Edible mushroom strain preservation technology
The bacteria species are the main biological resources and the primary production materials for the production of edible fungi. After a good strain has been selected, it must maintain its good traits or change as slowly as possible, so as not to reduce the production performance, and it can be used in production for a long time. Therefore, the preservation of bacteria in the production of edible fungi is of great significance. First, there are many methods for the preservation of bacteria strains, but the principle is much the same. The first thing to do is pick a good pure breed. Use of spores, spores, and vegetative bodies of microorganisms; secondly, based on their physiological and biochemical properties, artificially create conditions such as low temperature, dryness, or lack of oxygen, inhibit the metabolism of microorganisms, and reduce their life activities to extremely low levels or be dormant. , so as to extend the life of the bacteria and to maintain the original traits of the bacteria and prevent variation. No matter which method of preservation is used, no death, no contamination of bacteria, and no degradation are required during preservation of the strain. Second, the preservation of bacteria, a low temperature, regular transplantation preservation method. The strains that need to be preserved are inoculated on a suitable slant culture medium and incubated at a suitable temperature. When the mycelium is robustly covered with slopes, they are taken out and stored at a low temperature of 3-5°C or in a refrigerator or freezer at 4°C. Transplantation should be carried out once every 4-6 months, depending on the nature of the strain. During the preservation, it should be noted that the ambient temperature should not be too high to prevent the mold from entering the tube through the tampon. Therefore, if a tampon is used, the tampon can be wrapped with clean sulfated paper or kraft paper to reduce the chance of contamination and to prevent the medium from drying out. In addition to straw mushroom species, other edible mushroom species can be preserved by this method. 2, liquid paraffin preservation method. Take chemically pure liquid paraffin (requires no moisture, no mildew), put it in a triangular bottle, add a tampon and wrap it, sterilize for 1 hour at 1 kg/cm2, and put it in a 40°C incubator for several days. To evaporate the water until the paraffin oil is completely transparent. The treated paraffin oil was transferred to a blank slope and cultured at 28-30° C. for 2-3 days, which proved that no bacteria could grow. The liquid paraffin is then aseptically injected into the beveled test tube to be preserved. The injection volume was 1 to 1.5 cm above the slope of the culture medium, and the rubber plug was plugged in and sealed with solid paraffin and stored upright at a low temperature and dry place. The preservation time is more than one year. At low temperatures, the preservation time can be extended. 3, sand and soil preservation. The river sand was soaked and washed several times with water, and the coarse particles were removed through a 60-mesh sieve and then soaked in 10% hydrochloric acid for 2-4 hours. The organic substances were removed, and the water was rinsed until the pH of the running water reached neutrality and was dried for use. At the same time, the poor soil or vegetable garden soil is soaked in water to make it neutral, and after the sedimentation, the supernatant is discarded, dried and ground, sieved with a 100-mesh sieve, and the treated sand and soil are treated as (2-4): 1 mix, with the magnet out of the iron, and then sub-installed in a small test tube or ampoule, the amount of each tube 0.5-2 grams, stuffed tampon, with a paper wrap sterilization (1.5 kg/cm2, 1 hour), Then dry heat sterilization (160 °C, 2-3 hours) 1-2 times for sterility testing, use after passing. The spore-forming beveled strains were inoculated into 3-5 ml of sterile water under aseptic conditions to scrape the bacteria to form a bacterial suspension, and then the bacteria was sucked into a sand tube with a sterile pipette to soak the sand. So far. The inoculated sand tube is placed in a vacuum desiccator containing desiccant and pumped by a vacuum pump for several hours until the sand is dry. The vacuum drying operation needs to be completed within 48 hours after the spores are inserted to avoid spore germination. The prepared sand tubes are sealed with paraffin and can be preserved for 2-10 years at low temperatures. 4, filter paper preservation method. Take a white (collect dark spores) or black (collect white spores) filter paper, cut into 40.8 cm small pieces of paper, lay flat in a petri dish and wrap paper for sterilization (1 kg/cm2, 30 minutes). Spores were collected by hooking and allowing the spores to fall on the filter paper. The spore-laden filter paper strips are placed in a preservation tube, and the preservation tube is placed in a desiccator for 1-2 days. The filter paper is removed to make the filter paper moisture content reach about 2%, and then stored at a low temperature. 5. Preservation of Natural Substrates (1) Preservation of Wheat Grains: Take wheat without pods and impurities for panning, soak for 12-15 hours, add boiling water for 15 minutes, and continue hot dipping for 15 minutes to make the wheat bulge without breaking. Dry moisture is spread out to dry, so that the moisture content of wheat is about 25%. Mix calcium carbonate and gypsum into cooked wheat grains (grain, calcium carbonate, and gypsum: 10 kg: 133 g: 33 g), mix well and put into test tubes, about 2-3 g each, and then clean the test tube. , tampon, sterilization 1.5 (1.5 kg / square centimeter, 2 hours), after passing the sterile examination, spare, the test tube substrate cooling after inoculation, suitable temperature culture, after the mycelium covered with matrix coated paraffin tampon , low temperature preservation. Transfer once in about 2 years. (2) The bran song preservation method. Fresh bran was taken and sieved through 60 mesh to remove coarses. Mix the bran and tap water by 1:1 and put them into small test tubes. Each tube is about 1/3 of the height. Add tampon to the paper and autoclave (1.5 kg/cm2, 30 minutes). After the test is qualified, reserve the robust strains grown on the slant medium and transfer them to the sterile bran tube. Note that even though the bran in the small test tube is loose, it is loose and is cultivated at a suitable temperature. When the mycelium is full of bran, the bran microtubules are placed in a desiccator and stored at low or moderate temperatures. 6, saline preservation method. Take 0.7-0.9 grams of pure sodium chloride, put into 100% milliliter of distilled water, stir and evenly pack test tubes, each tube 5-10 milliliters, sterilized (1 kg/cm2, 30 minutes), pass the sterility test After the reserve, the bacteria to be preserved are put into a potato dextrose liquid medium and shaken at a suitable temperature for 5-7 days. Aseptically, a small amount of culture bacteria is inoculated and injected into a test tube of a qualified physiological saline, stuffed with a sterile rubber stopper, sealed with paraffin wax, and preserved at room temperature or at a low temperature. 7, frozen vacuum drying method. The cultured, well-grown bacteria or spores were suspended in sterile serum, egg white, and skim milk to prepare a bacterial suspension. The suspension was aseptically dispensed in sterile glass ampoule bottles, each about 0.3. - 0.5 ml, and then connected to a freeze-drying apparatus using a pressure-resistant rubber tube. The ampoule bottle was rapidly frozen in a freezer at -30°C to -40°C, and was evacuated and dried in a frozen state and melted in a vacuum. , Stored at -20 °C, generally can be stored for more than a decade, but the cost is higher. 8, liquid nitrogen ultra-low temperature preservation method. Firstly, the strains to be preserved shall be made into a suspension of bacteria; secondly, ampoules shall be prepared, each bottle shall be filled with 0.8 ml of cryoprotectant, 10% (by volume) glycerin distilled water solution, and sterilized with a tampon (1 kg/cm2, 5). minute). After sterility inspection, access to the species to be preserved, flame sealing the bottle mouth, check for leaks, put the sealed ampoules bottle in the freezer, slow down by 1 °C per minute, to ensure The collection gradually and evenly freezes until -35°C. After that, the freezing speed does not need to be controlled. The ampoule is immediately frozen in a liquid nitrogen tank and stored at -150 to -196°C. This method is only used by a few scientific research institutes.
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