Technical regulation for production of edible bacteria

Edible strains refer to artificially cultured pure mycelium of edible fungi, which are usually divided into mother species, original species and cultivated species. Mother species are pure mycelia identified as valuable for production through strict screening. The mother seed is cultured with test tube slant medium, which is also called test tube species or first-class species. The mother seed is propagated and transferred to sawdust or grain culture medium to obtain the strain, which is called the original seed or secondary species. The culture container is mostly a can bottle or a strain bottle. The strain propagated from the original seed is called cultivated species or tertiary species, which can be directly used for production and cultivation.

1. Preparation of mother seed

1. Prepare mother seed slant culture medium. The most commonly used is PDA culture medium, the preparation method is: take a few disease-free potatoes, wash and peel, cut into thin slices, weigh 200 grams, add 1000 ml of water to boil, boil until the potato crisp and not into degrees, the resulting liquid is filtered with 6 layers of gauze, the filtrate is made up to 1000 ml with water, then add 20 grams of agar and glucose each, continue to heat to melt, sub-package into test tubes while hot, about 8 ml per tube. The test tubes are 180×10 mm in size and cleaned with a front brush. After subpackaging, plug with cotton plug; bundle every 10 pieces into a bundle, put them vertically in a small iron wire cage, sterilize them with high pressure or earth steamer, and then put them into an inclined plane when they are hot. When the culture medium solidifies, they can be used for inoculation.

2. Organize the separation of legal parent species. The fruiting bodies with strong growth, regular appearance, large flesh thickness, no diseases and insect pests and characteristics of the variety are selected and collected at the maturity of seven or eight minutes as separation materials. Cut off the mushroom stem, wipe the mushroom surface with 75% alcohol cotton ball, and move to the inoculation box. The inoculation box was fumigated with formaldehyde in advance. Wipe the fruiting body material with alcohol cotton again, tear it longitudinally from the cap, take the material from the junction of the cap and the cup with a sterile blade or small tweezers, cut the tissue of wheat grain size, transfer it to the middle of the inclined culture medium of the test tube with an inoculation needle, and transfer one piece from each tube. During inoculation, the mouth of the test tube should always be above the flame of alcohol lamp, and the inoculation needle should also be burned red on the flame and cooled at the mouth of the test tube before taking the inoculation material. Dozens of tubes can be inoculated each time, and then sent to the incubator or incubator for cultivation. The temperature is maintained at 23~26℃, and the relative humidity of the air is maintained at about 70%. After 3 days of culture, white villous hyphae grew around the tissue block, and the test tube could be filled in about one week. If green, red, yellow, black and milky white colonies appear on the slant medium, they are contaminated by miscellaneous bacteria and should be eliminated in time. The strains overgrown with slant are strictly selected, and the parent species can be obtained by selecting the strains with fast growth and thick white hyphae and then transferring the tubes once.

3. Transfer culture of mother seed. The transfer of tubes is carried out in an inoculation box, in which a number of slant media are put in advance, and the inoculation box is routinely disinfected. Operators wash their hands, wear white coats and hats, cover their hair, and wipe their hands and test tube walls with 75% alcohol cotton wool. Take the mother seed test tube and culture medium test tube in your left hand, take the inoculation needle in your right hand, open the cotton plug of the test tube above the alcohol lamp flame, burn the inoculation needle and the test tube mouth on the flame at the same time, and the test tube mouth is always near the flame. Cool the heated inoculation needle on the wall of the tube mouth, then cut a small piece of medium about the size of wheat grain full of hyphae with the needle tip, quickly take it out from the tube mouth, connect it to the middle of the medium slope, then burn the tube mouth on the flame, quickly plug the cotton plug, and then connect the next tube. After all inoculation, send to incubator or incubator.

II. Preparation method of original seed

1. Preparation of stock medium. Formula: hardwood sawdust 78%, wheat bran 20%, sucrose and gypsum powder 1% each, pH 6.5, water 60%. Also useful corn or wheat seed culture medium system, but the cost is high. According to the production quantity, weigh the materials according to the proportion of the formula, fully mix them to make the water content suitable, that is, hold the materials with hands, but do not drip, and then bottle them until the shoulder of the bottle, punch a round hole in the center with a thick stick of fingers to increase ventilation, tie the bottle mouth with polypropylene film, and sterilize them in time. High pressure or earth steamer can be used.

2. Inoculation and culture. Routine disinfection of inoculation room and inoculation box. Take the sterilized seed culture medium can bottle or seed bottle into the inoculation box, each time you can take 200 bottles, and then fumigate with formaldehyde once. The operator strictly implements aseptic operation. Wipe the outer wall of the mother seed test tube with alcohol cotton, take the inoculation box, open the cotton plug on the flame, take the inoculation shovel or big tweezers with your right hand, and burn it red on the flame to cool, then pick out a piece of hyphae (with culture medium attached) of bean size from the mother seed tube, open the film of the original seed culture medium bottle, quickly put the hyphae of the mother seed into the center of the original seed culture medium, make the hyphae surface adhere to the medium surface in the bottle, immediately cover the film, and then connect the next bottle in turn. A mother seed can be connected to 15 - 20 bottles of original seeds. The inoculated strain bottles shall be labeled in time, indicating the variety, grade (several grades), inoculation date, inoculator, etc. of edible fungi. Send them to the incubator in time. The temperature is maintained at 23~26℃, and the relative humidity of the air is maintained at about 70%. Indoor shading or weak scattered light can be. After culturing for about 30 days, the hyphae can be used for propagation and cultivation. If not used temporarily, it should be stored in dry, low temperature and dark conditions for a short time.

III. Method for preparing cultivated seeds

Cultivated seeds are strains directly used for production and cultivation, and the preparation method of the original seeds is basically the same. One bottle of original seed can be expanded and cultivated in about 100 bottles. The culture medium is the same as the original seed. After sterilization, inoculate in an inoculation box. Under the control of alcohol lamp flame, take out a fingernail-sized mycelium from the original seed bottle, put it into the center of the culture medium, cover it with film, then take another bottle, and send it to the culture room for shelf culture after all the inoculation. After culturing for 30 days in dark, the mycelium can be used for cultivation after the bottle is full.