Enhanced Production of Bacteriocin from Probiotics Using Optimization Techniques by Response Surface Methodology

Bacteriocin, antibacterial peptides, are regarded as potential alternatives to conventional antimicrobials from probiotics tested against different test organisms. They are a family of microbial defense systems which may inhibit the invasion of strains and act as biopreservatives. Antimicrobial activity of crude bacteriocin was assessed and characterized by the Agar Well Diffusion Assay and the cell-free extract was collected by centrifugation of broths, poured into the wells and tested against test organisms. The activity of the culture filtrate was measured by the zone of inhibition. Statistical screening of variables for enhancement of the production of biopreservatives was carried out. Bacteriocin was effective against E. coli and Staphylococcus from the analysis; dextrose, sodium acetate, ammonium citrate and NaCl were screened as important variables using the Placket-Burman Design. Low-cost nitrogen sources often used in production were used to replace their expensive counterparts in De Man Rogosa and Sharpe (MRS) constituents were used in lower concentration than in the MRS medium for control. Three variables were chosen from the seven variables considered in the Placket-Burman (PB) design. Response surface method was used to study and optimize their interaction and to determine their optimimum levels. Central composite design (CCD) was used to move toward the optimum response and determined the maximum activity of the bacteriocin. By solving the regression equation and analyzing the response surface, the optimal concentrations of dextrose (15 g/L), sodium acetate (4.5 g/L), ammonium citrate (2.5 g/L) and NaCl (1.5 g/L) stimulated the production of bacteriocin and the optimal equation was then verified. Under optimized conditions, microbial growth had two-fold higher production than common MRS. The molecular mass of purified bacteriocin was approximately 2kD by SDS-PAGE. All experiments were done at room temperature for two days under stationary condition. The protein extraction was completed by dialysis, followed by precipitation, purification by ammonium sulphate precipitation and then chromatography. The new medium was less expensive. Therefore, it has more significance for application as a biopreservative in food products. The improvement in production was achieved due to this optimized medium. The results indicated the importance of statistical tools for the enhanced production of bacteriocin. The potential of preservation could be achieved either by a bacteriocin starter culture or by bacteriocin as a food additive.