[0036] Embodiment three
[0037] a. Cut into five-section awns 76% with a diameter of 1-2cm, 19% bran, 3% sugar, 1% gypsum powder, and 1% lime;
[0038] b. Stir the ingredients in step a evenly, and then add water to make a cultivation material with a moisture content of 75% and a pH of 7.8;
[0039] The above percentages are mass percentages.
[0040] The cultivation method of Pleurotus eryngii includes the following steps:
[0041] Example one
[0042] 1. Making cultivation material: Put the cultivation material with 65% moisture content and pH 7.5 into the cultivation bottle, then seal the cultivation bottle with paper, and then put the cultivation bottle at a pressure of 0.984kg/cm 2 , Sterilize at a temperature of 124°C for 2 hours or sterilize at a temperature of 124°C for 18 hours at atmospheric pressure, then cool it for use;
[0043] 2. Inoculation: Open the sealing paper and use the inoculation tool to plant the bacteria into the cultivation material of the cultivation bottle; then move the cultivation bottle after the inoculation into the germination room for cultivation in the dark, and control the humidity of the germination room at 60%; The temperature in the bacteria room should be controlled at 25°C; the temperature in the bacteria room should be controlled at 24°C in the second week; the temperature in the third week should be controlled at 22°C until the mycelium is full of the culture bottle;
[0044] 3. Invigorate buds: When the mycelium in the cultivation bottle is full, continue to cultivate for 9 days in the germination room at a temperature of 22°C, then control the temperature of the germination room to 10°C and the humidity to 90%. The germ room increases the stimulation of scattered light, strengthens ventilation, and keeps the air fresh. After 3 days, a white primordium appears in the cultivation bottle, which gradually forms mushroom buds;
[0045] 4. Fruiting management: When the primordium differentiates to form small mushroom buds of 1-2 cm, the fruiting management should be carried out in time, leaving 3-4 buds in each bottle mouth. During the fruiting stage, the temperature of the mushroom room should be maintained At 10℃, the initial relative humidity of the air in the mushroom room should be maintained at 89%. When the diameter of the mushroom cap grows to 2-3cm, spray water to cool and increase the humidity, but you cannot spray water on the mushroom body until the mushroom body is collected. Harvest, 2 days before harvest, the relative humidity of the air in the germ room should be controlled at 84%;
[0046] The above percentages are mass percentages.
[0047] The cultivation methods of the above-mentioned strains are:
[0048] I. Preparation of bacterial culture medium: select five-piece mango granules with a diameter of 1-1.2cm and soak them in 4.8% lime water for 40 hours. After the five-piece mango granules are softened and fermented, they are removed, and then rinsed with water. Adjust the PH value to 7.3-7.7, then dry the washed five-piece mango granules, select 72% five-piece mango granules, 25% bran, 2% glucose, and 1% gypsum powder, and then mix the ingredients evenly according to the above , And then add water to the evenly stirred ingredients until the moisture content of the culture medium is 58%. Put the culture medium with 58% moisture content into the culture flask, and then seal with sealing paper, and then put the culture flask under pressure 0.984-1.12kg/cm 2 , Sterilize at a temperature of 120°C for 2 hours or at a temperature of 100°C for 10 hours at normal pressure, then cool for use;
[0049] II. Inoculation: In the inoculation box or inoculation room, first open the sealing paper near the alcohol lamp, and the flame is 1-1.5cm away from the mouth of the bottle. Avoid directly burning the mouth of the flask. Take the mother plant in the test tube and grow it. Then, move the inoculated culture bottle into the hair growth room to protect from light. The humidity of the hair growth room is controlled at 60% and the temperature is controlled at 23°C. After 25 days of cultivation in the hair growth room, the hyphae grow Fill the surface of the culture medium and remove non-normal individuals in time,
[0050] The above percentages are mass percentages.
[0051] The above-mentioned method for the expansion and propagation of the mother species, the mother species use the PDA medium test tube slant culture method. The formula of the PDA medium is: 200g of potatoes cut into small pieces of 0.8-1.2cm in side length, 20g of glucose, and agar 20g, 1000ml water, constant volume, boil in a pot for 9-11 minutes until the potato chips are soft and not rotten, then filter, take the filtrate, add 20g agar to the filtrate, continue to boil until the agar is completely melted, add 20g glucose, Then add water to 1000ml of culture medium, and then divide the 1000ml of culture liquid into test tubes while it is hot. The volume of each aliquoted culture liquid is 1/5 to 1/4 of the length of the test tube, and then put a cotton plug on the mouth of the test tube Seal, and then put the test tube containing the culture liquid at a pressure of 1kg/cm 2 , Sterilize in a sterilization pot at 120℃ for 29-31min. After sterilization, take out the test tube in an oblique position. After the medium has solidified and cooled, move it into the inoculation box, take 3-5 test tubes and place them at 29℃ for cultivation Cultivate in the box for 3 days. If no bacteria appear, PDA medium is formed for inoculation.
[0052] Embodiment two
[0053] 1. Making cultivation material: Put the cultivation material with 75% moisture content and pH of 8 into the cultivation bottle, then seal the cultivation bottle with paper, and then put the cultivation bottle under the pressure of 1.12kg/cm 2 , Sterilize at 128℃ for 2.5 hours or at 128℃ for 18 hours at normal pressure, then cool for use;
[0054] 2. Inoculation: Open the sealing paper and use the inoculation tool to plant the bacteria into the cultivation material of the cultivation bottle; then move the cultivation bottle after the inoculation into the bacteria room for cultivation in the dark, and control the humidity of the bacteria room at 70%; The temperature in the bacteria room should be controlled at 28°C; the temperature in the bacteria room should be controlled at 26°C in the second week; the temperature in the third week should be controlled at 24°C until the mycelium is full of the culture bottle;
[0055] 3. Invigorate buds: When the hyphae in the cultivation bottle are full, continue to cultivate for 11 days in the germination room at a temperature of 24°C, and then control the temperature of the germination room to 18°C and the humidity to 95%. The germ room increases the stimulation of scattered light, strengthens ventilation, and keeps the air fresh. After 6 days, a white primordium appears in the cultivation bottle, which gradually forms mushroom buds;
[0056] 4. Fruiting management: When the primordium differentiates to form small mushroom buds of 1-2 cm, the fruiting management should be carried out in time, leaving 3-4 buds in each bottle mouth. During the fruiting stage, the temperature of the mushroom room should be maintained At 20℃, the initial relative humidity of the air in the mushroom room should be kept at 91%. When the diameter of the mushroom cap grows to 2-3cm, spray water to cool down and increase humidity, but do not spray water on the mushroom body until the mushroom body is collected. Three days before harvesting, the relative humidity of the air in the germ room should be controlled at 86%;
[0057] The above percentages are mass percentages.
[0058] The cultivation methods of the above-mentioned strains are:
[0059] I. Preparation of bacterial culture medium: select five-piece mango granules with a diameter of 1-1.2cm and soak them in 5.2% lime water for 48 hours. After the five-piece mango granules are softened and fermented, they are removed and rinsed with water. Adjust the PH value to 7.3-7.7, then dry the washed five-piece mango granules, then select 74% five-piece mango granules, 24% bran, 1% glucose, and 1% gypsum powder, and then mix the ingredients evenly according to the above , And then add water to the well-mixed ingredients until the moisture content of the culture medium is 62%. Put the culture medium with the moisture content of 62% into the culture bottle, then seal it with a sealing paper, and then put the culture bottle under the pressure 0.984-1.12kg/cm 2 , Sterilize at 122℃ for 2.5 hours or at 100℃ for 10.5 hours at normal pressure, then cool for use;
[0060] II. Inoculation: In the inoculation box or inoculation room, first open the sealing paper near the alcohol lamp, and the flame is 1-1.5cm away from the mouth of the bottle. Avoid directly burning the mouth of the flask. Take the mother plant in the test tube and grow it. Then, move the inoculated culture bottle into the hair growth room to avoid light and cultivate. The humidity of the hair growth room is controlled at 70% and the temperature is controlled at 25°C. After 35 days of cultivation in the hair room, the hyphae grow Fill the surface of the culture medium and remove non-normal individuals in time,
[0061] The above percentages are mass percentages.
[0062] The above-mentioned method for the expansion and propagation of the mother species, the mother species use the PDA medium test tube slant culture method. The formula of the PDA medium is: 200g of potatoes cut into small pieces of 0.8-1.2cm in side length, 20g of glucose, and agar 20g, 1000ml water, constant volume, boil in a pot for 9-11 minutes until the potato chips are soft and not rotten, then filter, take the filtrate, add 20g agar to the filtrate, continue to boil until the agar is completely melted, add 20g glucose, Then add water to 1000ml of culture medium, and then divide the 1000ml of culture liquid into test tubes while it is hot. The volume of each aliquoted culture liquid is 1/5 to 1/4 of the length of the test tube, and then put a cotton plug on the mouth of the test tube Seal, and then put the test tube containing the culture liquid at a pressure of 0.984-1.12kg/cm 2 , Sterilize in a sterilization pot with a temperature of 122°C for 31 minutes. After sterilization, take out the test tube in an oblique manner. After the medium has solidified and cooled, move it into the inoculation box, and take 3-5 test tubes and place them in the 31°C incubator After 3 days of cultivation, if no bacteria appear, a PDA medium is formed for inoculation.
[0063] Embodiment three
[0064] 1. Making cultivation material: Put the cultivation material with 75% moisture content and pH of 8 into the cultivation bottle, then seal the cultivation bottle with paper, and then put the cultivation bottle under the pressure of 1.12kg/cm 2 , Sterilize at 128°C for 2-2.5 hours or sterilize at 128°C at normal pressure for 18.5 hours, then cool for use;
[0065] 2. Inoculation: Open the sealing paper and use the inoculation tool to plant the bacteria into the cultivation material of the cultivation bottle; then move the cultivation bottle after the inoculation into the bacteria room for cultivation in the dark, and control the humidity of the bacteria room at 70%; The temperature in the bacteria room should be controlled at 28°C; the temperature in the bacteria room should be controlled at 26°C in the second week; the temperature in the third week should be controlled at 24°C until the mycelium is full of the culture bottle;
[0066] 3. Invigorate buds: When the mycelium in the cultivation bottle is full, continue to cultivate for 9-11 days in the germination room at a temperature of 22-24℃, and then control the temperature of the germination room to 10-18℃ and humidity control It is 90%-95%, and increase the scattered light stimulation to the germ room, strengthen ventilation, and keep the air fresh. After 3-6 days, white primordium appears in the cultivation bottle, and the primordium gradually forms mushroom buds;
[0067] 4. Fruiting management: When the primordium differentiates to form small mushroom buds of 1-2 cm, the fruiting management should be carried out in time, leaving 3-4 buds in each bottle mouth. During the fruiting stage, the temperature of the mushroom room should be maintained At 10-20℃, the initial relative humidity of the air in the mushroom room should be maintained at 89-91%. When the diameter of the mushroom cap grows to 2-3cm, spray water to cool and humidify, but you can't spray water on the mushroom body. Until the mushroom bodies are harvested, the relative humidity of the air in the germ room should be controlled at 84-86% 2-3 days before harvest;
[0068] The above percentages are mass percentages.
[0069] The cultivation methods of the above-mentioned strains are:
[0070] I. Preparation of bacterial culture medium: select five-section mango granules with a diameter of 1-1.2cm and soak them in 5% lime water for 45 hours. After the five-section mango granules are softened and fermented, they are removed, and then rinsed with water. Adjust the PH value to 7.3-7.7, then dry the washed five-piece mango granules, then select 73% five-piece mango granules, 25% bran, 1% glucose, and 1% gypsum powder, and then mix the ingredients evenly according to the above , And then add water to the well-mixed ingredients until the moisture content of the culture medium is 60%. Put the culture medium with 60% moisture content into the culture flask, and then seal with sealing paper, and then put the culture flask under the pressure 0.984-1.12kg/cm 2 , Sterilize at 121°C for 2 hours or at 100°C for 10 hours at normal pressure, then cool for use;
[0071] II. Inoculation: In the inoculation box or inoculation room, first open the sealing paper near the alcohol lamp. The flame is 1-1.5cm away from the mouth of the bottle. Avoid directly burning the mouth of the flask. Take the mother plant in the test tube and grow it. Then, move the inoculated culture bottle into the hair growth room to avoid light and cultivate. The humidity of the hair growth room is controlled at 60-70% and the temperature is controlled at 23-25°C. After 30 days of cultivation in the hair room, Until the hyphae overgrown the surface of the medium, and remove non-normal individuals in time,
[0072] The above percentages are mass percentages.
[0073] The above-mentioned method of expansion and propagation of the mother species, the mother species use the PDA medium test tube slant culture method. The formula of the PDA medium is: 200g of peeled potatoes cut into small pieces of 0.8-1.2cm in side length, 20g of glucose, and agar 20g, 1000ml of water, constant volume, boil in a pot for 9-11 minutes until the potato chips are soft and not rotten, then filter, take the filtrate, add 20g agar to the filtrate, continue to boil until the agar is completely melted, then add 20g glucose, Then add water to 1000ml of culture medium, and then divide the 1000ml culture liquid into test tubes while it is hot. The volume of each aliquoted culture liquid is 1/5 to 1/4 of the length of the test tube, and then put a cotton plug on the mouth of the test tube Seal, and then put the test tube containing the culture liquid at a pressure of 1.12kg/cm 2 , Sterilize in a sterilization pot with a temperature of 121℃ for 30 minutes. After sterilization, take out the test tube in an oblique manner. After the culture medium has solidified and cooled, move it into the inoculation box. Take 3-5 test tubes and place them in the 30℃ incubator After 3 days of cultivation, if no bacteria appear, PDA medium is formed for inoculation.
[0074] In the following, the present invention will be further described in conjunction with the embodiments;
[0075] The harvested five-section awns are quickly dried, and cut into 1-2cm granules with a small straw cutter as raw materials for use. Soak the five-piece awn granules in about 5% lime water for 40-48 hours, wait for it to soften and ferment, remove it, rinse with clean water, adjust the pH to about 7.5, and dry the forage after soaking in water for cultivation The water content of the material is controlled at about 60%-70% for use.
[0076] The mother species of Pleurotus eryngii (also called the first-level strain) introduced from the regular strain factory are expanded and cultivated. The mother species (first-level strains) are grown by using the PDA culture medium test tube slant culture method, which is produced according to the conventional method. The formula of PDA medium: 200g potatoes (peeled); 20g glucose; 20g agar; constant volume with 1000ml water. Wash the fresh potatoes, peel them, and cut them into small pieces of about 1 cm. Place them in a small aluminum pot and boil for 10 minutes until the chips are soft and not rotten. Filter with double gauze and take the filtrate. Add 20g of agar to the filtrate, continue to boil until the agar is completely melted, add 20g of glucose, and make up to 1000ml of water. Dispense it into the test tube while it is hot. Pack 1/5 to 1/4 of the length of the test tube. Wipe the mouth of the tube and put a tampon. The tampon should be tight enough to prevent any gaps. Use an autoclave with a pressure of 0.984-1.12kg/cm 2 , Sterilize at 121℃ for 30min. After sterilization, place the test tube obliquely so that the front end of the culture medium spreads to 1/2 of the length of the test tube. Cover it with gauze or sterilized newspaper (to prevent excessive condensation water). After the culture medium has solidified and cooled, move it into the inoculation box. Take 3-5 test tubes and place them in an incubator at 30°C for 3 days. If no bacteria appear, it means that the sterilization is complete and it can be used for inoculation.
[0077] All operations of inoculation should be performed in the inoculation box or the inoculation room. To be carried out under aseptic conditions, place the inoculation rake on the flame of the alcohol lamp and burn it. After cooling, pull off the tampon of the test tube near the flame of the alcohol lamp, and hook a small piece of mother species with culture medium (first-level strain ), transfer it to the inclined plane and place it in the middle of the culture medium, sterilize the test tube mouth on an alcohol lamp, and put a cotton plug immediately. Each female species (first-level bacterial species) can expand and breed about 30. Placed in an incubator at 25°C for 7-15 days, the mother hyphae can grow over the slope. After the hyphae grow up and continue to cultivate for 5-7 days, the original seed can be produced.
[0078] The original species is also called the secondary species. Pleurotus eryngii substituting material for cultivation of Pleurotus eryngii seed culture medium formula is pentagonal awn particles 73%, bran 24%, glucose 2%, gypsum powder 1%, water 120-130%; Stir the granules, bran and gypsum powder evenly, then add the dissolved sugar water and water, the moisture of the culture material is about 60%, (that is, it is best when the culture material has water seeping out between the fingers by hand) , While filling the material into the bottle while bottling, hold the neck of the bottle by hand and vibrate continuously until the material is filled to the bottle mouth. Rake the surface flat with a flat iron hook, press and ram to the shoulder of the bottle (4/5 of the whole bottle), and seal it. The original sealing paper should be thicker than kraft paper. Cut the kraft paper into a square about 5cm larger than the diameter of the bottle mouth. Cover the bottle mouth with cotton rope or high temperature resistant rubber ring. The medium is autoclaved and the pressure is 0.984-1.12kg/cm 2 , The temperature is 121 ℃ (2 ~ 2.5 hours) or the normal pressure sterilization is maintained at 100 ℃ for 10 hours, the original seed culture flask is quickly put into a clean room after the pot is heated, and the inoculation can be carried out after cooling.
[0079] During inoculation, operate in the inoculation box or inoculation room. First open the sealing paper near the alcohol lamp. The flame is 1-1.5cm away from the bottle mouth. Do not directly burn the mouth of the flask. Take the oblique mother seed, wipe the outer wall with an alcohol cotton ball, remove the cotton plug of the mother seed test tube near the flame of the alcohol lamp, burn the test tube mouth on the flame, burn the inoculation rake on the flame of the alcohol lamp, and place it inside the test tube to cool Later, cut the oblique surface of the mother species (first-level strain) into 6-8 sections, be sure to remove the tip section, and connect the remaining sections together with the culture medium to the original seed bottle culture medium. Inoculate 1-2 segments per bottle. After inoculation, quickly burn the mouth of the flask and the sealing paper with the flame of an alcohol lamp. Move the original bacteria bottle into the bacteria room to avoid light and cultivate, the indoor humidity is 60-70%, and the indoor temperature is set to 23-25°C. During the vegetative growth period of Pleurotus eryngii, a small amount of air exchange is sufficient. Mycelium can grow over 25-35 days at about 25℃. When the hyphae grow up, it can be used to transfer cultivated species (third-level strains). The initial stage of mycelium culture should be checked once a day. When the mycelium covers the surface of the culture medium and goes down 1-2cm, it can be checked once a week. Timely removal of non-normal individuals is an important means to improve the purity of strains.
[0080] 7-10 days after the original seed bottle is full of hyphae, the hyphae are in the optimal growth period. After inoculation with new culture material, they can show strong adaptability. At this time, it is most suitable for expanding cultivated species.
[0081] Cultivation (third-level strain) cultivation
[0082] Cultivation (three-level strain) culture medium: adopt bag cultivation. The formula is 75% of five-piece mango granules, 20% bran, 3% sugar, 1% gypsum powder, 1% lime; according to the formula, stir the five-piece mango granules with bran and gypsum powder evenly, and then add to dissolve it. The water content of the culture material is about 70%. Before bagging, the pH of the culture material is measured with pH test paper and adjusted to 7.5-8. The culture material in the bag must be tight enough. It is best that the material surface is about 1cm away from the mouth of the bottle after being lightly compacted with a ramming wood. The upper part of the culture material is required to be slightly tight and the bottom part is slightly loose. The sealing paper of the cultivated species can be kraft paper or newspaper, and the method is the same as that of the original species. The culture medium is autoclaved, the pressure is 0.984-1.12kg/cm2, the temperature is 126℃ (2~2.5 hours) or the normal pressure is sterilized for 18 hours, and the autoclave sterilization: requires single layer placement, 126℃ maintenance time 2-2.5 Hours; the basic requirements of normal pressure sterilization: within 4-5 hours after the temperature rises, it is required to reach 100 ℃, if the time is too long, the material will become sour. The temperature stabilizes to 100°C, and the timer starts. After 18 hours, you can uncover the pot immediately, and take out the pot after proper cooling. The bacteria bag enters the cooling room as soon as possible, and it is required to take it out of the pot while it is hot (necessary for continuous production) to avoid contact between the bacteria bottle and the pollution source, and increase the humidity of the cooling room (combined with spray disinfection).
[0083] The sterilized cultivation bag is inoculated after cooling, and the inoculation disinfection method is conventional. Wipe the outer wall of the selected stock with an alcohol cotton ball, burn the sealing paper with a flame, and discard the upper part with an inoculation rake. When the cultivated species are inoculated, it can be operated by one person or two persons. In single operation, use the left hand to hold the bacteria bottle, remove the sealing paper with the right hand, seal the bottle mouth with the alcohol lamp flame, and then use the right hand to hold the inoculation tool. After burning, take the original seed in the original seed bottle. Fix broad bean large and small bacteria in the inoculation hole, and sprinkle a small amount of debris on the surface. Each bottle of original species (secondary bacteria species) can receive 50-60 bottles of cultivated species.
[0084] For two-person operation, one person is responsible for holding the original seed bottle and clamping the strain inoculation; the other person is responsible for opening the sealing paper of the cultivation bag to 45 degrees, and burning the bottle mouth and sealing paper with flame after inoculation.
[0085] After inoculation, the bacteria bag is moved into the bacteria room to avoid light and cultivate. The humidity of the germination room should be controlled at 60-70%; the indoor temperature setting: the temperature should be controlled at 25-28℃ in the early period of culture (within one week); the temperature should be controlled at 24-hours in the early and middle period of culture (7-15 days) 26℃; Late cultivation period: the temperature should be controlled at 22-24℃ after 15 days. CO during vegetative growth of Pleurotus eryngii 2 It has a promoting effect on the growth of mycelium, so a small amount of air exchange during the cultivation period is enough. The hyphae can grow over the culture flask for about 35 days.
[0086] Invigorate buds, after the mycelium is full of bags, continue to cultivate for about 10 days to further mature and accumulate more nutrients. When the temperature is controlled at 10-18℃, the budding can be managed. Pleurotus eryngii can produce mushrooms without scratching it. However, in order to produce the mushrooms neatly and uniformly, the bacteria can be scratched first. The specific method is: use a small spoon to scrape off the old bacteria on the surface of the bottle mouth and make the material surface of the bag mouth flat. Scratching time is usually done when the bottle is full of hyphae. In about 10 days, when new white aerial hyphae grow on the surface of the material, water can be sprayed to humidify the buds. During bud intensification, the temperature should be controlled between 10-18°C. It is difficult to form primordium when it is lower than 8°C or higher than 20°C. The best bud intensification is to maintain the humidity at 90%-95% during bud intensification at 10-15°C. Increase the scattered light stimulation, strengthen ventilation, and keep the air fresh. After 3-6 days, white primordium will appear, which further differentiates into mushroom buds.
[0087] Fruiting management When the primordium differentiates to form 1-2cm small mushroom buds, the filter paper at the mouth of the bag should be removed in time for fruiting management. Leave 3-4 in each bag to keep the mushrooms beautiful and big, and the commodity rate is high.
[0088] At the fruiting stage, attention must be paid to the regulation of temperature, humidity, light and ventilation in order to obtain high yields.
[0089] Temperature control The temperature should be kept at 10-20℃, preferably at 15-18℃. Under this temperature condition, the growth and development of the fruit body is normal and healthy.
[0090] Humidity regulation Water and temperature management are very important for the differentiation and development of fruit bodies. The relative humidity of the air at the initial stage should be kept at about 90%. When the diameter of the mushroom cap grows to 2-3cm, water can be properly sprayed to cool and humidify, and water should not be sprayed on the mushroom body. In 2-3 days before harvest, in order to extend the preservation period after harvest, the relative air humidity should be controlled at about 85%. When the tide of mushrooms is over, you can pour water into the bag.
[0091] Light adjustment: The fruit body needs to scatter light in both the generation and development stages.
[0092] Air conditioning The fruit body development stage requires increased ventilation, and always keep the air in the mushroom room fresh.
[0093] Harvesting and processing When the cap is flat, the spores have not yet ejected, and the cap diameter is the same as the stalk or slightly smaller than the stalk, it must be harvested. After the first tidal mushrooms are harvested, the material surface should be cleaned in time, the water should be stopped for 4-5 days, and the temperature, humidity and ventilation conditions of the mushroom room should be adjusted. After about 14 days, the second tide mushrooms can be harvested. The output of Pleurotus eryngii is mainly concentrated in the first tide mushroom, which accounts for more than 70% of the total output. For harvesting, pick large and leave small at the right time, and harvest in stages. When harvesting single mushrooms, hold the base of the stalk and pull up, and cut the clump mushrooms with a knife. Touchao mushrooms are harvested for about 15 days before the second wave can be harvested.