What is the isolation culture and strain preservation of medicinal fungi?

(Ran Yanzhu)

I. Isolation and cultivation of pure strains

In order to obtain pure strains of a certain medicinal fungus, it is necessary to isolate it from other organisms or microorganisms around it. The isolation work should be based on the characteristics of medicinal fungi, from the isolation method, the type of medium, the control of culture conditions and whether to add some kind of inhibitor in the medium and so on comprehensive consideration, otherwise the effective purpose of isolation of strains can not be achieved. There are three methods of isolation of pure strains: tissue isolation, spore isolation and host isolation.

(A) tissue separation method

The use of young tissue mycelium, in sterile conditions, inoculated on the medium, and revert to no tissue differentiation of the mycelium, this method of isolation of the strain obtained by the vitality of the mycelium grows fast, get the pure bacterial life rate is high. This method is adapted to a wider range, especially for some spores are not easy to germinate or germination rate is not consistent species as well as mycelium, mycelium and other tissues, the use of this method is appropriate.

Tissue separation, prior to the separation of the material to be selected, the material must be pure strain, good quality, young and fat without pests and diseases, and then surface disinfection. First wash the outside with water to clean the dirt or debris attached, put into the sterile operating room (cabinet) in the sterile vessels, with surface disinfectant (such as 0.1-0.2% mercuric acid, 10% bleach, 70-75% alcohol, 5% Lysol or 2% formaldehyde, etc.) solution soaked for a certain period of time. The disinfection time depends on the characteristics of the disinfectant and the texture and size of the mushroom tissue. Generally, such as mushrooms (shiitake, honey ring fungus) fresh and tender tissues, disinfection time should be short, controlled in 0.5-1.0 minutes, so that neither harm the tissue and can achieve the purpose of sterilization of the tissue surface; like the epidermal tissues of the thicker Poria coccinea, soaked in 5% Lysol solution for more than half an hour, without affecting the vitality. Mercuric solution is stronger bactericidal force, the organization by the surface disinfection, must then be rinsed by sterile water, to remove the attached residual drugs, so as not to affect the bacterial germination and growth. After rinsing with sterile water, the water should be removed with sterilized filter paper, and the tissue should be cut into small pieces of about 0.5cm2 with a sterile knife, and placed in the pre-inactivated and medium-filled petri dishes or test tubes, generally about 5 pieces per dish, and the rows should be kept at certain intervals, and it is appropriate to put only 1 piece in the test tube. Cultivation of the bottom of the dish up, 24-28 ℃ at a constant temperature for a few days, the tissue around the block can grow new mycelium, and then can be purified to get pure strains.

Surface disinfection, such as the method of mastering the improper or aseptic operation is not strict, often cause contamination, disinfection is excessive, the loss of the bacterium itself and the separation of pure strains of bacteria, the separation of porous fungi or mycorrhizal flesh is more fertile umbrella fungi, but also without the use of medicinal soaking, as long as the outer skin with 70-75% alcohol cotton wipe, remove the skin, pick the internal tissue blocks Direct isolation. In order to prevent bacterial infection, the small pieces of tissue to be isolated can be dipped in 0.1-0.5% gentamycin (or streptomycin) and then transferred. To prevent bacterial contamination on the medium, 0.125mg of chloramphenicol can be added to each milliliter of medium, which is more stable than the first two kinds of high-temperature resistant, and can be autoclaved together with the medium.

Separation of medicinal fungi, usually used in the medium composition is as follows:

Potato (peeled) 200g (chopped and boiled for half an hour to filter the dregs)

Sucrose 20g

Potassium dihydrogen phosphate 3g

Magnesium sulphate 1.5g

Vitamin B1 10mg

Agar 18g (add water to 1000ml)

Agar 18g (add water to 1000ml). to 1000 ml)

In addition to the above media, Sartre's medium or potato, glucose, agar medium is also commonly used.

(2) Spore separation method

This is a method to utilize the spores embedded in the hyphae or pores of medicinal fungi, so that they can be ejected on the medium suitable for growth under aseptic conditions and germinate and grow into mycelium to obtain pure strains. This method is used in the isolation of medicinal fungi such as shiitake mushroom, silver fungus, Poria and so on.

The sexual spores of medicinal fungi are formed by the nuclei of opposite sexes, after nuclear mating, with biparental heredity, high variability, strong vitality, suitable for breeding work, in order to prevent infection by stray fungi, the isolation of materials should be strictly sterilized, for the fruiting layer of the fruiting body is not exposed, can be immersed in a 0.1-0.2% mercuric solution for 2-3 minutes for surface disinfection. For the fruiting bodies that are not exposed, they can be immersed in 0.1-0.2% mercuric solution for 2-3 minutes for surface sterilization. If the fruiting layer is exposed, such as shiitake mushroom, in order to avoid spore killing, 70-75% alcohol should be used to disinfect the surface of the cap and stalk, and the fruiting entities should be cut off the stalk, inserted in the bracket made of metal wire, and put into the Petri dish, and the outside of the dish should be covered with a glass jar or a bell-shaped cover, and a large amount of spores can be collected in the Petri dish after a few hours. In addition can also take the hanging method. Such as silver fungus, the ear flap washed with water, put into a sterile triangular bottle or a large test tube, with sterile water oscillation rinse three times, take out and put in a sterile glass dish, with filter paper to absorb excess water, cut a piece of ear flap in the sterilized fine wire hooks, and then hung in a thin layer of medium poured into the triangular bottle, the ear flap from the base of 2-3cm. 24-26 degrees Celsius, after 1-26 hours, the ear flap can be collected. -26 ℃, after 1-2 days of culture, can be seen in the base of the vaguely white spores. Then continue to cultivate 2-3 days can form a milky white paste-like protruding colonies. That is, psyllid spore strains. Because the strain so obtained in the wood chips mycelium can not germinate, so can not be directly as a bottle planting of the strain.

Poria cocos fungus stramonium spore isolation is used in the emission of spore clouds of its substrate. With the aerial capture method, to test tube mouth against the spore cloud, so that the spores fall into the test tube medium. Or, with the attachment method, the rising spore cloud is attached to the top of the medium and incubated at 25℃.

If the purpose is hybridization breeding, the single spore separation method is to be used. The use of single spore separator for single spore separation, is currently an advanced means, but the equipment is expensive, only a few units in the country to use. The more widely used is the bacterial liquid continuous dilution method. The method is to use sterile water to disperse the spores, minimize the distribution density of spores in sterile water, and finally make each drop of water contain only 1-2 spores. Then use a sterile syringe to inhale the spore suspension, drop on the slant base surface, turn the test tube, the bacterial liquid will be uniformly distributed on the slant, cultured at the appropriate temperature, selecting the best colonies for purification. The single spore isolate of heterozygous combined aspergillus, although it can reproduce mycelium, but will not form a substrate, so it can not be used for cultivation and production.

(C) host isolation method

This method is also known as the base mycelium isolation method. This is the use of growth of a certain medicinal fungi mycelium substrate to do the separation of materials and obtain pure strains of a method. It is generally applied only when the above two methods fail to achieve the purpose. Selected as the seed of the substrate, if the spores have been released, the substrate began to dry or rot, mycelium inactivation, difficult to use spore isolation and tissue isolation method to obtain pure strains, or some varieties of the substrate of gelatinous (silver fungus, fungus), leathery (Yunzhi), corky (Ganoderma lucidum), woody (needle cracked hoof) and other taxa can be used in this method.

Criteria for selecting mycorrhizal fungi: excellent traits of growing substrates, vigorous mycelial development in the base, mycorrhizal fungi are not decayed without contamination by stray bacteria. In order to prevent bacterial contamination, in addition to the use of acidified medium or add antimicrobials, should make the fungus wood air-dry and lose water. Such as ear wood, mushroom wood and ling wood. Take silver fungus as an example, after selecting the ear wood, in the parts of the growth of the fruiting body, cut off a piece of fungus wood 1-1.5cm thick, remove the bark, put the fungus wood piece into 0.1% ascending mercury solution for a few minutes of surface disinfection, rinse off the liquid, suck out the surface moisture, put it into the aseptic room (cabinet) of the Petri dish, cut off the outer layer of the wood piece, and cut the middle of the part of the growth of mycelium with aseptic knife into 0.5cm2 size small pieces, into the medium surface for the appropriate temperature for culture and purification of transplantation.

Because of the different species of medicinal fungi, the appearance and structure of the colonies formed are also different. Therefore, the identification of the isolated species, in addition to experience, more accurate is after a period of cultivation, pending the formation of some special structures, such as mycelial bundles, mycelium, mycelium, especially the fruiting bodies and spores before judging.

Second, the preservation and rejuvenation of strains

Medicinal fungi strains are important resources and natural wealth of the country. It is also the basis of medicinal fungi production and scientific research. Medicinal fungi of different "species", its medicinal value and culture conditions are different, is the same "species" of different strains, there are also differences. In production practice, we should choose strains that meet the production requirements, should have good efficacy, short production cycle, high yield, strong life force and other characteristics. For the active ingredient is clear, should choose the strain with high content, for the active ingredient is not determined, should choose the strain with significant pharmacological effect, no toxic reaction and good clinical efficacy, high yield. Production can also be based on certain requirements of the purpose, the application of the natural variation of the bacterium to select, and constantly consolidate its characteristics into new strains or artificial directional cultivation to improve the characteristics of the strain. Such as through hybridization or mutation breeding selection of good strains, so that the production or effective substance content to reach a new level.

A strain of pure, good quality strains, often due to poor management and too many generations as well as improper storage methods and other reasons, and caused by the degradation of good traits, this phenomenon is called strain degradation. In order to prevent the degradation of strain traits or restore the original traits of strains that have been degraded, should take the scientific storage methods and effective rejuvenation measures.

(A) the preservation of strains

Medicinal fungal strains in the dry low temperature, freezing or reduce the oxygen content and other conditions of preservation, so that its metabolic intensity is reduced, to maintain the dormant state of the strain, you can prevent the strain degradation.

1. oblique low temperature preservation

In addition to grass mushrooms and other individual high temperature type strains, suitable for preservation at about 15 ℃ conditions, the vast majority of species are suitable for low temperature preservation. That is, the preserved strains are put in the refrigerator at about 4-5 ℃, and transferred to the tube for one generation after 3-6 months. In order to restore the ability of the strain to live, need to be removed from the refrigerator 1-2 days before use, placed at room temperature for activation of the transfer tube, can not be used directly.

2. Mineral oil sealing method

The above oblique strains, infused with a layer of sterilized liquid paraffin oil, sterilized in a triangular flask due to the entry of steam, which will affect the quality of the preserved strains, and therefore need to be placed in the 40 ℃ temperature box so that the water vapor evaporation before use. Cold paraffin oil is injected with a sterile pipette, and the amount injected should be enough to cover the whole slant and 1cm above the culture, and the cotton plug at the mouth of the test tube should be wrapped tightly with cellophane or plastic cloth, and then placed in the refrigerator or stored at room temperature. This preservation method can prevent the culture medium from losing water and the entry of external air, which can reduce the metabolism and maintain the vitality of the strain, thus playing a role in prolonging the service life of the strain. Pick the mycelium from the paraffin tube and transfer it to the new slant for activation culture, which can be used for production later. This method can save the strain 1-2 years without losing vitality.

3. Freeze-drying method

This method is the use of vacuum, drying and low-temperature three-combined preservation of strains of fungi, preservation period can be up to 10-20 years. Specific method: the preservation of fungi spores made of suspension, loaded into the ampoule tube, and then suddenly cooled down and frozen, and pumped out the air into a vacuum, so that the culture in a solid state sublimation and dehydration, melting seal the bottle mouth, preserved at low temperature or room temperature.

4. Liquid nitrogen ultra-low temperature preservation method

Ultra-low temperature can make the metabolic function reduced to the lowest level and the strain does not mutate. Experiments have proved that the use of this new technology can preserve all strains of microorganisms. Liquid nitrogen ultra-low temperature refrigerator temperature up to -196-130 ℃. Ultra-low temperature preservation of strains with high efficiency, wide range of use, easy to operate and other advantages, but the instrument is expensive, the application is not yet common.

In addition to the above methods of preservation of strains of bacteria, but also found that Ganoderma lucidum bacteria (Chi Chi, thin cover Ganoderma lucidum) in the wheat bran and millet on the slant, in the low temperature (4 ℃) preservation for more than a year still have vitality. If the cotton plug of the preservation tube is replaced by a rubber plug, it can be preserved for more than 4 years.

(B) strain rejuvenation

Medicinal fungi of various species, biological characteristics of different, any one selected from the breeding of fine varieties, are often due to multiple generations of culture, long-term artificial culture or long-term storage without transfer of the tube as well as preservation of the conditions of discomfort, etc., the strains of bacteria will be a natural mutation, degradation phenomenon.

The main manifestations of strain degradation include slow growth of mycelium, deterioration of substrate traits, decreased yield, and reduction of active ingredients. Too many generations, due to the mycelium in the long-term active nutrient growth, life reproduction is inhibited, so the number of generations should be controlled between 2-3 times. Has produced degradation of the strain, it is necessary to continue to retain and apply, then the strain must be re-strengthened. Strain rejuvenation, in addition to improving the culture conditions, the cultivated species have been domesticated, the most reliable method is to carry out sexual reproduction (spore isolation).

Mycelium strain of silver fungus, after many generations, there will be weak vitality, low ear rate degradation phenomenon. Can use the method of adding spores to re-strengthen the strain. That is, take a spore test tube injected with 10 ml of sterile water, the spore suspension into the sawdust strain, so that the strain ear rate will be improved. Spores can not germinate mycelium in the wood chip base, such as adding 1-2% calcium superphosphate and 1-2% bone meal can germinate mycelium. Reinforcement can also be achieved by reinoculating the original host.