The application of liquid strains to the factory production of enoki mushroom is the inevitable tren
Enoki mushroom is one of the main varieties of factory cultivation of edible fungi in my country. At present, factory cultivation has become the main production mode of enoki mushroom, and it is an inevitable trend of future development for liquid strains to gradually replace solid strains for the production of enoki mushroom. Carrying out the research and development of enoki mushroom liquid strains and industrial production applications can effectively improve the economic benefits of enterprises and promote the sustainable development of the industry.
1. Culture medium
The preparation of enoki mushroom liquid strain mainly involves three kinds of culture media, namely PDA solid medium, shake flask fermentation medium and fermenter medium. A variety of shake flask fermentation medium and fermentation tank medium formulas have been reported, and they have achieved obvious results.
1.1 Shake flask fermentation medium
Formula 1: Glucose 20g, peptone 2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, trisodium citrate 1g, calcium chloride 0.1g, ferrous sulfate 1mg, manganese sulfate 1mg, VB10.1μg, VB20.1μg, water 1000mL.
Formula 2: Soluble starch 40g, glucose 10g, peptone 3g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, VB110μg, VB250μg, water 1000mL.
1.2 Fermenter culture medium
Recipe 1: 150g potato, 30g bran, 20g sucrose, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 1000mL water.
Formula 2: 40g soybean meal powder, 10g white granulated sugar, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, VB1100μg, VB2500μg, 1000mL water.
Recipe 3: 50g corn flour, 30g bran, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, VB10.1μg, VB20.5μg, water 1000mL.
In the fermentation tank culture medium, generally add an appropriate amount of antifoaming agent, such as polydimethylsiloxane emulsion, etc., which can effectively prevent the formation of foam and avoid the influence of foam on the fermentation process of liquid bacteria.
2. Liquid strain preparation process
In the preparation process of the industrialized liquid strain of enoki mushroom, in order to effectively shorten the growth cycle and avoid unnecessary pollution, companies often use shake flask liquid seeds directly into the fermentation tank culture medium. The specific process is: test tube or flat solid strain → Shake flask fermentation → fermentation tank strains → inoculation of cultivation materials. The specific process is as follows:
2.1 Plate inoculation of bacteria
Choose PDA plate medium, inoculate a bacterial seed block with a diameter of about 0.5 cm in the center of the plate, and cultivate it at 25°C. The aerial hyphae of enoki mushroom are well developed, and the whole plate can be covered in 6~7 days. It is recommended to use a flat plate (9cm petri dish) to cultivate solid strains. First, it is helpful to observe the growth of aerial hyphae of strains, and the other is to punch holes from the edge of the colony.
2.2 Shake flask fermentation culture mycelium
Use a 1000mL shake flask to fill 600mL fermentation medium and sterilize it for later use. Use a hole puncher to punch holes from the edge of the plate colony and inoculate them into the shake flask fermentation medium. The inoculation amount is 8-10 solid strains (diameter 5mm). Rotate at 160r/min and incubate at 23°C for 6-7 days. In order to prevent the mycelium balls from condensing together, a magnetic stirrer can be used to break up the mycelium blocks in the seed liquid.
2.3 Fermentation tank inoculation strains
A 1 000L pneumatic fermenter is used to load 800L of fermentation medium, which is autoclaved and steam sterilized for later use. The inoculum amount is 1%~2%. After 6~7 days of cultivation at 23℃, it can be placed in the pot and used for inoculation of cultivation material.
3. Matters needing attention
3.1 Prevent contamination by bacteria
Liquid strain seed production has high efficiency and is easy to realize factory operation, but the contamination of bacteria is a problem that cannot be ignored in the liquid strain preparation process. In particular, small and medium-sized enoki mushroom production enterprises have weak anti-risk capabilities. Once contamination by bacteria occurs, it is very likely to cause bankruptcy. There are three main reasons for the contamination of bacteria: one is technical operation problems; the other is leakage of fermentation tanks and auxiliary equipment; the third is bacterial contamination.
In response to the above problems, it is recommended that companies take the following measures to control the risks to the lowest extent. Regularly train technical operators to improve their theoretical and operational levels; establish operating specifications for fermentation workshops and guide technicians to operate in accordance with the regulations; regularly inspect and maintain equipment to avoid leakage of equipment; regularly carry out strain purification and inspection work, and use Use plate culture, microscope observation and other methods to determine whether the bacteria are infected with other bacteria in time.
3.2 Maintain stable strains
Strains are the core of an enterprise's production, and maintaining the stability of strains is the key to high and stable production. Enterprises should conduct sampling inspections of liquid bacteria once a week. The detection methods include back-up plate culture and microscopic observation. The quality of the bacteria can be preliminarily judged by observing the production status of aerial hyphae and whether there is lock-like association or not, so as to prevent the degradation of the bacteria. Cause irreparable losses to the enterprise. The study found that the use of LBL liquid medium with bromothyfen blue can eliminate severely degraded strains, and the severely degraded strains have poor decolorization ability. It is recommended that manufacturers can promote it.