JPH0398575A - Freeze preservation of basidiomycetes or the like - Google Patents

Abstract

PURPOSE:To preserve Basidiomycetes for a long period of time stably and safely by inoculating mycelia of Basidiomycetes on a wood powder-based medium for preservation containing sawdust sterilized with a polyhydric alcohol as an autifreezing agent, culturing and freezing at an ultralow temperature. CONSTITUTION:A wood powder-based medium for preservation containing at least sawdust is mixed with a polyhydric alcohol (e.g. glycerol or ethylene glycol) as an antifreezing agent and sterilized. Then mycelia of Basidiomycetes (e.g. Lentinus edodes) or a mold (e.g. koji mold) capable of growing in a wood powder-based medium are inoculated into the medium and cultured. Then the mycelia are multiplied to a given range, frozen at an ultralow temperature and stored. Consequently, the mycelia can be stored in a frozen state for a long period of time and the mycelia are optionally thawed, the thawed mycelia are partially used as an inoculation source for a medium for subculture or culture and the rest can be frozen again.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cryopreservation method for basidiomycetes and the like, which is particularly suitable for use with basidiomycetes to which mushrooms belong.

[Prior art and its issues]

Conventionally, as a method for preserving basidiomycetes to which mushrooms such as shiitake and enokitake belong, a preservation method using a slant culture medium is known.

This method involves forming an agar medium slant in a tilted test tube, growing basidiomycete hyphae on this slant, and then
Slants are stored at low temperatures, and the storage temperature is usually 5℃.
Before and after.

However, in this method, water evaporates from the medium during storage, so the storage period is usually limited to 6 months to 1 year. In addition, subculturing causes expansion of physiological and genetic variations in hyphae, which often impedes the production of edible mushroom seeds and the production of pharmaceuticals and other useful substances using basidiomycetes. There were also problems with the preservation of bacterial strains.

To solve this problem, we cut the mycelia from the slant together with the agar and placed the pieces in a container containing an aqueous glycerol solution.
There is also a method of suspending the material in an ampoule and freezing it at a low temperature using liquid nitrogen or the like.

This method allows for long-term storage without worrying about the spread of mutations in basidiomycetes; however, it requires the inclusion of ampoules of bacterial samples and programmed freezing, i.e., the purpose of delaying the crystallization of water inside and outside the cells and the production of water. In order to keep the crystals in a fine state, complicated operations are required, such as using special equipment such as a program freezer to slowly lower the temperature to a low temperature range of around -30°C with a temperature gradient of 1 to 2°C per minute. shall be. Moreover, if the temperature rises due to failure to replenish liquid nitrogen or equipment failure, the hyphae will die. In addition, samples from the same ampoule are limited to one use, and there is a disadvantage in that preservation work is not easy and safe.

Note that a method for preserving basidiomycete strains by freeze-drying has also been proposed, but at present the challenge is to improve the survival rate, and this has not been put to practical use.

The object of the present invention is to provide a method for cryopreservation of basidiomycetes, etc., which solves the problems existing in the prior art.

[Means to solve the problem]

The cryopreservation method for basidiomycetes, etc. according to the present invention is to
The basic idea is that it can be rapidly frozen at low temperatures without any special precautions, and even if it is thawed naturally after long-term storage, it can be restored without damaging the mycelium. Although the principle of cryopreservation using wood flour-based media is not fully understood, ■ Most of the water in wood flour-based media is dispersed or absorbed inside and outside of the sawdust, so it is difficult to prevent ice formation due to freezing. The formation of this occurs mainly in sawdust, and ■ Because the water content of the medium is low (usually around 60 to 70%), the water activity is low and there is little damage to the hyphae even if the medium freezes rapidly. Conceivable.

In addition, mycelia cultured in a wood flour-based medium with polyhydric alcohols such as glycerol added as a cryoprotectant have significantly improved resistance to damage caused by freezing treatment, and by repeating the treatment more than 10 times. It was also confirmed that hyphal survival was ensured. This means that even if a temporary failure occurs in an ultra-low temperature storage device, the safety of the stored bacterial strain can be guaranteed.

In addition, when a cryoprotectant is not added, if freezing and thawing are repeated two or more times after culturing mycelia, the survival rate of mycelia gradually decreases.

A water powder-based medium for storage is prepared by mixing sawdust with nutrients such as rice bran and wheat bran, depending on the type of basidiomycete or filamentous fungi that can be grown in the wood-based medium to be preserved, and then adding the above-mentioned cryoprotectant. is added to adjust the moisture content, and the mixture is placed in a small culture container such as a test tube and sterilized.

As for the tree species for sawdust, broad-leaved trees such as beech are suitable. In the case of coniferous trees such as cedar, the sawdust is piled up in the open, sprinkled with water, and left for about 6 months to remove any substances that inhibit the growth of mycelium or decompose it.

As the nutritional material, rice bran, bran, corn plan, etc., which are commonly used for mushroom cultivation, or a mixture thereof are mixed according to the type of basidiomycete to be preserved. If necessary, medium materials, organic compounds, and inorganic compounds used for culturing microorganisms, such as yeast extract, are added.

As cryoprotectants, polyhydric alcohols such as glycerol and ethylene glycol are effective.

These enhance the protective effect in proportion to the added concentration, but when added in an amount of 5% or more per dry weight of the medium, mycelial growth during the culture process is suppressed in proportion to the concentration, prolonging the culture. Ethylene glycol has a stronger inhibitory effect than glycerol. Therefore, the appropriate concentration of the cryoprotectant added is about 5 to 12%, preferably about 10%.

Regarding the moisture content of wood flour-based media, the lower the moisture content of the medium, the better the resistance of mycelium to freezing and thawing treatments, but a medium with a low moisture content retards the growth of mycelia. Therefore, the moisture content of the medium is approximately 56-70% based on the dry weight of the medium.
Preferably, it is adjusted to about 60 to 64%.

On the other hand, the thus prepared and sterilized wood flour-based medium is inoculated with mycelia of basidiomycetes or filamentous fungi that can grow in this wood-flour-based medium.

Mycelium grown on a slant medium can be used as an inoculum for the fungal strain. In the case of edible mushrooms, hyphae grown in a medium with a composition used for cultivation may be used. Also,
Types of filamentous fungi that can be cultured and preserved in wood flour-based media include basidiomycetes and other fungi that lack wood assimilation ability, such as mycorrhizal basidiomycetes such as matsutake and shimeji. It can also be used for preservation.

After inoculation of mycelium, culture is performed. and a predetermined range of medium,
Preferably, when mycelium has spread over the entire area, it should be frozen and preserved.

As a cryopreservation device, an ultra-low temperature storage (deep freezer) is convenient, and the cryopreservation temperature is -60°C or lower, preferably at a standard temperature. Set in the range of 80°C to -95°C. Note that a liquid nitrogen storage container can also be used. Such cryopreservation does not require any special temperature program, and it is sufficient to simply transfer the culture container itself from room temperature to a storage container. When using a frozen bacterial strain, remove the sample (culture container) from storage and thaw it naturally. After inoculating onto a slant culture medium, the remainder can be frozen and stored again.

〔Effect of the invention〕

By this cryopreservation method, many filamentous fungi such as wood-decaying basidiomycetes such as shiitake mushrooms, mycorrhizal basidiomycetes such as matsutake, saprophytic basidiomycetes such as mushrooms, ascomycetes, deuteromycetes, and zygomycetes can be preserved. It can be frozen and preserved for a long period of five years or more, and can be used by repeatedly thawing and freezing the same strain as necessary.

In addition, no special temperature program operations for freezing and thawing are required, and it is possible to achieve a particular simplification of operations for preserving bacterial strains.

Furthermore, even in the event of an accident in a frozen storage facility, the strain can be stored stably without damage or other problems, ensuring safety and reliability, and the quality and yield are equivalent to cultivation using non-preserved bacteria. obtain results.

〔Example〕

Preferred embodiments of the present invention will be specifically shown below.

(Example 1) (A) Mix 1.5 g of beech sawdust and 0.48 g of rice bran, add an aqueous solution containing glycerol corresponding to 10% of the water content of the medium, and make the final concentration of water content of the medium 64. Prepare a wood flour-based medium with %. At this time, the moisture content was adjusted by correcting the moisture content of sawdust and rice bran, which were separately dried at a constant temperature of 80° C. and weighed.

Then, this medium was packed into a fraction collector test tube with an inner diameter of 15.5 mm to a depth of about 5 cm.
It was sealed with a silicone stopper and sterilized at a temperature of 120°C for 10 minutes.

(B) Inoculation and cultivation On the top of the medium obtained in (A), Enokitake (Flama+uii
na veltipes) and oyster mushroom (Pleur
A small piece of 3 to 5 mm square cut out from the mycelium of S. otus ostreatus was inoculated and cultured for 3 weeks at a temperature of 25°C.

(C) Freezing and restoring hyphae The test tubes after culture in (B) were transferred to a deep freezer set at a temperature of -85°C, frozen, and stored for 5 years. During this storage period, the test tubes were taken out every 6 months and left to thaw at room temperature, and a small amount of the sawdust mycelial mass was inoculated onto a PDA slant medium (agar medium using potatoes) at a temperature of 25°C. When cultured at ℃, vegetative hyphae were recovered from all hyphal masses. After the thawed test tube was used as such an inoculum, it was cryopreserved again and the hyphae restoration was repeated.

(D) Results The enokitake and oyster mushroom mycelia restored by (C) after 3 years of cryopreservation were cultivated in an edible mushroom sawdust medium stored in an 850 cc bottle, and compared with those subcultured with the same strain for 3 years. As a result, yields equivalent to those obtained through subculture were obtained in all cases, and no changes in the shape of the mushrooms were observed due to cryopreservation.

(Example 2) (A) Medium Same as Example 1 (A).

(B) Inoculation and cultivation In place of the test strain of Example I, as wood-decaying basidiomycetes, shiitake mushroom (Lentinus edodes), Nameko mushroom (
Ph-o1iota nameko), Favolus arcu arius, Coriolus versicolor,
Ganoderma lucidum
, Trametes sanguine
a) As a saprophytic basidiomycete, Agaritake
cus bisporus)
Coprinus cinereus), mycorrhizal basidiomycete, Boletus pulver
ulentus), Ibarishimeji (Lyophyll)
un+ tylicolor), Shimejimodoki (Rh
odopyllus clypeatus), ascomycetes, Neurospora cra
ssa), Aspergillus spp.
gillus oryzae), Aspergillus niger (A
spergillus niger).

In addition, Example 1 (except that the culture period was 2 to 4 weeks)
Same as B).

(C) Results of freezing and restoring hyphae Using the preserved strain obtained in Example (B), Example 1 (
When the cells were cryopreserved for 3 years in the same manner as in C), thawed, and inoculated onto a PDA slant medium, restored hyphae were generated from all of them.

(Example 3) (A) Medium Glucose Ig, fructose Ig, and 0.5 g of dried yeast (Ebios) were dissolved and suspended in 100 ml of water,
A liquid medium adjusted to pH 5.6 is added to 1.5 g of sawdust to adjust the water content to 68%, and glycerol is added to the final concentration of 5%. Then, it was placed in a test tube, sealed with a silicone stopper, and sterilized at a temperature of 120° C. for 10 minutes.

(B) Inoculation and culture (A) The mycelium of Matsutake, which had been cultured in advance on a glucose-Ebios slant medium (medium composition: 2% glucose, 0.5% Ebios, 1.5% agar), was cut out from the top of the medium in (A). Small pieces were inoculated and cultured for 3 months at a temperature of 25°C.

(C) Cryopreservation and restoration of hyphae. Results Using the preserved strain obtained in this example (B), Example 1 (C
) was frozen for one year, thawed, inoculated onto a glucose/shrimp slant medium, and cultured. Restored mycelium was obtained from the mycelial mass.

Although various embodiments have been described above, the present invention is not limited to these embodiments, and can be modified as desired without departing from the gist of the present invention.

Claims (1)

[Scope of Claims] [1] A preservation wood flour-based medium containing at least sawdust sterilized with polyhydric alcohol as a cryoprotectant is inoculated with hyphae of a basidiomycete or a filamentous fungus that can grow in a wood flour-based medium. A cryopreservation method for basidiomycetes, etc., which comprises culturing the basidiomycetes, spreading the mycelium over a predetermined area, and then freezing the basidiomycetes at an ultralow temperature. [2] After thawing the cryopreserved hyphae and using a portion of the thawed hyphae as an inoculum for passage or cultivation medium,
2. The cryopreservation method for basidiomycetes, etc. according to claim 1, characterized in that the remaining portion is refrozen. [3] The cryopreservation method for basidiomycetes, etc. according to claim 1, characterized in that glycerol or ethylene glycol is used as the polyhydric alcohol. [4] The cryopreservation method for basidiomycetes, etc. according to claim 1, wherein the ultra-low temperature is set to -60°C or lower. [5] The cryopreservation method for basidiomycetes, etc. according to claim 4, wherein the standard temperature at ultra-low temperature is set at -80°C to -95°C.