Study On L-lactic Acid Fermentation By Lactobacillus Casei

Lactic acid was one of the few industrialized bio-based chemicals, lactic acidwas produced by fermentation processing, which had advantages of high yields andlow cost. However, at present, the main problem was that L-lactate had low opticalpurity, which could not meet the requirement that polylatic acid was synthesized byL-lactic acid. To solve the problem, we studied the optimization of L-lactatefermentation medium and fermentation conditions, L-lactic acid with high opticalpurity of the mutant strain wsa achieved using the methods of UV and DESmutagenesis.Firstly, a wild type strain which producing L-lactic acid was identified using16SrRNA, and to construct phylogenetic tree by BLAST for sequence similaritycomparision.It was named as Lactobacillus casei ZW-63ASecondly, L-lactic acid fermentation medium and fermentation conditions wereoptimized by single factor experiments and response surface method. The bestL-lactic acid fermentation conditions were obtained, to achieve the highest L-lactateproduction, the seed culture was statically incubated at37℃for12h, and thenincubated on a rotary shaker at37℃,150rpm for8h.10g Calcium carbonateneutralizer was supplemented, adding glucose in the medium as carbon source, addingyeast in the medium as nitrogen source, the best inoculum was10%(v/v), the bestliquid medium volume was100mL. The factors of glucose, yeast,sodium acetate wereoptimized using Box-Behnken center-composite experimental design and responsesurface method, the optimum fermentation medium for L.casei ZW-63A wasdetermined as follows(g/100mL): glucose17.85, yeast1.27, sodium acetateanhydrous0.19. Under the optimized conditions in flask fermentation, the opticalpurity of L-lactic acid was improved to96.21%, which improved6.09%beforeoptimization, and the L-lactic acid production was improved to146.67g/L, which was2.03times of the production before optimization.Tween80and betaine were used as osmoprotectant in the fermentation ofL-lactic acid by L.casei ZW-63A. The result show that, comparing to the controlfermentations with Tween80addition, the L-lactate production was obviouslyimproved by betaine, and the highest L-lactate production was obtained when batainewas added2g/L. In shake flask level, L.lactis, L.buchneri, L.rhamnosus were used astest strains in the L-lactate fermentation, comparing to the control fermentations withTween80addition, the results show that, the L-lactate production and lactatedehydrogenase were all improved by betatine supplementation.Fed-batch lactate fermentation by L.casei was tested in5-L fermenter withbetaine or Tween80supplementation. The result show that, the betaine showedhigher L-lactic acid productivity comparing to the Tween80. Fed-batch fermentationwith betaine supplementation,L.casei ZW-63A produced up to190g/l L-lactic acid,with the yield of95.47%, the productivity of2.64g/l h, and the optical purity of 97.03%; all were higher than those of L-lactate production in Tween80control(withthe titer of170g/l, yield of91.15%, productivity of2.36g/l h, optical purity of96.78%).Finally, based on the results of fermentation medium and fermentation conditionsfor L-lactate fermentation, then UV and diethyl sulphate(DES) were carried on theoriginal strain. The best mutated time was60seconds, the best broth dilution was10-5,a mutated strain with L-lactate optical purity of97.75%was obtained. After that, thestrain(UV) was mutated by diethyl sulphate(DES). The best mutated time was30minutes, and the best mutated volume fraction was1%. After that, the L-lactic acidproduction of mutation strain was140g/L, the opyical purity of the mutation strainwas98.83%, and then by genetic stability tested, the mutated strain showed a goodstability, and it was named as Lactobacillus casei ZW-63B.