①Choose Bacillus subtilis GDGR-1006 as the preserved strain; ②Preparate glucose, dipotassium hydrogen phosphate, magnesium sulfate, malic acid, yeast powder, agar, and water in proportion to make primary strain culture medium and put it into an eggplant bottle;
③Inoculate the preserved strains in the primary strain culture medium described in step ②, the inoculum amount is 0.1%±0.02%, and at a temperature of 37°C±0.5°C, culture them statically for 22h±2h to obtain the primary strains;
④Put the primary strains into the shake flask culture medium with an inoculum size of 0.1%±0.02%, and cultivate them on a reciprocating shaker with a rotating speed of 160±10 rpm, a temperature of 37°C±0.5°C, and a time period of 12 -14h, when the microscopic examination of the strain and OD and other indicators reached the standard, the liquid strain of the shake flask was obtained.
①Preparate glucose solution, dipotassium hydrogen phosphate, magnesium sulfate, malic acid, yeast powder, and water in proportion to make a secondary culture medium, and pump it into the secondary culture medium after steam sterilization at 121°C±1°C for 15min±1min. After the seed tank is cooled to 37°C±0.5°C, insert the shake flask strain described in step (1) ④, the inoculation amount is 0.1%±0.02%, and at the same time, compress and filter the sterile The air is input into the secondary seed tank. At this time, the ventilation ratio is 1:0.2~0.4v/V.min, the pH is 6~8, the pressure is 0.03~0.1MPa, and it is cultivated for 10h~12h. That is, to obtain secondary strains;
② Mix glucose solution, dipotassium hydrogen phosphate, magnesium sulfate, malic acid, yeast powder, and water in proportion to make a fermentation medium, and then pump it into the fermenter after steam sterilization at 121°C±1°C for 15min±1min, and cool down to 37°C After ℃ ± 0.5 ℃, insert the secondary bacteria, and the inoculum amount is 10%; at the same time, according to the above step (2) ①, under the condition of inputting sterile air under pressure, the bacteria will be extinguished by steam at 120 ℃ ± 1 ℃ for 8 min ± 1 min. The post-bacteria glucose solution and malic acid are fed continuously at the same time; at this time, the ventilation ratio is 1:0.3~0.6v/V.min, the pH is 6~8, the pressure is 0.04MPa~0.1MPa, and the fermentation is 50h~60h; When it reaches 45g/L±2g/L and the conversion rate is 28%±2%, the Methyl Guanosine fermentation broth is obtained.
