A method for producing Beta Leucine by fermentation
(1) Activation culture: Inoculate the Beta Leucine producing bacteria (i.e. Corynebacterium glutamicum ACCC16522, purchased from Shanghai Fuxiang Biotechnology Co., Ltd.) onto 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 culture: Take a ring of well-growing activated bacteria and inoculate it into a shake flask containing seed medium for seed culture. The liquid volume is 200mL/500mL. The conditions for seed culture are 32°C, 150r/min shaker culture After 20 hours, the concentration of bacteria in the fermentation broth was measured with a spectrophotometer. If the OD620 value of the bacteria reached 1.2, it could 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%, get the seed liquid that step (2) obtains, inoculate in the 10L fermentor that contains fermentation medium, the filling amount is 6L/10L, and culture temperature is 30 ℃-35 ℃, the stirring speed is 600rpm/min, feed medium is added during the cultivation process, the ventilation volume is 80-100L/h in the first 12h, and the ventilation volume after 12h is 100-120L/h, and the fermentation process is controlled by automatic flow of ammonia water Medium pH=6.6±0.05, cultured for 30-32 hours to obtain fermentation broth. The end time of fermentation was judged by the fact that acid production no longer increased. Before and after fermentation, the content of Beta Leucine was determined by high performance liquid chromatography; the content of residual sugar in the fermentation liquid was detected by a biosensor 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 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 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: 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 load The sample volume is 260mL of fermentation broth, the eluent used is 3.0mol/L ammonia water, the elution flow rate is 0.1-1.0BV/h, the effluent is collected, and the Beta Leucine in the fermentation broth and effluent is determined by high performance liquid chromatography The content of the calculated yield.
