The Study On Mutagensis Of Strain For Higher Nisin Production And Its Bioprocess Optimization

Nisin , a safe aseptic worldwide , is a natural product from microorganism. Nisin is a polypeptide composed with 34 amino acids. It could inhibit most Gram-positive bacteria, especially spores of bacterium. Nisin's stability depends mostly on temperature, pH substance and other factors. It is quite sensitive to protease, and could be broken down byα-chymotrypsin in alimentary canal which shows that nisin is harmless to human. It doesn't bring about intersectant drug resistence with antibiotics.In this study , Lactococcus lactis subsp. lactis ATCC11454 was mutanted by UV and EMS. The mutants were screened by nisin as antibiotic agent combined with agar diffusion method, then, a high-yielded mutant LD2 was got . The strain has a titer of 2500 IU/mL, which is as 2.13 compared with Lactococcus lactis subsp.lactis ATCC11454 . The strain LD2 is stable.The culture condition of LD2 in flask was investigated , then the optimal fermention medium and conditions were confirmed . Three statisticd methods, Plackett-Burman experiment , path of steepest ascent and response surface methodology were applied to optimize LD2 culture medium. With the Plackett-Burman method , the factors affected the nisin titer were studied , and four important components were got , which are glucose , peptone , yeast extract and NaCl.Path of steepest ascent was used to find the markable responsible area . After, Box-Behnken experiment were used to optimize the medium , the optimal medium were got as : glucose 19.3 g/L,peptone 28.6 g/L, yeast extract 24.1 g/L,KH2PO4 10 g/L,NaCl 5.9 g/L,MgSO4·7H2O 0.2 g/L . Under the optimal condition , the max predicted nisin titer is 10531 IU/mL ,while the experimental results 10070IU/mL relative error 4.3 % , which is increased 3.9 times than the non-optimization.result.The effects of the medium pH,temperature,rotate speed,medium amount,inoculum size and culture time were studied in this work . The result suggests the optimal culture condition as : medium pH 6.5 , temperature 37℃, rotate speed 150 r/min , inoculum size 3 % , medium amount 100 mL(250 mL) . Under this condition , after fermention of 8 h , the titer of the Nisin got to 10100 IU/mL .The pilot-scale fermentation was carried in the Biomate 10 L fermenter . Anaerobic and aerobic batch fermentation was studied , and the results showed that nisin titer was correlated withμ. The biomass and nisin titer were higher in aerobie fermentation , which were 6.78 g/L and 11932 IU/mL respectively . The pH feed-back controlled fed-batch fermentation was carried out in this work . The glucose content was kept at the level of 18~22 g/L , and max biomass got 1.36 g/L , while the titer of nisin got 11583 IU/mL . The results of exponential feeding shows that the titer and biomass could got 15367 IU/mL and 6.20 g/L respectively , while the glucose content was kept at the low level of 0.2 g/L during the fermentation.