CN112961786A

CN112961786A - Freezing preservation method of edible fungi

Info

Publication number: CN112961786A
Application number: CN202110216824.2A
Authority: CN
Inventors: 孙姣; 李洁; 张振宇
Original assignee: Shandong Xiangyu Seed Technology Co ltd
Current assignee: Shandong Xiangyu Seed Technology Co ltd
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2021-06-15

Abstract

The invention discloses a freezing preservation method of edible fungi, and relates to the technical field of edible fungi preservation. Which comprises the following steps: the perlite is used as a carrier, the mycelium of the edible fungi to be preserved is spread and grown to the internal pores of the perlite, the perlite on which the edible fungi to be preserved are grown is frozen and preserved, and a freezing protective agent is added before the freezing preservation. The method uses perlite as carrier, and the perlite has porous structure and does not contain available nutrient components, so that the perlite can be used as mycelium attachment matrix. Therefore, hypha of the edible fungi can spread and grow into the gap structure of the perlite, and meanwhile, the freezing protective agent can also enter the gap in large quantity, so that the strain is effectively protected, and the preservation survival rate of the strain can be improved. The perlite does not contain water, the perlite grown with the edible fungi is directly frozen and preserved, and the damage of a culture medium with high water content to mycelium cells can be avoided, so that the survival rate and the germination rate of the thalli are improved.

Description

Freezing preservation method of edible fungi

Technical Field

The invention relates to the technical field of edible fungus preservation, in particular to a freezing preservation method of edible fungi.

Background

In the field of edible fungi, the strains are the key for edible fungi production, are also important germplasm resources in China, and are basic materials for production and scientific research. The aim of strain preservation is to ensure that the strain to be preserved keeps biological activity, does not generate fading and mutation, and does not generate strain pollution because of the need of regular transfer for keeping the strain alive. The strain preservation can be as close as possible to the state of the original strain to meet the requirements of research, use and the like.

At present, the preservation of the edible fungi is mainly carried out by using a hypha preservation method and a liquid nitrogen preservation method. The liquid nitrogen preservation method is mainly characterized in that edible fungi are inoculated on culture media with different matrixes, the culture media full of hyphae are punched or diced, 5% -10% of glycerol is used as a protective agent, and the culture media are placed in a liquid nitrogen tank for long-term preservation after being cooled to-90 ℃ by a programmed cooling instrument.

The strains preserved by the liquid nitrogen preservation method can keep the biological activity after being recovered, so the strains are widely adopted.

However, the problems of low survival rate and low germination rate of the strains after the strains are recovered after the strains are frozen at present exist.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a freezing preservation method of edible fungi to solve the technical problems.

Aiming at the problems of low survival rate and low germination rate of the frozen strain after recovery, the inventor finds that the low survival rate of the frozen strain after recovery is caused by the fact that part of a culture medium with the strain to be preserved is placed in a freezing tube for preservation in the existing freezing method, and taking a PDA culture medium as an example, because the water content of the PDA culture medium is high, mycelium cells are easy to expand due to ice crystals formed by water under a low-temperature condition in the process of program cooling or recovery and temperature return, and the cells are damaged due to extrusion of external ice crystals on the cells, so that the survival rate of thalli is reduced, and the germination rate is low.

The inventor provides a new idea to solve the technical problems and provides a new preservation substrate to ensure the survival rate of the strains after recovery.

The invention is realized by the following steps:

a freezing preservation method of edible fungi comprises the following steps: the perlite is used as a carrier, the mycelium of the edible fungi to be preserved is spread and grown to the internal pores of the perlite, the perlite on which the edible fungi to be preserved are grown is frozen and preserved, and a freezing protective agent is added before the freezing preservation.

The perlite is used as a carrier, and the perlite has a porous structure and does not contain available nutrient components, so that the perlite can be used as a hypha attachment matrix. Therefore, hypha of the edible fungi can spread and grow into the gap structure of the perlite, and meanwhile, the freezing protective agent can also enter the gap in large quantity, so that the strain is effectively protected, and the preservation survival rate of the strain can be improved.

The perlite does not contain water, the perlite grown with the edible fungi is directly frozen and preserved, and the damage of a culture medium with high water content to mycelium cells can be avoided, so that the survival rate and the germination rate of the thalli are improved.

Compared with the conventional method, the thalli preserved by the freezing preservation method provided by the invention has the advantages that the germination speed is high, and the survival rate is greatly improved.

The method provided by the invention has the following beneficial effects: the survival rate of the mycelium is high, the germination speed is high, the mycelium grows vigorously, and the problems that the edible fungi are easy to pollute, mutate and even die in the using and passage processes, so that the strains are declined and the strains are lost are solved.

In one embodiment, the cryopreservation method comprises: the method comprises the steps of firstly placing perlite on a culture medium, then inoculating the edible fungi to be preserved on the culture medium, culturing until hyphae overgrow and grow to the internal pores of the perlite, and then freezing and preserving the perlite growing the edible fungi to be preserved.

Optionally, culturing until mycelium overgrows to the inner pores of the perlite, forming mycelium particles taking the perlite as a support structure, and then freezing and preserving the perlite which is wrapped with the edible fungi to be preserved on the periphery.

The hyphae are observed to cover the exterior of the perlite particles by naked eyes, and the hyphae are considered to have spread and grow to the interior of the perlite particles after being cultured for 3-5 days; and judging the wrapping degree of the edible fungus hyphae on the perlite, and if the edible fungus hyphae finish the full wrapping of the perlite, judging that the hyphae overgrows and grows to the internal pores of the perlite. In other embodiments, the determination may be made based on the growth time.

In one embodiment, the edible fungi to be preserved are cultured in the culture medium at a temperature of 15-30 ℃. Alternatively, the culture temperature may be 15 ℃, 18 ℃, 20 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or 30 ℃.

In one embodiment, the incubation time is 8-25 days.

In a specific embodiment, the culture temperature and culture time are adaptively adjusted according to the type of the edible fungi to be cultured, and are not limited to the temperature and time defined in the present invention.

In one embodiment, the cryopreservation method comprises: inoculating the edible fungi to be preserved to a culture medium for primary culture, then placing perlite in the culture medium after the primary culture, carrying out secondary culture, and after the hypha of the edible fungi is spread and grown to the internal pores of the perlite, carrying out freezing preservation on the perlite grown with the edible fungi to be preserved. This embodiment requires special care to prevent excessive hyphal aging.

In one embodiment, the time for the first culture is 3-20 days, and the time for the second culture is 7-20 days.

In one embodiment, the temperature of the first culture is 15-30 ℃ and the temperature of the second culture is 15-30 ℃.

In other embodiments, the temperature and time of the primary culture and the secondary culture can be adaptively adjusted according to the need of culturing the strain, and are not limited to the temperature and time ranges given above in the present invention.

In one embodiment, the medium is a plate medium or a slant medium. The medium may be a plate or a slant medium, depending on the enrichment requirement.

In one embodiment, the culture medium is a PDA culture medium, a potato complex culture medium, a wood chip leaching culture medium, a bean sprout juice culture medium, or a cottonseed hull culture medium.

The PDA culture medium is potato glucose agar culture medium, and can be prepared by itself or selected from commercially available PDA culture media. PDA culture medium is suitable for most edible fungi.

The potato comprehensive culture medium is suitable for culturing and preserving mushroom, oyster mushroom, agaricus bisporus, needle mushroom, hericium erinaceus, lucid ganoderma, agaric and the like.

The wood chip leaching culture medium can be used for culturing strains of wood rotting fungi.

The bean sprout juice culture medium is suitable for culturing strains such as black fungus, hericium erinaceus and oyster mushroom.

The cottonseed hull culture medium is suitable for culturing Hericium Erinaceus, Auricularia, etc.

In addition, in other embodiments, other culture mediums capable of culturing edible fungi may be selected, and any culture medium that can satisfy the growth of edible fungi is within the scope of the present invention. Such as other natural media, semi-synthetic media, and synthetic media.

In one embodiment, the perlite is expanded perlite, surface treated expanded perlite, perlite ore. In other embodiments, the type of perlite may be selected as desired.

In one embodiment, the perlite is a post-sterilized perlite; the aseptic processing step comprises the steps of washing the perlite to be processed with water, drying and sterilizing.

In one embodiment, sterilization is performed at 119-; preferably, the sterilization time is 30-60 min.

In one embodiment, the perlite has a particle size of 3 to 8 mm.

The edible fungi are sterilized to avoid the pollution of the mixed fungi carried by the perlite. In other embodiments, commercially available perlite that has been sterilized may also be selected. Preferably, the surface is white and the perlite particles are dried with high quality.

In other embodiments, the sterilization method may be selected as needed, and is not limited to the steam sterilization method described above.

The inventor finds out through practice that the perlite with the grain size just meets the requirement of freely taking and placing the frozen pipe, and in other embodiments, the size of the perlite can be selected according to the requirement, for example, the perlite with the diameter larger than 8mm or smaller than 3mm is also feasible and is also within the scope of the invention concept.

In one embodiment, the freezing preservation of the perlite for growing the edible fungi to be preserved comprises: placing the perlite growing the edible fungi to be preserved in a freezing storage tube, adding a freezing protective agent into the freezing storage tube, then placing the freezing storage tube in a program cooling instrument for cooling, and placing the freezing storage tube at a low temperature for preservation after cooling.

It should be noted that the present invention provides only one specific freezing and temperature-reducing method, and in other specific practical processes, the temperature can be controlled for freezing and preservation as required, and is not limited to the method of temperature reduction in a program temperature-reducing instrument.

In one embodiment, the temperature reduction program of the programmed temperature reduction instrument is: at normal temperature, the temperature is reduced to 4 ℃ at a speed of 4-5 ℃/min, and then the temperature is reduced to-90 ℃ at a speed of 0.8-2 ℃/min.

The cooling program is only a cooling method provided by the invention, and in a specific practical process, the cooling program can also automatically select to increase the cooling rate or delay the cooling rate according to needs.

In one embodiment, the freezing tube is stored in a liquid nitrogen tank after the temperature is reduced. In other embodiments, storage in other low temperature environments may be selected, such as ultra-low temperature refrigerators.

In one embodiment, the cryoprotectant is at least one of glycerol, dimethyl sulfoxide, tween 80, glucose, trehalose, inositol and xylitol; preferably, the lyoprotectant is added to the vial at a concentration of 5-15% by volume.

In other embodiments, the cryoprotectant may also be selected from acidic materials such as glutamic acid, aspartic acid, or lactic acid; can also be selected from neutral substances such as lactose, sucrose, raffinose, sorbitol D, L-threonine, etc.; it may also be selected from high molecular substances such as gelatin, peptone, dextrin, soluble starch, hydroxymethyl cellulose, pectin gum arabic, peptone, etc.

In one embodiment, the cryopreservation method further comprises a strain recovery step, wherein the strain recovery step comprises the following steps: and taking the freezing tube out of the liquid nitrogen tank, carrying out constant-temperature water bath, and transferring to a culture medium for recovery culture of the strain after perlite grown with the edible fungi is completely melted.

In one embodiment, the constant temperature water bath is a water bath at 36-38 ℃; the constant temperature water bath time is 1-10 min. In other embodiments, the perlite can be melted completely by hot water directly at higher temperature or at ambient temperature.

In one embodiment, the culture medium used for the above recovery culture is PDA culture medium, potato integrated culture medium, or wood chip leaching culture medium.

In addition, in other embodiments, other culture mediums capable of culturing edible fungi may be selected, and any culture medium that can satisfy the growth of edible fungi is within the scope of the present invention.

The recovery culture of the strain is carried out at 15-30 ℃.

The edible fungus is selected from any one of the following: straw mushroom, stropharia rugoso-annulata, agaricus bisporus, pleurotus nebrodensis, coprinus comatus, shiitake mushroom, dictyophora phalloidea, malassezia, oyster mushroom, flammulina velutipes, pleurotus eryngii, grifola frondosa, hericium erinaceus, agrocybe cylindracea, russula vinosa, tricholoma matsutake and pleurotus geesteranus.

In another embodiment, the selection of the edible fungi may be performed as needed, and is not limited to the above-mentioned ones. For example, other edible fungi may be used: auricularia, Boletus, Ganoderma, and Cordyceps.

The invention has the following beneficial effects:

the invention provides a freezing preservation method of edible fungi, which takes perlite as a carrier, and can be used as a hypha attachment matrix because the perlite has a porous structure and does not contain available nutrient components. Therefore, hypha of the edible fungi can spread and grow into the gap structure of the perlite, and meanwhile, the freezing protective agent can also enter the gap in large quantity, so that the strain is effectively protected, and the preservation survival rate of the strain can be improved. The perlite does not contain water, the perlite grown with the edible fungi is directly frozen and preserved, and the damage of a culture medium with high water content to mycelium cells can be avoided, so that the survival rate and the germination rate of the thalli are improved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the growth of Lentinus edodes strain No. seven river 2 before liquid nitrogen preservation in mixed pearlite/PDA culture medium;

FIG. 2 shows the growth of strain 808 of Lentinus edodes before liquid nitrogen preservation on mixed perlite/PDA medium;

FIG. 3 shows the growth of Pleurotus ostreatus 3015 strain before liquid nitrogen preservation on perlite/PDA mixed culture medium;

FIG. 4 shows the growth of Flammulina velutipes strains before liquid nitrogen preservation in a perlite/PDA mixed culture medium;

FIG. 5 shows the growth of Pleurotus eryngii strain before liquid nitrogen preservation on perlite/PDA mixed culture medium;

FIG. 6 shows the growth of Lentinus Edodes strain QIHE No. 2 after recovery at low temperature with perlite as matrix and liquid nitrogen preservation for 1 year;

FIG. 7 shows the growth of Lentinus edodes L808 strain after low-temperature storage in liquid nitrogen for 1 year with perlite as matrix;

FIG. 8 shows the growth of Pleurotus ostreatus 3015 after recovery from cryopreservation with perlite as matrix and liquid nitrogen for 1 year;

FIG. 9 shows the growth of Flammulina velutipes (Fr.) Sing strain after low-temperature storage for 1 year with perlite as matrix and recovery in liquid nitrogen;

FIG. 10 shows the growth of Pleurotus eryngii strains after low temperature storage for 1 year with perlite as matrix and liquid nitrogen recovery;

FIG. 11 shows the growth of Lentinus edodes strain No. seven-river 2 after recovery on PDA culture medium after cryo-preservation with perlite as preservation medium in liquid nitrogen for 1 year;

FIG. 12 shows the growth of Lentinus edodes strain 808 on PDA medium after its recovery in liquid nitrogen cryopreservation for 1 year using perlite as preservation medium;

FIG. 13 shows the growth of Pleurotus ostreatus 3015 on PDA medium after 1 year of recovery from cryopreservation in liquid nitrogen with perlite as the storage medium;

FIG. 14 shows the growth of Flammulina velutipes (Fr.) Sing strain in PDA culture medium after low-temperature storage with perlite as storage medium and liquid nitrogen for 1 year;

FIG. 15 shows the growth of Pleurotus eryngii strain on PDA culture medium after 1 year of recovery by liquid nitrogen cryopreservation with perlite as preservation medium.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a cryopreservation method for the Lentinus edodes strain No. Heqi river 2 and Lentinus edodes strain L808. The method comprises the following steps of:

taking expanded perlite as a preservation substrate, wherein the particle size of the expanded perlite is 3-8mm, and carrying out cleaning, drying and sterilization treatment; and under the aseptic condition, the perlite matrix is spread on a PDA plate to prepare a mixed culture medium of the perlite and the PDA.

Then, the mycelium to be preserved is inoculated on the mixed culture medium in a multi-point inoculation mode, and the mixed culture medium is placed in an incubator at 25 +/-1 ℃ for culture for 17 days until the mycelium overgrows and grows into perlite to form mycelium particles. (Lentinus Edodes strains hepta 2 and Lentinus Edodes strain L808, respectively, were cultured individually) growth conditions before preservation are shown in FIGS. 1 and 2.

Placing the above mycelium particles (i.e. pearlite with mycelium) in aseptic freezing tube, injecting 10% glycerol into the freezing tube, making into glycerol tubule, cooling with programmed cooling instrument, and rapidly placing in liquid nitrogen tank for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out from a liquid nitrogen tank, and is subjected to constant-temperature water bath at 36 ℃ for 5min, hypha particles are completely melted and then transferred to a PDA culture medium, and the PDA culture medium is placed at the temperature of 25 +/-1 ℃ for culture until hypha germinates.

Example 2

This example provides a method for cryopreservation of Pleurotus ostreatus strain 3015. The method comprises the following steps of:

Then, the mycelium to be preserved is inoculated on a mixed culture medium in a multi-point inoculation mode, and is placed in an incubator at 24 +/-1 ℃ for culture for 14 days until the mycelium overgrows into perlite to form mycelium particles. The growth of Pleurotus ostreatus strain 3015 before preservation is shown in FIG. 3.

And then placing the mycelium particles (namely the perlite with the mycelium) into a sterile freezing storage tube, injecting 5% of glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out from a liquid nitrogen tank, and is subjected to constant-temperature water bath at 36 ℃ for 5min, hypha particles are completely melted and then transferred to a PDA culture medium, and the PDA culture medium is placed at the temperature of 24 +/-1 ℃ for culture until hypha germinates.

Example 3

The embodiment provides a method for freezing and preserving flammulina velutipes. The method comprises the following steps of:

Selecting golden mushroom with white hypha, no pollution and no bacteria degradation, inoculating the mycelium to be preserved on a mixed culture medium in a multipoint inoculation mode, and culturing in an incubator at 24 +/-1 ℃ for 14 days until the mycelium grows into perlite in a spreading mode to form mycelium particles. The growth of needle mushroom before preservation is shown in FIG. 4.

Example 4

The embodiment provides a method for freezing and preserving pleurotus eryngii. The method comprises the following steps of:

Selecting pleurotus eryngii strains with white, dense and pollution-free mycelia and without bacteria degradation, then inoculating mycelia to be preserved on a mixed culture medium in a multipoint inoculation mode, and culturing for 14 days in an incubator at 23 +/-1 ℃ until the mycelia overgrow and grow into perlite to form mycelia particles. The growth of Pleurotus eryngii before preservation is shown in FIG. 5.

When the culture medium is recovered in the preservation period of 1 year, the freezing tube is quickly taken out from a liquid nitrogen tank, and is subjected to constant-temperature water bath at 36 ℃ for 5min, hypha particles are completely melted and then transferred to a PDA culture medium, and the PDA culture medium is placed at the temperature of 23 +/-1 ℃ for culture until hypha germinates.

Example 5

Compared with the example 3, the difference is that in the example, the edible fungi to be preserved are firstly inoculated on the culture medium, and are placed in the incubator at 24 +/-1 ℃ for primary culture for 5 days, then the perlite is placed in the culture medium after the primary culture, and are placed in the incubator at 24 +/-1 ℃ for secondary culture for 14 days until the hypha spreads and grows to the interior of the perlite to form mycelium particles. The remaining preservation method and the recovery method were the same as in example 3.

Example 6

This example is compared to example 4, with the only difference that the cryoprotectant in this example is sucrose.

Example 7

This example differs from example 1 only in that the cryoprotectant in this example is trehalose.

Example 8

This example is compared with example 1 except that the medium in this example is a wood chip leaching medium and the preservation method is the same as in example 1.

Comparative example 1

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 25 +/-1 ℃ for 17 days until the mycelium grows to form mycelium particles. (Lentinus Edodes strains Qihe No. 2 and Lentinus Edodes strains L808 separately cultured)

And then placing the mycelium particles into a sterile freezing storage tube, injecting 10% of glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

Comparative example 2

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 24 +/-1 ℃ for 14 days until the mycelium grows to form mycelium particles.

And then placing the mycelium particles into a sterile freezing storage tube, injecting 5% of glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year. The cooling procedure is as follows: firstly, the temperature is reduced to 4 ℃ at the normal temperature of 25 ℃ at the speed of 5 ℃/min, and then the temperature is reduced to-90 ℃ at the speed of 1.2 ℃/min.

Comparative example 3

Comparative example 4

and (3) taking PDA as a preservation substrate, inoculating the mycelium to be preserved on the PDA plate according to a multi-point inoculation mode, and culturing in an incubator at 23 +/-1 ℃ for 14 days until the mycelium grows to form mycelium particles.

Experimental example 1

The average survival rates of the mycelia after the resuscitations in examples 1 to 6 and comparative examples 1 to 6 were respectively measured, and the average survival rates of the mycelia are shown in table 1, and it can be seen from table 1 that the survival rate of the mycelia after the strain preservation using perlite as the substrate is higher than that of the mycelia after the strain preservation using PDA.

FIG. 6 shows the growth of Lentinus Edodes strain QIHE No. 2 after recovery at low temperature with perlite as matrix and liquid nitrogen preservation for 1 year; FIG. 7 shows the growth of Lentinus edodes L808 strain after low-temperature storage in liquid nitrogen for 1 year with perlite as matrix; FIG. 8 shows the growth of Pleurotus ostreatus 3015 after recovery from cryopreservation in liquid nitrogen for 1 year using perlite as matrix; FIG. 9 shows the growth of needle mushroom after low-temperature storage in liquid nitrogen for 1 year with perlite as matrix; FIG. 10 shows the growth of Pleurotus eryngii after 1 year recovery by cryopreservation in liquid nitrogen with perlite as substrate.

FIG. 11 shows the growth of Lentinus edodes strain Qihe No. 2 after recovery at low temperature with PDA as matrix and liquid nitrogen for 1 year; FIG. 12 shows the growth of Lentinus edodes L808 after recovery from cryopreservation in liquid nitrogen for 1 year using PDA as substrate; FIG. 13 shows the growth of Pleurotus ostreatus 3015 after recovery from cryopreservation in liquid nitrogen for 1 year using PDA as substrate; FIG. 14 shows the growth of needle mushroom after low temperature storage in liquid nitrogen for 1 year using PDA as substrate; FIG. 15 shows the growth of Pleurotus eryngii after 1 year resuscitation by using PDA as substrate and liquid nitrogen cryopreservation.

Comparing fig. 6 and fig. 11, it can be seen that the strain growth is better when the method of preserving perlite as a substrate by liquid nitrogen is adopted than the method of preserving PDA substrate of comparative example 1 under the same recovery time.

Similarly, the same conclusion can be drawn by comparing fig. 7 and fig. 12.

Table 1 hypha average survival statistical table.

Experimental example 2

In the experimental example, feasibility experiments are carried out on different edible fungus strains by using perlite with different granularity as a liquid nitrogen preservation matrix.

Three kinds of perlite with different granularities of 3-5mm, 5-6mm and 6-8mm are respectively designed to be used as preservation matrixes for liquid nitrogen preservation of edible fungus strains, and liquid nitrogen preservation is respectively carried out on the champignon Qihe No. 2, the champignon L808, the oyster mushroom, the pleurotus eryngii and the flammulina velutipes, and the hypha germination rate is revived and observed after 1 year.

Specifically, according to the preservation method of example 1, the piece-shaped or block-shaped oak perlite substrates with the granularity of 3-5mm, 5-6mm and 6-8mm are respectively cleaned, dried and sterilized; respectively spreading perlite matrixes with different granularities on a PDA plate under the aseptic condition to prepare a mixed culture medium of the perlite and the PDA; inoculating mycelia to be preserved on a mixed culture medium/PDA according to a multi-point inoculation mode, and culturing for 17d in an incubator at 25 +/-1 ℃ until the mycelia overgrow and grow into a perlite/PDA substrate to form mycelia particles; then placing the mycelium particles into a sterile freezing storage tube, injecting 10% glycerol into the freezing storage tube to prepare a glycerol tubule, cooling by a programmed cooling instrument, and quickly placing the glycerol tubule into a liquid nitrogen tank for storage for 1 year.

Storing in liquid nitrogen tank for 1 year, taking out the freezing tube from the liquid nitrogen tank, thawing in 36-38 deg.C water bath for 5min, transferring to PDA culture medium after mycelium granule is completely melted, and culturing at 25 + -1 deg.C until mycelium germinates. And finally, counting the survival rate of hyphae. As shown in table 2 below, it can be seen from table 2 that the size of perlite has no significant effect on the survival rate of the strain.

Table 2 results of feasibility experiments were performed on quercus robur of different particle sizes as a liquid nitrogen preservation substrate.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A freezing preservation method of edible fungi is characterized by comprising the following steps: the method comprises the steps of taking perlite as a carrier, after hypha of edible fungi to be preserved spreads and grows to the inner pores of the perlite, carrying out freezing preservation on the perlite growing the edible fungi to be preserved, and adding a freezing protective agent before freezing preservation.

2. The method for cryopreservation of edible fungi as claimed in claim 1, wherein the method for cryopreservation comprises: firstly, placing perlite on a culture medium, then inoculating the edible fungi to be preserved to the culture medium, culturing until hyphae overgrow and grow to the internal pores of the perlite, and freezing and preserving the perlite on which the edible fungi to be preserved grow;

preferably, after hypha grows to the internal pores of the perlite in a spreading way, mycelium particles taking the perlite as a supporting structure are formed, and then the perlite wrapped with the edible fungi to be preserved is frozen and preserved.

3. The method for the cryopreservation of edible fungi according to claim 2, wherein the culture temperature of the edible fungi to be preserved on the culture medium is 15-30 ℃;

preferably, the culture time is 8-25 d.

4. The method for cryopreservation of edible fungi as claimed in claim 1, wherein the method for cryopreservation comprises: inoculating the edible fungi to be preserved to a culture medium for primary culture, then placing perlite in the culture medium after the primary culture, carrying out secondary culture, and after hyphae of the edible fungi spread and grow to the internal pores of the perlite, carrying out freezing preservation on the perlite growing the edible fungi to be preserved.

5. The cryopreservation method of edible fungi as claimed in claim 4, wherein the time for the primary culture is 3-20d, and the time for the secondary culture is 7-20 d;

preferably, the temperature of the primary culture is 15-30 ℃, and the temperature of the secondary culture is 15-30 ℃.

6. A method for the cryopreservation of edible fungi according to any one of claims 2 to 5, wherein the culture medium is a plate culture medium or a slant culture medium;

preferably, the culture medium is a PDA culture medium, a potato comprehensive culture medium, a wood chip leaching culture medium, a bean sprout juice culture medium or a cottonseed hull culture medium.

7. The method for cryopreservation of edible fungi according to any one of claims 1 to 6, wherein the perlite is expanded perlite, surface-treated expanded perlite or perlite ore;

preferably, the perlite is a post-sterile perlite; the aseptic processing step comprises the steps of washing perlite to be processed with water, drying and sterilizing;

preferably, sterilization is performed at 119-122 ℃; preferably, the sterilization time is 30-60 min;

preferably, the perlite has a particle size of 3-8 mm.

8. The method for cryopreservation of edible fungi as claimed in claim 1, wherein the step of cryopreservation of perlite grown on edible fungi to be preserved comprises: placing perlite growing the edible fungi to be preserved in a freezing storage tube, adding a freezing protective agent into the freezing storage tube, then placing the freezing storage tube in a program cooling instrument for cooling, and preserving the freezing storage tube at a low temperature after cooling;

preferably, the temperature reduction program of the program temperature reduction instrument is set as follows: reducing the temperature to 4 ℃ at a speed of 4-5 ℃/min, and then reducing the temperature to-90 ℃ at a speed of 0.8-2 ℃/min;

preferably, after cooling, placing the freezing tube in a liquid nitrogen tank for preservation;

preferably, the lyoprotectant is at least one of glycerol, dimethyl sulfoxide, tween 80, glucose, trehalose, inositol and xylitol; preferably, the volume concentration of the lyoprotectant added to the cryopreservation tube is 5-15%.

9. The method for the cryopreservation of edible fungi according to claim 8, wherein the method for cryopreservation further comprises the recovery of the strain, and the recovery of the strain comprises the following steps: taking the freezing tube out of the liquid nitrogen tank, carrying out constant-temperature water bath, transferring the perlite on which the edible fungi grow to a culture medium after the perlite is completely melted, and carrying out recovery culture on the strains;

preferably, the thermostatic water bath is a water bath at 30-38 ℃; the constant-temperature water bath time is 1-10 min;

preferably, the recovery culture of the strain is performed at 15-30 ℃.

10. The method for cryopreservation of edible fungi according to claim 1, wherein the edible fungi is selected from any one of the following fungi: straw mushroom, stropharia rugoso-annulata, agaricus bisporus, pleurotus nebrodensis, coprinus comatus, shiitake mushroom, dictyophora phalloidea, malassezia, oyster mushroom, flammulina velutipes, pleurotus eryngii, grifola frondosa, hericium erinaceus, agrocybe cylindracea, russula vinosa, tricholoma matsutake and pleurotus geesteranus.