Statistical optimization of glucose, sodium citrate and potassium chloride supplementation for enhanced bacteriocin production by Lactococcus lactis MT186647 and Lactobacillus fermentum MT186598 isolated from fermenting Pentaclethra macrophylla benth

Abstract

Bacteriocin production by lactic acid bacteria (LAB) is strongly conditioned by growth-medium composition, and adjusting ingredients offers a low-cost route to enhanced yield. Response surface methodology (RSM) based on a three-factor, five-level central composite design (CCD) was used to optimize supplementation of an African oil bean seed ( Pentaclethra macrophylla Benth) extract broth with glucose, sodium citrate and potassium chloride for two LAB isolated from the seed and identified by 16S rRNA sequencing, Lactococcus lactis MT186647 and Lactobacillus fermentum MT186598. Twenty runs per organism were performed; bacteriocin activity was determined by the zone of inhibition against Staphylococcus aureus ATCC 19095 by agar well diffusion. For both isolates, glucose exerted a significant negative linear effect and all three supplements significant negative quadratic effects, while no two-factor interaction reached significance, indicating an additive response. The second-order models accounted for 71.85% ( L. lactis ; p = 0.060) and 73.15% ( L. fermentum ; p = 0.050) of the variation, with significant lack-of-fit; the regression was therefore only marginally significant, and the predicted R² was 0.00%, indicating no demonstrated ability to predict new runs. The desirability-based optima (6.35/0.55/1.78 g/L glucose/sodium citrate/potassium chloride for L. lactis ; 6.44/0.55/1.59 g/L for L. fermentum ; predicted zones 13.12 and 12.74 mm) are best interpreted as an estimated optimal region rather than a quantitatively predictive optimum. Validation under these optima combined with the previously established optimal culture conditions (1.9% NaCl, 31 °C, initial pH 6.0) produced zones of 15.22 mm ( L. lactis ) and 14.96 mm ( L. fermentum ), exceeding the supplementation-only model estimates by 16.0% and 17.4% and the previously reported culture-condition-only outcomes (13.33 and 11.75 mm) by 14.2% and 27.3%. Because these differences were not tested statistically and partly reflect the fixed 1.9% NaCl (optimized separately and absent from the supplement CCD), the two optimization layers are interpreted as combining additively rather than demonstrating an established synergy. Moderate, balanced supplementation with inexpensive, food-grade additives can substantially enhance bacteriocin yield, supporting further development, subject to confirmatory multi-point validation, of L. lactis MT186647 and L. fermentum MT186598 as starter or protective cultures for the controlled fermentation of P. macrophylla to Ugba .