A method for producing L-Leucine Powder by fermentation
(1) Activation culture: Inoculate the L-Leucine Powder producing bacteria (i.e., Corynebacterium glutamicum ACCC16522, purchased from Shanghai Fuxiang Biotechnology Co., Ltd.) on the activation medium, and activate the culture in an incubator at 32°C for 12 hours to obtain the activated bacteria.
Activation medium: anhydrous glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, NaCl 2.5g/L, agar powder 20g/L, pH=7.0-7.2.
(2) Seed cultivation: Take a ring of well-growing activated bacteria and inoculate them into a shaker flask containing a seed medium for seed cultivation. The liquid volume is 200mL/500mL. The conditions for seed cultivation are 32°C and 150r/min shaker culture for 20h. The concentration of bacteria in the fermentation broth is measured with a spectrophotometer. If the OD620 value of the bacteria reaches 1.2, it can be used as a qualified seed solution to obtain a seed solution.
Seed medium: anhydrous glucose 20g/L, corn steep liquor powder 25g/L, ammonium sulfate 2g/L, KH2PO4·3H2O 1.5g/L, vitamin B1 300μg/L, biotin 200μg/L, pH=7.0-7.2.
(3) Fermentation culture: according to the inoculum amount of 10%, take the seed liquid obtained in step (2), inoculate it into a 10L fermenter containing fermentation medium, the liquid volume is 6L/10L, the culture temperature is 30°C-35°C, the stirring speed is 600rpm/min, and the feeding medium is added during the cultivation process. The ventilation rate in the first 12h is 80-100L/h, and the ventilation volume after 12h is 100-120L/h. Add ammonia through automatic flow Water controls pH=6.6±0.05 during the fermentation process, cultures for 30-32 hours, and obtains a fermentation liquid. The end time of fermentation was judged by the fact that acid production no longer increased. Before and after fermentation culture, the content of L-leucine is measured by high performance liquid chromatography; the content of residual sugar in the fermentation liquid is detected by a biosensing analyzer.
Fermentation medium: sodium malate 2.0-4.0g/L, anhydrous glucose 140-160g/L, corn steep liquor dry powder 35g/L, ammonium sulfate 20g/L, KH2PO4 3H2O 1.0g/L, MgSO4 7H2O 0.4g/L, MnSO4 H2O 0.1g/L, vitamin B1 300μg/L, biotin 50μg/L, CaCO3 30g/L, defoamer 0.5%, pH=7.0-7.2.
Feed medium: sodium malate 1.0g/L, inositol 1.0-2.0g/L, corn steep liquor dry powder 12g/L, ammonium sulfate 25g/L, KH2PO4·3H2O 2.0g/L, MgSO4·7H2O 0.6g/L, MnSO4·H2O 0.3g/L, vitamin B1 400μg/L, biotin 60μg/L, CaCO3 20g/L, defoamer 1.0%, pH=7.0-7.2.
(4) Extraction of L-Leucine Powder: take the fermentation broth after fermentation, centrifuge at 8000r/min for 10min, collect the supernatant, extract and purify the supernatant through an ion exchange column, load the sample at a flow rate of 0.5BV/h, and the loading volume is 260mL of fermentation broth. Calculate the yield of L-leucine in the effluent.
