Preservation Technology of Edible Fungi

Strain is the main biological resources, but also the primary means of production of edible fungi. After an excellent strain is selected, it must keep its excellent characters unchanged or change slowly as much as possible, so as not to reduce production performance and be used in production for a long time. Therefore, culture preservation is of great significance in the production of edible fungi.

I. Principle of strain preservation

There are many methods of culture preservation, but the principles are similar. First of all, we must select good purebred. Secondly, according to its physiological and biochemical properties, artificially create conditions such as low temperature, dryness or hypoxia to inhibit the metabolism of microorganisms, so that their life activities are reduced to a very low level or in a dormant state, thereby prolonging the life of the strain and keeping the original characteristics of the strain to prevent variation. No matter which preservation method is adopted, it is required not to die, pollute miscellaneous bacteria and degrade in the process of strain preservation.

II. Methods of culture preservation

1. Low temperature periodic transplantation preservation method. Inoculate the strain to be preserved on an appropriate slant culture medium, culture at an appropriate temperature, take it out when the mycelium grows strongly on the slant, store it in a low temperature drying place of 3-5 DEG C or a refrigerator or freezer at 4 DEG C, transplant and transfer the tube once every 4-6 months, and the details should be determined according to the characteristics of the strain. When preserving, pay attention to the environmental temperature not to be too high, in case mold enters the tube through the tampon. Therefore, if a tampon is used, the tampon can be wrapped with clean sulfuric acid paper or kraft paper to reduce the chance of contamination and prevent drying of the medium. In addition to straw mushroom strains, other edible fungi strains can be preserved by this method.

2. Liquid paraffin preservation method. Take chemically pure liquid paraffin (no moisture and mildew required), put it in a conical flask, add cotton plug and wrap it with paper, sterilize it under the pressure of 1 kg/cm2 for 1 hour, and then put it into a 40℃ incubator for several days to evaporate the water until the paraffin oil is completely transparent. Transfer the treated paraffin oil onto the blank slant, culture at 28-30℃ for 2-3 days, and prove that there is no growth of impurities before use. Liquid paraffin is then aseptically injected into the slant tubes to be preserved. The injection volume should be 1-1.5 cm higher than the culture medium slope, rubber stopper, sealed with solid paraffin, and stored upright in a low temperature dry place. The preservation time is more than one year, and the preservation time can be prolonged at low temperature.

3. Sand tube preservation method. soak river sand in wat for several times, sieving with 60 mesh sieve to remove coarse particles, soak in 10% hydrochloric acid for 2-4 h to remove organic substances, washing with water until pH of running water reaches neutral, and drying for later use. soak poor ridge soil or vegetable garden soil in water to make it neutral, deposite, discarding supernatant, drying, grinding, sieving with 100-mesh sieve, mix that sand and soil at (2-4):1, sucking iron with magnet, packaging into small test tubes or ampoules, filling with cotton plug, wrapping with paper for sterilization (1.5 kg/cm2, 1 hour), sterilizing with dry heat (160 DEG C, 2-3 hours) for 1-2 times, performing sterility test, and using after passing the test. Inject 3-5 ml sterile water into the slant strain which has formed spores under sterile condition, scrape the lawn, prepare bacterial suspension, and then suck the bacterial suspension with sterile pipette and drop it into the sand tube to soak the sand. Put the inoculated sand tube into a vacuum dryer filled with desiccant, connect it to a vacuum pump for several hours until the sand is dry. Vacuum drying should be completed within 48 hours after spore inoculation to avoid spore germination. The prepared sand tube is sealed with paraffin and can be preserved for 2-10 years at low temperature.

4. Filter paper preservation method. Take white (collect dark spores) or black (collect white spores) filter paper, cut it into small strips of 4×0.8 cm, spread it in a petri dish and wrap it with paper for sterilization (1 kg/cm2, 30 minutes). Spores were collected by hook suspension and dropped onto filter paper strips. Put the filter paper strip carrying spores into a preservation test tube, and then put the preservation test tube into a dryer for 1-2 days, remove the moisture from the filter paper, so that the moisture content of the filter paper reaches about 2%, and then store it at low temperature.

5. Natural substrate preservation method

(1) Wheat preservation method: take@#@245@#@without shriveled grains and impurities, wash it, soak it for 12-15 hours, add water to boil for 15 minutes, continue to soak it for 15 minutes, so that the wheat grains will swell without breaking, drain the water and spread it out for drying, so that the moisture content of the wheat grains is about 25%. Calcium carbonate and gypsum are mixed into cooked wheat grains (the ratio of wheat grains, calcium carbonate and gypsum is 10kg: 133g: 33g), mixed evenly and put into test tubes, each tube is about 2- 3g, then the test tubes are cleaned, cotton plugs are stuffed, sterilized for 1.5 (1.5kg/cm2, 2 hours), and the test tubes are reserved after passing the sterility test. The test tube substrate is cooled and inoculated. The proper temperature culture is carried out. After the hyphae are fully grown, the cotton plugs are coated with paraffin, and the hyphae are stored at low temperature. Transfer once every 2 years or so.

(2) bran koji preservation method. Take fresh bran, sift through 60 mesh sieve to remove coarse grains. Mix bran and tap water according to the ratio of 1:1, put them into small test tubes, fill each tube with about 1/3 height, wrap them with cotton plug paper, sterilize them under high pressure (1.5 kg/cm2, 30 minutes), and prepare them after passing sterile inspection. Transfer the healthy strains growing on the inclined medium to sterile bran koji tubes. When transferring seeds, pay attention to beating the bran in the small test tubes evenly, and cultivate them at appropriate temperature until the hyphae are full of bran. Put the bran koji tubes into a dryer and preserve them at low temperature or appropriate temperature.

6. Normal saline preservation method. take 0.7- 0.9g of pure sodium chloride, putting into 100% ml of distil water, stirring evenly, subpackaging into test tube, sterilizing (1kg/cm2, 30min), passing sterility inspection, and then preparing for later use, inoculating that strain to be preserved into potato glucose liquid culture medium, shaking and culturing at appropriate temperature for 5-7 days. Aseptically suck a few culture strains and inject them into the normal saline test tube which has passed the inspection, plug it with sterile rubber stopper, coat it with paraffin, and preserve it at room temperature or low temperature.

7. Freeze-vacuum drying method. suspending cultured and grown thalli or spores in sterilized serum, egg white and skim milk to prepare bacterial suspension, aseptically packaging the suspension in sterilized glass ampoules, each tube being about 0.3-0.5 ml, connecting the ampoules with a freeze drying device by a pressure-resistant rubber tube, rapidly freezing the ampoules in a freezing tank at-30 DEG C to-40 DEG C, evacuating and drying under freezing, melting and sealing the ampoules under vacuum, and storing at-20 DEG C, Generally, it can be stored for more than ten years, but the cost is higher.

8. Liquid nitrogen cryopreservation. First, prepare the bacteria suspension to be preserved for later use; secondly, prepare ampoules, add 0.8 ml cryoprotectant 10%(volume ratio) glycerol distilled water solution to each bottle, and plug them for sterilization (1 kg/cm2, 5 minutes). After sterility test, insert the strain to be preserved, melt the bottle opening with flame, check whether there is air leakage, put the ampoule bottle with sealed mouth in the freezer, slowly cool down at the rate of 1℃ per minute, so that the preserved product is gradually frozen evenly until-35℃, and then the freezing speed does not need to be controlled. After freezing, immediately put the ampoule into a liquid nitrogen tank and store it at-150 to-196℃. This method is only used by a few scientific research institutes.