Box Behnken Desing for the Optimization of the Antimicrobial Substance Production by Lactic Acid Bacteria

Abstract

Abstract In this research, we employed a 3-factor Box-Behnken experimental design (BBD), which falls within the category of response surface methods. The objective was to ascertain the optimal conditions for the production of antimicrobial substances by lactic acid bacteria (LAB). We utilized the MINITAB 19 trial version software package (MINITAB Statistical Software, State College, Pennsylvania, USA) for our data analysis. Throughout our analyses, a significance level of α=0.05 was maintained. The experimental parameters encompassed three variables: temperature, incubation time, and substrate concentration. Three levels of temperature were applied in the experiments: 30°C, 35°C, and 37°C. Incubation times were set at 24 hours, 48 hours, and 72 hours, while substrate (glucose) concentrations were established at 1%, 2%, and 3%. In addition to these variables, LAB isolates included in the trials were isolates coded as F2, 40, 50, O2 and Pediococcus pentosaceus ATCC 43201, which was the reference bacteria. Optimal conditions for bacteria coded as O2 and 40 could not be determined using the BBD method. We observed that the production of antimicrobial substances by F2 bacteria was influenced by the incubation period, with the most effective production occurring after an incubation period exceeding 70 hours. Similarly, P. pentosaceus ATCC 43201 exhibited optimal antimicrobial substance production after an incubation period exceeding 70 hours. Bacteria coded as 50 displayed varying antimicrobial activity in response to all three model parameters. The highest antimicrobial substance production was achieved at a temperature of 37°C, an incubation period of 72 hours, and a substrate concentration of 2% in the environment. These results from our trial suggest that the antimicrobial effectiveness of LAB generally increases beyond the logarithmic phase in the growth curve. This observation aligns with the notion that bacteria tend to exhibit a more competitive nature as environmental factors become more limiting, which is often associated with the production of secondary metabolites.