CN113207553A - Flammulina velutipes strain medium preservation method for improving strain stability and medium used by method - Google Patents

Abstract

A needle mushroom strain medium preserving method for improving strain stability and a medium used by the method, the medium formula is as follows: 59-69% of wood chips, 20-25% of rice bran, 10-15% of corncobs, 1% of calcium carbonate, 63-65% of water content and 6.0-6.5 of pH value. The matrix is used for storing strains, so that the flammulina velutipes strains can be stored in a refrigerator at 4 ℃ for 6-12 months. No fruiting phenomenon exists in the preservation process. Production and cultivation tests are carried out on the substrate strains preserved for 6 months and 12 months, and the results show that the characteristics such as hypha growth speed, germination time, yield quality and the like are stable.

Description

Flammulina velutipes strain medium preservation method for improving strain stability and medium used by method

Technical Field

The invention belongs to the technical field of edible mushroom cultivation, and relates to a needle mushroom strain medium preservation method for improving strain stability and a medium used by the method.

Background

Flammulina velutipes, alias Lentinus edodes, hackberry fungi, and Desmodium villosum belonging to the phylum Eumycota, Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, and Desmodium. The flammulina velutipes is a common edible fungus, has smooth and tender pileus, crisp and refreshing stipe, rich nutrition and delicious taste, and is popular with the public.

Flammulina velutipes is composed of two major parts, namely, vegetative organ (mycelium) and reproductive organ (fruiting body). The mycelium is formed by spore germination, and under the condition of artificial culture, the mycelium is usually in a white villiform shape and has transverse septa and branches, and a plurality of hyphae are gathered together to form the mycelium. Different from other edible fungi, the hyphae can form a large amount of unicellular powder spores (also called conidia) when growing to a certain stage, and can germinate into mononuclear fungi, flammulina velutipes or binuclear hyphae under proper conditions. Tests prove that the quantity of the powdery spores in the hypha stage of the flammulina velutipes is related to the quality of the flammulina velutipes, and the strains with more powdery spores have poor quality. The needle mushroom belongs to low-temperature fruiting fungi, mycelia can grow within the range of 5-32 ℃, but the optimal temperature is 22-25 ℃, mycelia are resistant to low temperature and weak in resistance to high temperature, and even stop growing at 34 ℃ or above, and the mycelia die. Therefore, the reasonable preservation of the flammulina velutipes strains is very important.

In the industrialized production of needle mushrooms, the strains are usually cultured by adopting a PDA culture medium and then stored in a refrigerator at 4 ℃. However, the strains preserved by the method generally need to be transferred in 2-3 months, and phenomena of low-temperature fruiting and seed nature degradation are easy to occur.

Disclosure of Invention

The invention aims to provide a needle mushroom strain matrix preservation method for improving strain stability and a matrix used by the method.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a substrate comprises the following raw materials in percentage by mass:

the water content of the substrate is 63-65%, and the pH value is 6.0-6.5.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a needle mushroom strain medium preservation method for improving strain stability comprises the following steps:

step 1: mixing the raw materials according to the raw material formula of claim 1, putting the mixture into a container, flattening the material surface, punching a through hole from one end of the material surface to the other end of the material surface, inverting the container in water, enabling the material surface not to contact with the water surface to clean and remove redundant residual materials generated in punching, wiping a bottle opening with a towel, covering a silica gel plug, wrapping the bottle plug with tinfoil paper, sterilizing at 121 ℃ for 60-70 minutes, taking out of the pot, and cooling to room temperature on a sterile ultra-clean workbench;

step 2: cutting the flammulina velutipes strain preserved on the PDA culture medium into strain blocks, inoculating the strain blocks on the surface of the material and in the through holes, and covering with a silica gel plug; culturing in 18-20 deg.C biochemical incubator in dark, sealing the opening of the triangular flask with sealing film after the culture medium is filled with mycelia, and storing in 4 deg.C refrigerator for 6-12 months.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

the matrix culture medium strains adopted by the invention can realize that the flammulina velutipes strains are stored in a refrigerator at 4 ℃ for 6-12 months. No fruiting phenomenon exists in the preservation process. Production and cultivation tests are carried out on the substrate strains preserved for 6 months and 12 months, and the results show that the characteristics such as hypha growth speed, germination time, yield quality and the like are stable. The method can keep the strain stability of the flammulina velutipes, and avoid strain degeneration caused by frequent switching of PDA culture medium under low temperature and fruiting under low temperature.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Example 1: flammulina velutipes strain medium preservation method for improving strain stability and medium used by method

A needle mushroom strain medium preservation method for improving strain stability is characterized by comprising the following technical steps.

The formula of the matrix is as follows: 64% of wood chips, 22% of rice bran, 13% of corncobs, 1% of calcium carbonate, 64% of water content and 6.2 of pH value.

Quality standard:

note: all raw materials are required to be fresh, clean, dry, free of insects, mould and peculiar smell.

(2) The specific operation method comprises the following steps:

the preparation method of the substrate strain culture medium comprises the following steps: loading 150 g/bottle into a 250 ml triangular flask, flattening the charge level, punching a hole to the bottom (the brightness can be completely seen through the hole) in the center of the charge level by using an iron rod with the diameter of 8mm, inverting the triangular flask in clear water (the charge level is not in contact with the water surface), cleaning to remove redundant residual materials, wiping the bottle mouth with a towel, finally covering a silica gel plug, wrapping the bottle plug with tinfoil paper, placing at 121 ℃ for autoclaving for 60-70 minutes, taking out of the pot, and cooling to room temperature (below 20 ℃) on a sterile ultra-clean workbench.

(3) The substrate strain subculture method comprises the following steps:

according to the aseptic operation specification, dividing the PDA strains into strain blocks with the size of 5mm x 5mm, inoculating 3 strains on the material surface of a triangular flask, putting 1 strain in a material hole of the triangular flask, and covering a silica gel plug. Culturing in a biochemical incubator at 19 deg.C in the dark. Sealing the opening of the triangular flask with a sealing film after the culture medium is filled with mycelia, and storing in a refrigerator at 4 deg.C for 6-12 months.

Production test of substrate strain (strain stability verification test)

Taking out the substrate culture medium strain preserved in a refrigerator at 4 ℃, carrying out industrial production cultivation by adopting liquid strain, sequentially propagating to culture dish strain, shake flask strain and fermentation tank strain, and testing 2000 bottles according to the industrial production process specification of flammulina velutipes, wherein the conventional PDA culture medium strain and the production strain preserved in the refrigerator at 4 ℃ are used as a reference.

And (3) test results:

the two substrate strains are subcultured faster than the control in the aspect of hypha growth speed, and the growth speed difference between the treatments is small. From the single production point of view, the subcultures were all higher than the control species. Therefore, the substrate strain subculture technology has obvious advantages in production and is worth popularizing and applying in mass production.

Example 2: flammulina velutipes strain medium preservation method for improving strain stability and medium used by method

A substrate comprises the following raw materials in percentage by mass:

the water content of the matrix was 65% and the pH 6.5.

A needle mushroom strain medium preservation method for improving strain stability comprises the following steps:

step 1: mixing the raw materials according to the raw material formula of claim 1, loading into a container, flattening the material surface, punching a through hole from one end of the material surface to the other end of the material surface, inverting the container in water, keeping the material surface from contacting the water surface to clean and remove the excessive residual materials generated in punching, wiping the bottle mouth with a towel, covering a silica gel plug, wrapping the bottle plug with tinfoil paper, sterilizing at 121 ℃ for 60 minutes, taking out of the pot, and cooling to room temperature on a sterile ultra-clean workbench;

step 2: cutting the flammulina velutipes strain preserved on the PDA culture medium into strain blocks, inoculating the strain blocks on the surface of the material and in the through holes, and covering with a silica gel plug; culturing in 18 deg.C biochemical incubator in dark, sealing the opening of the triangular flask with sealing film after the culture medium is filled with mycelia, and storing in 4 deg.C refrigerator for 12 months. No fruiting phenomenon exists in the preservation process.

Example 3: flammulina velutipes strain medium preservation method for improving strain stability and medium used by method

A substrate comprises the following raw materials in percentage by mass:

the water content of the matrix was 63% and the pH was 6.0.

A needle mushroom strain medium preservation method for improving strain stability comprises the following steps:

step 1: mixing the raw materials according to the raw material formula of claim 1, loading into a container, flattening the material surface, punching a through hole from one end of the material surface to the other end of the material surface, inverting the container in water, keeping the material surface from contacting the water surface to clean and remove the excessive residual materials generated in punching, wiping the bottle mouth with a towel, covering a silica gel plug, wrapping the bottle plug with tinfoil paper, sterilizing at 121 ℃ for 70 minutes, taking out of the pot, and cooling to room temperature on a sterile ultra-clean workbench;

step 2: cutting the flammulina velutipes strain preserved on the PDA culture medium into strain blocks, inoculating the strain blocks on the surface of the material and in the through holes, and covering with a silica gel plug; culturing in a biochemical incubator at 20 deg.C in the dark, sealing the opening of the triangular flask with sealing film after the culture medium is filled with mycelia, and storing in a refrigerator at 4 deg.C for 6 months.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

Claims (2)

1. A substrate, characterized by: the raw material formula of the matrix comprises the following raw materials in percentage by mass:

59-69% of wood chips;

20-25% of rice bran;

10-15% of corncobs;

1% of calcium carbonate;

the water content of the substrate is 63-65%, and the pH value is 6.0-6.5.

2. A needle mushroom strain medium preservation method for improving strain stability is characterized by comprising the following steps: comprises the following steps:

step 1: mixing the raw materials according to the raw material formula of claim 1, putting the mixture into a container, flattening the material surface, punching a through hole from one end of the material surface to the other end of the material surface, inverting the container in water, enabling the material surface not to contact with the water surface to clean and remove redundant residual materials generated in punching, wiping a bottle opening with a towel, covering a silica gel plug, wrapping the bottle plug with tinfoil paper, sterilizing at 121 ℃ for 60-70 minutes, taking out of the pot, and cooling to room temperature on a sterile ultra-clean workbench;

step 2: cutting the flammulina velutipes strain preserved on the PDA culture medium into strain blocks, inoculating the strain blocks on the surface of the material and in the through holes, and covering with a silica gel plug; culturing in 18-20 deg.C biochemical incubator in dark, sealing the opening of the triangular flask with sealing film after the culture medium is filled with mycelia, and storing in 4 deg.C refrigerator for 6-12 months.