RESUMO
@#Objective To develop a shake-flask stage culture process for E.coli with higher biomass and higher bacterial viability based on Quality by Design(QbD)concept.Methods Using different shake-flask configurations as the investigation factors,and A600value of the bacterial suspension,wet weight of the culture and viability value of the bacteria as the indicators for investigation of the culture results,ANOVA was used for the analysis of culture results to obtain the third amplification flask configurations with high biomass and high bacterial viability.The two-factor two-level full-factor test was carried out with the shaker temperature and shaker speed as the test factors,the A value of bacterial suspension as the response value,the culture accumulation time as a variable factor,and the real-time online temperature and self-test speed of the shaker as the supplemented variable factors.The functional principal component analysis(FPCA)method was used to perform a generalized regression model to model the quasi-growth curve,and the optimized culture stop time and culture process were obtained by the growth curve model.The design space of the shaker culture process was optimized again using Monte Carlo simulation(MCS)with random noise added to the response value.The worst condition in the design space was selected as the setting condition for verification test,and serial 10 batch verification tests were performed in stages with different culture stop time.Results The third amplification shake-flask configurations:5 L disposable high-efficiency shakeflask and large area breathable film cover.The culture process design space:shaker temperature of 36.5-37.5 ℃,shaker speed of 220-230 r/min,and the design culture stop time of 18 h.The worst condition verification test showed that when the culture was stopped for 16 h,the culture results of higher cell viability value and lower biomass could be obtained,and when the culture was stopped for 18 h,the results of higher biomass and bacterial viability value could be obtained.Conclusion The shake-flask stage culture process for E.coli designed in this study has the characteristics of high biomass and high bacterial viability,and can be adjusted according to the adaptability of this culture process to meet different culture needs.
RESUMO
@#Objective To optimize a shake flask culture medium for Escherichia coli(E.coli)with high biomass and viability using artificial neural networks(ANN). Methods Using the proportion of glucose(Glu),yeast extract(YE),yeast peptone(YP),soy peptone(SP)and yeast nitrogen base(YNB)as the mixture component,and the A_(600)(A1)value of cell suspension,wet bacterial weight(G,g/L)of culture and cell viability(A2,A_(460))as the response values,the mixture design was used to screen the mixture components that had a significant effect on the response value. The ANN model was constructed with the test results of mixture design as training and verification data samples. The input variables were mixture components and restricted the upper and lower limits of the mixture components,and the output variables were mixture design response values. The optimized medium formula and reference values were obtained by the constructed ANN. The medium formula was further adjusted by Monte Carlo simulation to obtain the shake flask medium formula of E.coli,which was then verified for 10 times. Results The shake flask culture medium of E.coli was composed of Glu 26 g/L,SP 26 g/L,YNB13 g/L with the total concentration of 65 g/L. The verification results showed that the probability of A1 ≥ 14 was 60%,the probability of G ≥ 77 g/L was 50% and the probability of A2 ≥ 11 was 40%. The mean values of the incubation result data were equivalent to the reference values. Conclusion The shake flask culture medium of E.coli optimized in this study can obtain E.coli with high biomass and bacterial activity.