CN112088718A

CN112088718A - Preparation method of milin ganoderma lucidum strain

Info

Publication number: CN112088718A
Application number: CN201910520687.4A
Authority: CN
Inventors: 次仁罗布
Original assignee: Red Sun Technology Demonstration Family Farm In Milin Town Milin County
Current assignee: Red Sun Technology Demonstration Family Farm In Milin Town Milin County
Priority date: 2019-06-17
Filing date: 2019-06-17
Publication date: 2020-12-18

Abstract

The invention discloses a preparation method of a milin ganoderma lucidum strain, which comprises the following steps of preparing a culture medium, wherein the culture medium is prepared from the following components in parts by weight: 200 parts of peeled potato, 20 parts of glucose, 2 parts of monopotassium phosphate, 0.5 part of magnesium sulfate, 20 parts of agar and 1000 parts of water; preparing a mother strain: culturing the strain on a PDA culture medium until hyphae overgrow, and selecting the hyphae with high quality as a mother strain; preparing an original seed: preparing materials, stirring materials, subpackaging, sterilizing, cooling, inoculating, culturing, managing and inspecting quality; manufacturing cultivars: preparing materials, mixing the materials, bagging, sterilizing, cooling, inoculating, culturing, managing and inspecting quality. The potassium dihydrogen phosphate and the magnesium sulfate are added on the basis of the existing culture medium to promote metabolism of strains, synthesis of nucleic acid and conversion of phosphate, and on the basis of adjusting the pH value, the nutrition of the culture medium is improved, and the quality of the strains is improved.

Description

Preparation method of milin ganoderma lucidum strain

Technical Field

The invention relates to the technical field of fungus cultivation, in particular to a preparation method of a milin ganoderma lucidum strain.

Background

Ganoderma Lucidum, also known as Lingzhongling and Qiongzhen, is the fruiting body of Ganoderma Lucidum of Polyporaceae. Has effects in invigorating qi, tranquilizing mind, relieving cough and asthma, and prolonging life, and can be used for treating vertigo, insomnia, palpitation, short breath, neurasthenia, and cough and asthma due to asthenia. The strain is the basis of ganoderma lucidum production, the breeding of excellent varieties is an important link for ensuring the high quality and high yield of ganoderma lucidum, and the effect of the obtained ganoderma lucidum is higher than that of the ganoderma lucidum cultivated in a greenhouse by extracting wild ganoderma lucidum strains.

The preparation of the existing strains generally adopts the following method: preparing culture medium, preparing mother strain, preparing stock, and preparing cultivar. For example, application No. 201810984010.1 discloses a method for cultivating high-triterpene Ganoderma lucidum, which adopts PDA culture medium comprising potato 200 g, glucose 20 g, agar 15-20 g, and distilled water 1000 ml. The strain produced by the culture medium has low quality.

Disclosure of Invention

The invention provides a preparation method of a milin ganoderma lucidum strain for solving the technical problems.

The invention is realized by the following technical scheme:

a preparation method of the milin ganoderma lucidum strain comprises the following steps,

A. preparing a culture medium, wherein the culture medium is prepared from the following components in parts by weight: 200 parts of peeled potato, 20 parts of glucose, 2 parts of monopotassium phosphate, 0.5 part of magnesium sulfate, 20 parts of agar and 1000 parts of water;

B. preparing a mother strain: culturing the strain on a PDA culture medium until hyphae overgrow, and selecting the hyphae with high quality as a mother strain;

C. preparing an original seed: preparing materials, stirring materials, subpackaging, sterilizing, cooling, inoculating, culturing, managing and inspecting quality;

D. manufacturing cultivars: preparing materials, mixing the materials, bagging, sterilizing, cooling, inoculating, culturing, managing and inspecting quality.

The scheme adds the monopotassium phosphate and the magnesium sulfate on the basis of the existing culture medium to promote metabolism of strains, synthesis of nucleic acid and conversion of phosphate, and improves the nutrition of the culture medium and the quality of the strains on the basis of adjusting the PH value.

Preferably, the method for preparing the culture medium comprises:

slicing potato, placing into a pot, adding 1000ml water, and boiling until the potato is soft and not rotten;

filtering the liquid with 4-6 layers of gauze to remove filtrate for later use;

adding agar into the filtrate, continuously heating until the agar is melted, adding glucose, potassium dihydrogen phosphate and magnesium sulfate, and supplementing water to above ratio;

the liquid is subpackaged in test tubes, the length of the test tubes is 1/5-1/4, and the tube openings are wiped and sealed;

sterilizing at 121 deg.C and 0.11 MPa for 30 min;

the tube was tilted to allow the front end of the medium to spread to 1/2 th length, covered with a cover, and left to stand.

Preferably, the strain is a flesh tissue of a yellow-white ganoderma pileus growth area or a flesh tissue of the middle part of a pileus above a ganoderma stipe, and the strain is 5 mm by 5 mm.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention adds potassium dihydrogen phosphate and magnesium sulfate on the basis of the existing culture medium to promote metabolism of strains, synthesis of nucleic acid and conversion of phosphate, and improves the nutrition of the culture medium and the quality of the strains on the basis of adjusting the PH value.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.

Example 1

preparing a culture medium, wherein the culture medium is prepared from the following components in parts by weight: 200 parts of peeled potato, 20 parts of glucose, 2 parts of monopotassium phosphate, 0.5 part of magnesium sulfate, 20 parts of agar and 1000 parts of water;

preparing a mother strain: culturing the strain on a PDA culture medium until hyphae overgrow, and selecting the hyphae with high quality as a mother strain;

preparing an original seed: preparing materials, stirring materials, subpackaging, sterilizing, cooling, inoculating, culturing, managing and inspecting quality;

manufacturing cultivars: preparing materials, mixing the materials, bagging, sterilizing, cooling, inoculating, culturing, managing and inspecting quality.

Example 2

Based on the principle of the above embodiments, the present embodiment discloses a detailed implementation.

Preparing a culture medium, wherein the culture medium is prepared from the following components in parts by weight: 200 parts of peeled potato, 20 parts of glucose, 2 parts of monopotassium phosphate, 0.5 part of magnesium sulfate, 20 parts of agar and 1000 parts of water.

Specifically, the preparation method of the culture medium comprises the following steps:

slicing potato, placing into a pot, adding 1000ml water, and boiling until the potato is soft and not rotten; filtering the liquid with 4-6 layers of gauze to remove filtrate for later use; adding agar into the filtrate, continuously heating until the agar is melted, adding glucose, potassium dihydrogen phosphate and magnesium sulfate, and supplementing water to above ratio; the liquid is separately loaded into test tubes, the test tubes are loaded to 1/5-1/4 of the length of the test tubes, wiping is carried out, the tube openings are sealed by cotton plugs or silicon rubber plugs, the size of the cotton plugs is required to be standard, the tightness is moderate, and gaps cannot appear. Sterilizing at 121 deg.C and 0.11 MPa for 30 min; obliquely placing the test tube to make the front end of the culture medium spread to the length 1/2 of the test tube, covering the test tube with a covering material made of gauze or sterilized newspaper to prevent excessive condensation water, and standing to solidify the culture medium. 3-5 test tubes are placed at 30 ℃ for 3 days of culture, and if no foreign bacteria appear, the sterilization is complete and the test tubes can be used for inoculation.

Selecting strains, and selecting 6-7 min mature fresh Ganoderma without diseases and insect pests as seed Ganoderma. The surface of Ganoderma can be sterilized by ultraviolet irradiation and alcohol wiping. Tearing the pileus, dividing the pileus into two parts, and selecting flesh tissues in a yellow-white growing area of the ganoderma pileus or flesh tissues in the middle of the pileus above a ganoderma stipe as a seed taking part so as to improve the quality of strains; when the tissue is separated, firstly, the ganoderma lucidum flesh can be carefully cut by using a sterile scalpel, then, a small piece of flesh tissue with the diameter of 5 mm x5 mm is taken by using an inoculating needle and is inoculated to the middle upper surface of a test tube culture medium. Adjusting temperature to 28-30 ℃, and culturing in dark. After 2-3 days, the mushroom flesh tissue can start to germinate, and mycelium which is visible to naked eyes appears and gradually spreads on the surface of the culture medium. The test tube can grow over the inclined plane of the test tube in 7 to 10 days generally. If the flesh tissue is less than 2 days, mycelium which is visible to naked eyes appears, and various bacteria exist. If the mycelial flesh tissue still appears visible to the naked eye after being cultured for 4 days, it may be that a scalpel or an inoculating needle for cutting the ganoderma lucidum mycelial flesh tissue is not cooled sufficiently after being burned above an alcohol flame, or the mycelial flesh tissue loses activity and cannot germinate mycelium due to other reasons. The quality requirement selects the separated material with regular and normal growth speed, full appearance, vigorous growth vigor and pure white, fine, flat, compact and uniform aerial hyphae as the mother seed.

Attention points in the production process of mother seeds

(1) The proper strain production plan is specified, the stock seeds can be stored in a refrigerator for a period of time after the test tubes are full, but the stock seeds and the cultivated seeds are required to be used for production as soon as possible after the bottles (bags) are full.

(2) Both the strains obtained by tissue separation and the purchased strains need to be cultivated and tested, and the strains meeting the production requirements can be used for large-scale production. The wrong strain selection will cause immeasurable loss on production.

(3) The tissue isolation and tube transfer of the strain must be performed strictly following the requirements of aseptic manipulation.

(4) And repeated pipe rotation is avoided as much as possible. Gene mutation may occur during the tube transfer process, so that the strain is degenerated, and the life of the strain is reduced. Generally, after 3-4 times of tube transfer, the ganoderma lucidum mother seeds need to be separated and rejuvenated again, and cultivation tests are performed again, so that the ganoderma lucidum mother seeds are rejected and have good quality.

The Ganoderma stock is a strain obtained by inoculating Ganoderma mother strain onto natural matrix and performing amplification culture, and is a mixture of Ganoderma mycelia and natural matrix. The stock seed is used for propagation of cultivated species, and the Ganoderma cultivated species is a strain obtained by transferring Ganoderma stock seed to natural culture medium for amplification culture, and is also a mixture of Ganoderma mycelia and natural matrix. As the name implies, the ganoderma lucidum cultivar is the strain used for ganoderma lucidum cultivation.

Preparing a stock culture medium, wherein the stock culture medium comprises the following components in parts by weight: 76 parts of pine sawdust, 22 parts of wheat bran, 1 part of sugar, 1 part of gypsum, 55 parts of water, 10 parts of wheat bran, 2 parts of calcium superphosphate and 0.1 part of urea. Pine containing sterilizing substances such as rosin, essential oil, alcohol, ether, etc. is generally not used for cultivating edible fungi. The edible fungi are generally cultivated by using branches and wood chips of broad-leaved tree species such as tussah, willow, elm, maple, poplar, locust, mulberry, birch, maple, mulberry, toona sinensis, oak, peach, pear, apple, chinaberry, sycamore and the like. According to the scheme, pine trees in coniferous trees are used as the raw materials of the culture medium for the first time, and the quality of the strains can be guaranteed. During the preparation of the stock culture medium, fresh and dried pine sawdust subjected to solarization for 2-3 days is placed in a container, is soaked in boiling water, is added with 1-2% of lime powder and is stirred uniformly, and is covered with an old jute bag for heat preservation. Soaking for 1-2 days, controlling water, washing with clear water, spreading on plastic film, and drying to further volatilize harmful substances in sawdust. Spraying water to water content of 60-70% and pH 7-7.5, and wrapping the plastic film to ferment. Keeping for 2-3 days when the temperature of the materials reaches 65-69 ℃, then dispersing and stacking the materials, and cooling the materials for use. Or stacking and fermenting for 3-4 days after the temperature of the stack is raised to 60-70 deg.C and the stack is turned over after 3-4 days, turning the four sides to the middle. Mixing, subpackaging, sterilizing, cooling, inoculating, culturing, managing, and inspecting quality to obtain stock.

Preparing cultivated species, mixing, bagging, sterilizing, cooling, inoculating, culturing, managing and quality inspecting.

During the production of stock and cultivated species, the following aspects need to be noted: and (5) mixing and subpackaging the materials according to the production plan of the day and weighing the materials according to the formula. Uniformly stirring the dry materials (except sucrose), gradually adding water (sucrose dissolved in water), continuously stirring until the water content of the culture medium reaches the requirement, and subpackaging. In order to ensure the quality of the strain, the original strain (secondary strain) is produced by using a colorless or light-colored transparent 750 ml strain bottle as a strain container. Production of Ganoderma lucidum cultivars (three-stage species) PE bags (suitable for atmospheric sterilization) or PP bags (autoclavable) 15 cm by 30 cm by 0.055 mm (length by width by thickness) were used as seed containers.

During bottling, the material is filled into the bottle while the bottle neck is pinched by hand and continuously vibrated until the material is filled into the bottle neck. The surface is flattened by a flat iron hook and is pressed to the bottle shoulder. If the plastic bag is packed, the application force is uniform, the plastic bag can be lightly taken and placed, and the plastic bag is prevented from being damaged. The culture material should be slightly tight on the upper surface and slightly loose on the lower surface, and the tightness is proper. The air in the over-tight bottle is less, which affects the growth of hyphae, and the over-loose bottle has less hyphae and is easy to dry and shrink although the fungus grows fast. Washing off residual wood chips, wheat bran and other culture medium inside and outside the bottle mouth, drying, and then plugging with cotton plug or wrapping the bottle mouth with polypropylene film and kraft paper. The material bag can be sealed by a plastic ring and a breathable plastic cover or a sealing machine (similar to ham sausage sealing technology). The bottles (bags) filled with the material are called material bottles (bags).

And (3) sterilizing, namely filling the material bottles (bags) into a sterilizing pot, wherein two sterilization methods are adopted, namely high-pressure steam sterilization and normal-pressure steam sterilization. A small amount of stock seeds and cultivated seeds can be sterilized by a portable sterilization pot, but about 10 bottles can be sterilized each time, and if a large amount of stock seeds and cultivated seeds are produced, a large sterilization pot is used for sterilization. The normal pressure steam sterilization has high pollution rate, and the strain is preferably produced by using an autoclave (the specific sterilization method can be referred to ganoderma lucidum health preserving net).

The material bottle (bag) after cooling and sterilization can be cooled in a special cooling chamber or a receiving chamber. The cooling site must be disinfected 24 hours in advance. When the temperature of the culture material is reduced to below 30 degrees (obviously lower than the palm temperature), inoculation can be carried out. Before inoculation, sterile culture test is carried out to detect the sterilization effect.

Inoculation the stock or stock is transferred to a sterilized medium as required for aseptic processing, referred to as inoculation. The stock and the cultivated species are inoculated in a similar way, but the difference is that the stock is inoculated into the mother species, and the cultivated species is inoculated into the stock. Since opening the test tube or seed vial may cause contamination of the interior of the container by other microorganisms in the environment, all operations of inoculation should be performed in the inoculation chamber or clean bench. The inoculation box and the inoculation space are sterilized before use. Firstly, 4 g of smoke disinfectant (sodium dichloroisocyanuric acid) is used in each cubic meter of space, and the disinfection is carried out overnight. 4 g of smoke disinfectant is used for every cubic meter of space 4 hours before inoculation after preparation of each inoculation work, and then secondary disinfection is carried out. When the mother seed is inoculated with the original seed, under the aseptic condition, the inoculating needle is placed above the flame, fully burned and cooled, the test tube cotton plug is pulled out from the flame accessory of the alcohol lamp, a small piece of mother seed with the culture medium is hooked, the inclined plane of the mother seed can be transversely cut into 6-8 sections, the front end is discarded, and the rest sections and the culture medium are inoculated to the original seed culture medium.

When stock seeds are transplanted to the cultivated species, under the aseptic condition, a flat inoculation hook or an inoculation shovel is fully burnt and cooled above flame, a test tube cotton plug is pulled out from a flame accessory of an alcohol lamp, and surface stock seeds of the stock seeds which are close to a bottle opening by about 3 cm are discarded to reduce the mixed bacterial pollution of the cultivated species. Then transferring a proper amount of lower layer strains to the surface of the culture material of the cultivated species, wherein 50-60 bottles of cultivated species can be transferred from one bottle of stock seeds.

Although the material bottle (bag) is sterilized by steam, the wall of the bottle (bag) is contaminated by the bacteria in the moving process, and once the material bottle (bag) is opened in the inoculation process, the air flow and the respiratory air flow of the operator can infect the bacteria, so that the bacteria adhered to the wall of the bottle can contaminate the material bottle (bag). Therefore, strict aseptic operation, standard and convenient inoculation operation are required during inoculation, so that the pollution rate and the cost can be reduced. After the culture material is inoculated, plugging a cotton plug or covering a bottle cap, and moving to a constant temperature chamber or a culture chamber for culture.

Culturing the stock and the cultivated species, inoculating, culturing in a culture room, transferring the inoculated strain bottles (bags) to a constant temperature room or a culture room, cleaning the culture room before moving, keeping the environment clean, and transferring the strain bottles (bags) only after disinfection treatment in advance. The temperature of the culture room is maintained at about 25 ℃, and the relative humidity of air is 60-70%. Light-shielding and strong ventilation. In the early stage of strain culture, the strain is strictly checked every 2-3 days, and the mycelia are checked every 7-10 days after covering the surface of the culture medium. If red, green, yellow, black and other spores are found on the bottle wall, chat surface or inoculation block, which indicates that the mixed fungi such as neurospora erythraea, penicillium, aspergillus or rhizopus are polluted, the mixed fungi are eliminated, and if the detection time interval is too long, the bacterial colony of the mixed fungi can be covered by the mycelium of the vigorously growing edible fungi, so that the invisible pollution is caused. The plastic bag seed production is large in volume, so that the plastic bag seed production is not extruded when placed on a culture frame, and the plastic bag seed production is not required to be turned too frequently so as to avoid hypha damage or plastic bag breakage. The cultivation shelves in the cultivation room are mutually supported by battens so as to avoid the occurrence of dumping accidents.

Under the condition of proper environment, generally after 20-40 days of culture, hypha can be spread to the whole culture material, and then the hypha can be used for transferring cultivated species or used for production after 7-10 days of culture. The prepared strain can be cooled if it is temporarily not used

Dry, ventilated, light-proof and clean indoor short-term storage to ensure the quality of the strains. Even if the strain is stored at low temperature, the strain is not suitable to be stored for too long to prevent the strain from aging and activity reduction, so that the strain germinates slowly after inoculation and is easy to infect mixed bacteria.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A preparation method of the milin ganoderma lucidum strain is characterized by comprising the following steps,

2. The method of claim 1, wherein the method of preparing the culture medium comprises:

filtering the liquid with 4-6 layers of gauze to remove filtrate for later use;

sterilizing at 121 deg.C and 0.11 MPa for 30 min;

3. The method of claim 1, wherein said culture is a flesh tissue of a yellow-white ganoderma pileus growth region or a flesh tissue of a middle ganoderma pileus above a ganoderma stipe, and said culture is 5 mm by 5 mm.

4. The method for preparing the milin ganoderma lucidum strain according to claim 1, wherein the raw material comprises the following components in parts by weight: 76 parts of pine sawdust, 22 parts of wheat bran, 1 part of sugar, 1 part of gypsum, 55 parts of water, 10 parts of wheat bran, 2 parts of calcium superphosphate and 0.1 part of urea.

5. A method of producing a species of Ganoderma micrinum according to claim 4, wherein said species is selected from the group consisting of Ganoderma lucidum, Ganoderma sinense

Drying pine sawdust, soaking in boiling water, adding 1-2% lime powder, stirring, and keeping the temperature;

soaking for 1-2 days, controlling water, washing with clear water, spreading on plastic film, and air drying;

spraying water to make the water content reach 60-70% and the pH value reach 7-7.5, wrapping the plastic film around, and fermenting;

keeping for 2-3 days when the temperature of the materials reaches 65-69 ℃, dispersing and stacking the materials, and cooling.

6. A method of producing a species of Ganoderma micrinum according to claim 4, wherein said species is selected from the group consisting of Ganoderma lucidum, Ganoderma sinense

soaking for 1-2 days, controlling water, washing with clear water, spreading out, and air drying;

spraying water until the water content reaches 60-70% and the pH value is 7-7.5, stacking together, and fermenting;

when the temperature of the materials reaches 60-70 ℃, keeping for 3-4 days, turning the pile, turning the periphery to the middle, and fermenting again for 3-4 days.