Stochiometric and Kinetic Parameters for Bacteriocin and Biomass Formation from Local Isolate Lactobacillus Fermentum by Response Surface Methodology

Abstract

Response surface methodology (RSM) based on a Central Composite Design (CCD) was applied to optimize both biomass and bacteriocin production by Lactobacillus fermentum through modification of MRS medium composition. Glucose concentration, yeast extract concentration, and inoculum size were selected as independent variables and optimized using Design-Expert software. The developed quadratic models showed excellent predictive performance with coefficients of determination (R²) of 0.987 and 0.985 for biomass and bacteriocin production respectively. The optimized conditions for biomass production consisted of glucose at 39.9 g/L, yeast extract at 19.9 g/L, and an inoculum size of 2.0%, resulting in the highest biomass yield of 15 g/L. For the production of bacteriocin, the model predicted slightly varied different optimal values to be 40.1 g/L  of glucose, 20.0 g/L yeast extract and the inoculum size of 2.0% which collectively produced a maximum activity of 89.8 AU/ml. The validation experiments confirmed that the reliability and accuracy of the developed models, producing 15.8 g/L biomass and approximately 90 AU/mL of bacteriocin under optimized conditions.The Growth-kinetic analysis demonstrated that the production of bacteriocin was growth associated and it reaches to the maximum level after 48 hrs of incubation. The partial purification of the bacteriocin was achieved through using ammonium sulfate precipitation, dialysis, sucrose concentration, and gel filtration chromatography using Sephadex G-150. The specific activity was increased from 166.7 to 3000 U/mg protein in final purification process, corresponding to an 18- purification fold. The chromatographic profile showed a distinct activity peak related with the active bacteriocin fractions. These findings revealed the effectiveness of CCD-RSM for enhancing and improving bacteriocin biosynthesis by L. fermentum.