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Key Points in Preparing Shake-Flask Liquid Spawn

(1) Culture Medium Formula: Potatoes 200 g, glucose 20 g, peptone 5 g, potassium dihydrogen phosphate 2.0 g, magnesium sulfate 1.0 g, Vitamin B1 (1 tablet), pH 6.8–7.0, water 1000 mL.

(2) Medium Preparation

Simmer the potatoes in water for 30 minutes (until cooked but not mushy), then filter through six layers of gauze. Pour the filtrate into a pot, add the weighed ingredients according to the formula, and continue heating while stirring until everything is fully dissolved; then stop heating and top up with water to the original volume. Dispense the medium into Erlenmeyer flasks (150 mL of medium per 500 mL flask), add 10 glass beads to each flask, and seal the opening tightly with a breathable silicone stopper.

(3) Sterilization and Cooling: Place the flasks in a sterilizer and cover the tops with newspaper. After venting the air, begin timing once the temperature reaches 121°C and maintain this temperature for 30 minutes. Once the pressure drops to zero, release any remaining steam, open the lid, allow the flasks to cool to below 30°C, and then remove them to a clean bench for further cooling.

(4) Shake-Flask Inoculation: Select one freshly cultured test-tube slant culture. Under aseptic conditions, rapidly inoculate each flask with a 2–3 cm² piece of the mother culture (Figure 1-56); one mother culture tube can inoculate five shake flasks.

(5) Cultivation: Place the inoculated flasks on a shaker. For a rotary shaker, set the speed to 180 rpm and the temperature to 25°C; cultivate for approximately 5–6 days. Stop cultivation when mycelial pellets are evenly distributed throughout the transparent, orange-yellow nutrient medium. 2. Key Points of Fermenter Preparation

(1) Process Flow: Fermenter cleaning and inspection → Empty sterilization (sterilizing the fermenter vessel) → Liquid medium preparation → Filling → In-situ sterilization (sterilizing the medium) → Inoculation → Fermentation/cultivation → Sampling and testing → Determination of fermentation endpoint → Inoculation.

(2) Formula: Potato 100g, brown sugar 15g, glucose 10g, wheat bran 40g, peptone 2.0g, potassium dihydrogen phosphate 2.0g, magnesium sulfate 1.0g, Vitamin B1 (1 tablet), antifoam agent 0.3mL, pH 6.8–7.0, water 1000mL.

Filling and Sterilization: Load the culture ingredients into the fermenter according to the formula (e.g., 50L of culture medium for a 70L fermenter) and perform heat sterilization at 121°C for 1 hour.

(4) Inoculation: Allow the culture medium to cool to below 25°C. Perform inoculation strictly adhering to aseptic operation requirements; inoculate each fermenter with 1000–1500mL of master culture (spawn).

(5) Cultivation: Cultivation conditions: temperature 25°C, pH 6.5, cultivation time 72–90h, tank pressure 0.02–0.04 MPa, aeration ratio 1:0.8.

(6) Inoculation with Liquid Spawn: Once the liquid spawn is ready, it can be used to inoculate cultivation bags. Prior to inoculation—including the cooling phase for the bags—all air filtration and disinfection systems must operate according to startup protocols, and personnel entry/exit must strictly follow the disinfection procedures for cleanrooms. During inoculation, move the cooled bags (in baskets) from the sterilization cart to the dedicated inoculation line. Inside the inoculation room, positioned beneath the high-efficiency air filter, staff use an automatic liquid inoculation machine to spray the fermented liquid spawn onto the bag openings at a pressure of 0.20 MPa, applying approximately 25–30mL per bag. The inoculation environment requires absolute cleanliness, maintained at a Class 10,000 cleanliness level. After inoculation, the entire basket of mushroom bags is transported from the production line to the outdoors, where personnel use a trolley to move them to the incubation room for cultivation. Figure 1-59 illustrates the inoculation process using oyster mushroom liquid spawn.