Optimisation of fermentation conditions for cell growth of recombinant Lactococcus lactis ATCC11454-ldhΔnisRKOE using response surface methodology

Abstract

Food safety and shelf-life preservation remain major challenges in agroindustry due to the increasing prevalence of foodborne pathogens and the growing demand for natural alternatives to synthetic preservatives. Recombinant lactic acid bacteria capable of producing antimicrobial peptides have emerged as promising candidates for sustainable biopreservative development. In this study, a sequential Design of Experiment (DoE) approach was applied to optimise cell growth of recombinant Lactococcus lactis ATCC11454-ldhΔnisRKOE, a metabolically engineered strain developed for enhanced nisin biosynthesis and antimicrobial activity. A 12-run Plackett–Burman Design (PBD) was first employed to screen six fermentation variables, followed by a 20-run Central Composite Design (CCD) based on Response Surface Methodology (RSM) to optimise the significant factors. The PBD identified fermentation time, temperature, and initial cell density as influential factors for subsequent optimisation. The quadratic CCD model was statistically significant (p = 0.010), with an R² value of 81.45% and a non-significant lack-of-fit (p = 0.073), indicating an adequate fit to the experimental data. In the final reduced model, fermentation time and the quadratic effect of temperature significantly influenced cell growth (p < 0.05), whereas initial cell density showed a positive trend but did not reach statistical significance (p = 0.058). The optimum conditions predicted by the model were 2% inoculum density, 30°C fermentation temperature, and 24 h fermentation time, producing an experimental OD600 value of 1.650 that remained within the 95% confidence and prediction intervals. The findings demonstrated that metabolic engineering significantly altered the growth characteristics and fermentation behaviour of the recombinant strain compared with the wild-type. The optimised conditions obtained in this study provide an important foundation for future scale-up fermentation and sustainable production of recombinant L. lactis-based natural biopreservatives for food and agroindustrial applications.