Put the culture solution in a 500ml triangular flask, each with a capacity of 100ml, add 10 ~ 15 small glass beads or glass fragments with a diameter less than 0.8cm, seal them with cotton plugs and kraft paper, sterilize them at 12 1℃ for 40min, and take them out and cool them to 20℃. Each bottle was inoculated with a small piece of inclined mother seed, so that the aerial mycelium was suspended on the liquid surface with one side facing upwards, and it was still cultured at a constant temperature of 24 ~ 26℃ for 48 hours. When the aerial hyphae extend into the culture solution, they are placed on a reciprocating shaking table for oscillation culture, with the oscillation frequency of 80 ~ 100 times/min and the amplitude (stroke) of 6 ~10 cm. If a turntable is used, the eccentricity is 4 cm and the oscillation frequency is 150 ~ 220 rpm. Due to the high rotating speed, the mycelium ball is generally larger, and the surface of the mycelium ball is less fluffy, so it is not suitable for use as a strain. Therefore, the eccentricity of the general turntable should be properly adjusted before use. The temperature of the shaker is controlled at 24 ~ 26℃ and the culture time is 72 ~ 96 hours. The technical parameters of shaking table culture of different kinds of edible fungi are different. Grifola frondosa is generally at 25℃, 200 revolutions, with an eccentricity of 4 cm. After shaking the bottle for 3-4 days, the culture solution was yellow-brown, clear and transparent, with many mycelium balls. Under the microscope, the diameter of mycelium ball should be within 1-2 mm, the sample should be filtered by centrifugation (2500 rpm, 15 min), dried to constant weight at 105℃, and the dry weight of mycelium should be weighed to10g/L. Such strains can be inoculated in a triangular flask again. Because the mycelium pellets are in a vigorous growth state and have good dispersibility, the shake flask culture time can be shortened, and the shake flask culture can be terminated in 2 days at the earliest. If the culture medium is turbid, it is mostly the result of bacterial pollution. The culture solution can only be used for production after it has passed the microscopic examination. The liquid strain cultured in shake flask can be stored for 20 ~ 30 days in refrigerator at 4℃ and for 5 ~ 7 days at room temperature of 15 ~ 20℃, which has no adverse effect on inoculation success rate and yield.
In production practice, it is also possible to prepare liquid strains from large glass bottles, but there must be a better air filtration device.
2. The first-class seed tank for three-stage expanded culture of mycelium has a constant volume of 35 liters, and the shake-flask seeds are inoculated through an inoculation tube with a valve, and the shake-flask seeds are inoculated at a ratio of 5%-7% 1.5-2 liters, and are matured according to the fermentation process; Inoculate the first-class seeds into the second-class seed tank, with a constant amount of 350 liters, and inoculate 35 liters of the first-class seeds according to 10%, and cultivate them to maturity. No matter what kind of seed tank, before inoculation, the empty tank must be disinfected (135 ~140℃)1hour, and then injected with culture solution for sterilization. The procedure is to preheat the interlayer to 90℃ for 10 minute, then release steam into the inner layer to raise the temperature to about 1 15℃, and keep the temperature for 8 minutes, which should not be too long to avoid destroying nutrients. Then cold water is injected into the interlayer to cool to 26℃ and then inoculated. Pay attention to open the exhaust port and close the air inlet during inoculation, and reduce the pressure to 0.05 MPa. At the same time, samples were taken for microscopic examination, and the sterilization of the culture solution was checked with broth medium. Sampling at 0 o'clock to check miscellaneous bacteria, 0. D value (optical density) and initial sugar; 12, 24, 36 and 48 hours, take each sample to check 0. D value, residual sugar and pH value. After 48 hours of normal culture, the pH value dropped to 5.0 and the net length was 0. D value is about 0.5, sugar consumption is about one unit, and it can be transferred to a three-stage culture tank with large volume.
The volume of the three-stage culture tank is 10 ton, and the general constant volume is 7500 liters. At the same time, the culture medium and defoamer (polyoxypropylene glyceryl ether) were added, and the amount of defoamer was 2.5 liters, accounting for 0.03% of the culture medium. Inoculate strains according to 10% inoculation amount (two secondary seed tanks are connected 1 tertiary tank). First pour the jar (140℃) 1 hour, then actually pour the jar (115 ~118℃) for 50 ~ 60 minutes, and then drop it to 25℃ for inoculation. The pre-culture temperature is 25 ~ 26℃, and the post-culture temperature is 26 ~ 27℃. The pH is controlled at about 5.5, and urea is added when it is lowered, and the dosage is generally 0.2% of the culture solution. The ventilation rate in the early stage of fermentation is 1∶05 (volume/volume), and the ventilation rate can be increased to 1∶ 1 in the middle stage to prevent the pH from dropping too fast. After inoculation, the impeller speed was 180 rpm, and after 72 hours of culture, samples were taken every 12 hours to determine sugar, nitrogen, pH and miscellaneous bacteria. Intermediate feeding can improve the growth rate of mycelium, shorten the fermentation time and increase the bacterial concentration. Materials can be supplemented in 20 hours, 40 hours and 60 hours, and the amount of supplementary materials depends on the specific situation.
Pay attention to control the culture time, the yield of mycelium will be low if the tank is put too early, and the mycelium will age and autolyse if it is too late. Generally, it can be cultured in a three-stage tank for 3 ~ 4 days. Canning standard: pH is reduced to 5, residual sugar is about 2.5, ammonia nitrogen is not more than 30mg/ml, and the concentration of bacteria balls reaches 1000 ~ 1500/ml, or after 3000 revolutions/centrifugation 10 minute, the wet weight of bacteria mud per 100 ml.
Different technological conditions should be selected according to the growth requirements of fungi. Generally, the temperature of Grifola frondosa is controlled at 25℃, the tank pressure is 0.05 MPa, the ventilation rate (liquid: gas) 1: 0.3 ~ 0.5, and the stirring speed is 200 rpm.
The items to check the quality of fermentation broth include: purity check, no bacteria and mold were detected by microscope or plate culture; Vigor examination: Microscopic examination showed that the hyphae at the edge of the fungus ball were finely branched, and the hyphae were darker when stained with crystal violet, and there was no agglutination and vacuole in the hyphae protoplasm. After the culture medium was allowed to stand for 5 minutes, the bacterial sludge did not settle. 80% of the fungus balls are less than 65438 0 mm in diameter.
The culture solution is filtered by plate and frame or continuously centrifuged to obtain mycelium, which can be eaten directly or used as beverage and food additive after drying; You can also extract intracellular polysaccharide from Grifola frondosa, and concentrate the culture solution to obtain extracellular polysaccharide, which can be used as raw materials for preparing drugs.
The culture solution containing a large number of hyphae can be used to replace the mother seed and cultivated seed. The liquid strain has the advantages of fast growth, strong vitality, fast colonization and low pollution rate. In large-scale culture, mycelium liquid can be injected into sterilized culture bags and 5 tons fermentation tanks, and 3.5 tons of liquid strains can be produced within 3 days. According to the inoculation amount of 5%, 25 ml of liquid strains can be injected into 500 grams of dry material culture bags, and 3.5 tons of liquid strains can be inoculated into 70 tons of culture materials, about1.4000 culture bags.