CN111733108A

CN111733108A - Cooling method for liquid nitrogen preservation of needle mushroom strains

Info

Publication number: CN111733108A
Application number: CN202010656849.XA
Authority: CN
Inventors: 冯占; 邱丽雯; 李贺文; 余养朝
Original assignee: Jiangsu Chinagreen Biological Technology Co ltd
Current assignee: Jiangsu Chinagreen Biological Technology Co ltd
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-10-02

Abstract

The invention relates to a cooling method for liquid nitrogen preservation of flammulina velutipes strains, belonging to the technical field of ultralow temperature preservation of strains. The problem that the freezing speed is too fast or too slow and is not beneficial to cell survival in the prior art is solved, and the following technical scheme is provided: the method comprises the following steps: (1) placing the flat seeds cultured in a biochemical incubator at 20 ℃ in an ultra-clean workbench, and taking flat seed inoculation blocks into 2ml of protective solution of a freezing storage tube; (2) putting the frozen tube strain plate seeds obtained in the step (1) into a preservation box at 4 ℃ for 24H; (3) and (3) cooling the frozen tube strain plate seeds treated in the step (2) in a programmed cooling instrument at a speed of 2 ℃/min to-30 ℃, at the same time, at a speed of 3 ℃/min to-80 ℃, and then placing the frozen tube strain plate seeds in a liquid nitrogen tank for long-term storage. The activity and the characteristics of the liquid nitrogen preserved strains are ensured by setting a proper cooling rate, the result is superior to the same continuous subculture strains, the fruiting body quality is excellent, and effective help is provided for strain species collection and strain resource library establishment.

Description

Cooling method for liquid nitrogen preservation of needle mushroom strains

Technical Field

The invention relates to the technical field of ultralow-temperature preservation of strains, in particular to a cooling method for liquid nitrogen preservation of flammulina velutipes strains.

Background

The microorganism has the characteristic of easy variation, and the characters such as culture characters, agronomic characters, commodity characters and the like are reduced in the process of subculture and propagation production culture. The strain preservation method mainly adopts a method of controlling factors such as temperature, water activity, oxygen, nutrition and the like to reduce or inhibit the growth metabolism of the edible fungi, thereby achieving the aim of strain preservation. The programmed cooling liquid nitrogen freezing tube stores strains, liquid water is converted into ice in the freezing process, cell metabolism is stopped, and meanwhile, the freezing speed is too fast or too slow, so that water in cells is easily lost or large ice crystals are easily formed, and the cell survival is not facilitated.

Disclosure of Invention

The invention provides a cooling method for liquid nitrogen preservation of flammulina velutipes strains, which solves the problem that the freezing rate is too fast or too slow and is not beneficial to cell survival in the prior art, ensures the vitality and the characteristics of the liquid nitrogen preservation strains by setting a proper cooling rate, has a result superior to the same continuous subculture strains, has excellent fruit body quality, and provides effective help for strain type collection and strain resource library establishment.

In order to achieve the purpose, the invention provides the following technical scheme:

a cooling method for liquid nitrogen preservation of flammulina velutipes strains comprises the following steps:

(1) placing the flat seeds cultured in a biochemical incubator at 20 ℃ in an ultra-clean workbench, and taking flat seed inoculation blocks into 2ml of protective solution of a freezing storage tube;

(2) putting the strain plate seeds to be frozen into the tube strain plate seeds obtained in the step (1) into a preservation box at 4 ℃ for 24H storage;

(3) and (3) cooling the frozen tube strain plate seeds treated in the step (2) in a programmed cooling instrument at a speed of 2 ℃/min to-30 ℃, at the same time, at a speed of 3 ℃/min to-80 ℃, and then placing the frozen tube strain plate seeds in a liquid nitrogen tank for long-term storage.

The invention is different from the prior art in that:

the invention adopts precooling treatment for 24H under the condition of 4 ℃, then gradually reduces the temperature to minus 30 ℃ according to the cooling rate of 2 ℃/min, and finally gradually reduces the temperature to minus 80 ℃ according to the cooling rate of 3 ℃/min, thereby avoiding cell damage to the maximum extent. Taking out the strain for six months, activating, observing the hypha concentration from the growth vigor of the plate seeds for 9 minutes, uniformly and uniformly taking out the strain for 8 minutes, taking out the strain for twelve months, and taking out the strain for 6 months until the strain state is consistent, wherein the hypha form stability is 10 minutes.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto. The details not described in the examples are according to the prior art in the field.

Examples the strain growth description score was 10 points.

The first embodiment is as follows:

(2) placing the strain plate seeds of the frozen tube obtained in the step (1) into an incubator at 20 ℃ for standing for 2H;

(3) and (3) cooling the cryopreserved tubular strain flat seeds treated in the step (2) in a programmed cooling instrument, wherein the temperature is reduced to 4 ℃ at the speed of 1 ℃/min, then the temperature is reduced to-30 ℃ at the speed of 2 ℃/min, and then the flat seeds are placed in a liquid nitrogen tank for long-term storage, and the effects and effects are that after the flat seeds are taken out for 6 months and activated, the hypha concentration is observed from the growth vigor of the flat seeds for 6 minutes, and the growth speed is 8 minutes.

Example two:

(3) and (3) cooling the cryopreserved tubular strain flat seeds treated in the step (2) in a programmed cooling instrument, wherein the temperature is reduced to-30 ℃ at the speed of 2 ℃/min, then the temperature is reduced to-80 ℃ at the speed of 2 ℃/min, and then the flat seeds are placed in a liquid nitrogen tank for long-term storage, and the effects and effects are that after six months of taking out and activation, the hypha concentration is observed from the growth vigor of the flat seeds for 8 minutes, and the uniformity is 6 minutes.

Example three:

(2) and (2) cooling the cryopreserved tubular strain flat seeds treated in the step (1) in a programmed cooling instrument at a speed of 1 ℃/min to-80 ℃, and then placing the frozen tube strain flat seeds in a liquid nitrogen tank for long-term storage, wherein the action and the effect are that after six months of taking out and activation, the hypha concentration is observed from the growth vigor of the flat seeds for 6 minutes, and the uniformity is generally 6 minutes.

Example four:

(2) putting the frozen tube strain plate seeds obtained in the step (1) into a preservation box at 4 ℃ for 24H;

(3) and (3) cooling the frozen tube strain plate seeds treated in the step (2) in a programmed cooling instrument at a speed of 2 ℃/min to-30 ℃, at the same time, at a speed of 3 ℃/min to-80 ℃, and then placing the frozen tube strain plate seeds in a liquid nitrogen tank for long-term storage. The function and the effect are as follows: taking out for six months, activating, observing the hypha concentration from the growth vigor of the plate seeds for 9 minutes, uniformly and uniformly taking out for 8 minutes, taking out for twelve months again, wherein the strain state is consistent with that of taking out for six months, and the hypha form stability is 10 minutes.

Through experimental investigation, the invention of the fourth embodiment has the following advantages:

1. and in the strain stage, the strains are subjected to liquid nitrogen preservation through programmed cooling, the experimental preservation period is six to twelve months, the storage and taking-out operations are well-understood and easy to understand, the efficiency is high, and after the strains are taken out and activated, the growth vigor of the strain stage is superior to that of the strains in the same branch cultured in a subculture mode.

2. The number of buds in the bud stage is moderate in the growing room stage. The inhibition period is long and normal, and the proportion of the cap and the stem is coordinated and has no obvious difference with the same storehouse strain. After the pieces are inserted, the vitality of the mushroom buds is good, the sizes of the mushroom caps are uniform, and the growth vigor of the mushroom caps is better and is not different from that of the mushroom caps in the same storehouse. The mushroom caps in the harvesting period are uniform in size, good in compactness and viscosity and uniform in unit yield.

And (3) comparing the unit yield: the strain inoculation amount of the 1100CC culture bottle adopted in one factory is 32-35g, and the strain inoculation amount of the 1500CC culture bottle adopted in the fourth factory is 36-40g, as shown in the following table;

and (3) quality comparison:

and (3) liquid nitrogen preservation of strains: six baskets of samples are taken, the mushroom shape is better, the aerial hypha at the root is moderate, and the uniformity of the mushroom cap size is better. The root compactness, whiteness and viscosity are better, and the pores are slight. The uniformity is slightly poor, the layering is slightly large, the thicknesses of the mushroom stems are similar, and the number of small buds inside the mushroom stems is slightly large.

Subculture strains: six baskets of samples, the mushroom cap size homogeneity is better. The root has common compactness, a few of the roots crack slightly, the uniformity is poor, and the internal buds are few.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A cooling method for liquid nitrogen preservation of flammulina velutipes strains is characterized by comprising the following steps: