A Response Surface Methodological Approach for Large-Scale Production of Antibacterials from Lactiplantibacillus plantarum with Potential Utility against Foodborne and Orthopedic Infections

Abstract

A variety of bacteria, including beneficial probiotic lactobacilli, produce antibacterials to kill competing bacteria. Lactobacilli secrete antimicrobial peptides (AMPs) called bacteriocins and organic acids. In the food industry, bacteriocins, but even whole cell-free supernatants, are becoming more and more important as bio-preservatives, while, in orthopedics, bacteriocins are introducing new perspectives in biomaterials technologies for anti-infective surfaces. Studies are focusing on Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum). L. plantarum exhibits great phenotypic versatility, which enhances the chances for its industrial exploitation. Importantly, more than other lactobacilli, it relies on AMPs for its antibacterial activity. In this study, Response Surface Methodology (RSM) through a Box-Behnken experimental design was used to estimate the optimal conditions for the production of antibacterials by L. plantarum. A temperature of 35 °C, pH 6.5, and an incubation time of 48 h provided the highest concentration of antibacterials. The initial pH was the main factor influencing the production of antibacterials, at 95% confidence level. Thanks to RSM, the titer of antibacterials increased more than 10-fold, this result being markedly higher than those obtained in the very few studies that have so far used similar statistical methodologies. The Box-Behnken design turned out to be a valid model to satisfactorily plan a large-scale production of antibacterials from L. plantarum.

Keywords: Box–Behnken design; Lactiplantibacillus plantarum; antibacterial activity; bacteriocins; foodborne infections; orthopedic implant infections.

Figure 1

Response surface plots showing interactive effects between independent variables (temperature, pH and incubation time) for improved bacteriocin production by Lactobacillus plantarum.

Figure 2

Contour plots showing interactive effects between study parameters for enriched bacteriocin production by Lactobacillus plantarum.

Figure 3

Normal probability plot of the residuals of a regression model for increased bacteriocin production by Lactobacillus plantarum: (A) Correlation between actual and predicted values of the quadratic polynomial model; (B) Antibacterial activity of crude bacteriocin.

Figure 4

Inhibitory zone (mm) of bacteriocin produced by Lactobacillus plantarum against four pathogens. Each value is the mean ± SEM of five individual replicates. The differences differ significantly from each other (one-way ANOVA test; p < 0.001 and subsequent post hoc DMRT).