium with spherical shape was homogenized by means of a homogenization blender (manufactured by Sakuma Seisakujo) for 5 minutes and then subjected to a shear treatment, thereby largely disappearing. Cultivation was continued under the above conditions and 4 days later (main culture) the resulting globular mycelium was homogenized again for 5 minutes and then a treatment with effect of shear stress was subjected. At this stage, the concentration of cells of the fungus was 11 g / l.

1 ml of culture liquid containing the homogenized mycelium was added to the same substrate used in the first culture step and then subjected to the second culture step under the same conditions as in the first culture step. However, the growing period changed slightly, with the cultivation being carried out for 3 days and the main cultivation also for 3 days. The fungal cell concentration was of approximately the same level as in the first shaking culture step.

The third culture step was continued in a similar manner, with the fungal cell concentration reaching roughly the same level as in the first shaking culture step at 2 days cultivation and 3 days main cultivation.

Similarly, the fourth culture step was carried out to obtain a culture fluid with a fungal cell concentration of 12 g / l, which is higher than the concentration in the first culture step, at 2 days cultivation and 2 days main cultivation. The resulting mycelium did not take the form of pellets and was dispersed in the form of pulp and required no homogenization treatment. Observed under a microscope, the fungal cell did not show any islet cell shape, which is found in the usual dikaryotic mycelium, and its width was approx. twice the size of the dikaryotic mycelium.

By measuring the number of nuclei by the procedure described in Example 1, it was found that these cells are all mono-karyotic mycelium, as seen in the photomicrograph of FIG. Third

When the mycelium was further cultured under the same conditions as in the previous stage of cultivation, the propagated mycelium consisted of the monokaryotic mycelium and had all the properties of the monocaryotic mycelium shown in Table 1.

It was also found that the monokaryotic mycelium had a propagation rate that was more than twice the propagation rate of the dikaryotic mycelium.

10 g of the dried product of the monokaryotic mycelium obtained by the procedure described above was extracted with 300 ml of warm water at 95-100 ° C for 3 hours. The extract solution was concentrated under reduced pressure to 30 ° C and then pure ethanol was added to the concentration 90% and the resulting precipitate was dried to give 2 g of gray powder. A chemical analysis of this gray powder revealed a high molecular weight nitrogen-containing polysaccharide. When this drug is administered to mice transplanted with Sarcoma-180 (solid type), high anti-tumorigenic effect was demonstrated.

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EXAMPLE 3 100 ml of a liquid substrate containing 5% glucose and 0.75% yeast extract were added to a 500 ml volume conical flask, in which 8 g glass beads of 2-5 mm diameter were already placed and this mixed substrate was inoculated after heat sterilization of the same dikaryotic mycelium of Coriolus versicolor (Fr.) Quel., used in Example 1, using a platinum box and then subjected to 7 days of shaking at 25-2 ° C.

1 ml of culture liquid containing the mycelium thus obtained was added to the glass bead substrate as mentioned above and further subjected to another 6-day shaking culture. The resulting product was further subjected to a third shaking culture step over a period of 5 days.

The resulting mycelium had no cartilage cell shape and a width of approx. 1.5 times the original dikaryotic mycelium, and the result of nuclear counting in the manner set forth in Example 1 indicated that it was monokaryotic mycelium.

1 ml of this culture liquid was inoculated into 100 ml of a liquid medium containing 5% glucose and 0.75% yeast extract containing no glass beads and grown at 25 -2 ° C. The mycelium concentration formed 3 days after the start of cultivation was 10.5 g / l, while corresponding cultivation of the dikaryotic mycelium gave a concentration of 7 g / l 4 days after the start of cultivation.

Example 4 100 ml of a liquid substrate containing 5% glucose and 0.75% yeast extract were placed in a 500 ml conical flask and this substrate was inoculated after heat sterilization with the same dicharyotic mycelium of Coriolus versicolor (Fr.) Quel as used in Example 1. , using a platinum graft box and then subjected to shaking for 3 days. After a homogenization treatment, the entire fermenter was covered with a polyethylene bag and sealed against the ambient air, followed by 4 days of cultivation.

At the end of the cultivation, the fermenter atmosphere contained 5% CO 2 gas.

1 ml of culture fluid containing the mycelium thus prepared was inoculated into the substrate in the manner described in the first culture step and then subjected to the second shaking culture step in the same manner as the first step. This cultivation was further repeated 2 times.

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As a result of the same measurement performed in Example 1, it was found that the mycelium produced by this culture process was completely monokaryotic.

Example 5

A sample of the mycelium obtained from the cultivation of Coriolus versicolor (Fr.) Quel was taken. on an oblique substrate, and this mycelium was subjected to core assay according to the procedure described in Example 1, whereby it was found that the entire mycelium was dikaryotic and that no monokaryotic mycelium was present, as seen in the photograph of FIG. 4. This indicates that the mycelium obtained at this stage of culture is dikaryotic mycelium.

The fungus is Coriolus versicolor (Fr.) Quel. CM-103 and is deposited under the deposit number FERM-P No. 2414 on December 25, 1973 in the aforesaid Japanese government agency.

100 ml of a liquid substrate containing 5% glucose and 0.75% yeast extract was placed in a 500 ml volume conical flask and, after heat sterilization, inoculated the mycelium of Coriolus versicolor (Fr.) Quel. CM.103 using a platinum seed box, followed by 7 days of shaking at 25 -2 ° C. The mycelium in this culture was dikaryotic and present at a concentration of 10.8 g / l.

The dikaryotic mycelium thus obtained was inoculated with 0.01% in 20 liters of a liquid substrate in which 10% glucose and 1.5% yeast extract were dissolved, and the mycelium was subjected to submergence with stirring in a fermentor set to 25%. 2 ° C using a paddle-type stirrer at the rate of 500 rpm. minute. After 7 days of culture, mycelium was formed at a concentration of 10.2 g / l in the culture fluid. 20 ml of this culture liquid was inoculated into the same substrate and other culture steps were carried out under the same conditions over a period of 6 days.

A similar cultivation was repeated over a period of 5 days in the third cultivation step and over a period of 4 days in the fourth cultivation step.

At the end of the fourth culture step, the culture fluid was in the form of uniform pulp and contained the mycelium formed at a concentration of 11.5 g / l. The mycelium had no islet cell shape and was exclusively monokaryotic mycelium.