Optimization of the Culture Conditions of Lactic Acid Bacteria for Antimicrobial Activity and Mass Production of Cyclic Dipeptides.

Abstract

Cyclic dipeptides (CDPs) are secondary metabolites from lactic acid bacteria (LAB) known for their antibacterial, antifungal, and antiviral properties. Here, we investigated CDPs to optimize culture media and boost their production in LAB. The optimal conditions and ratio of Lb. plantarum LBP-K10 to Leu. mesenteroides LBP-K06 (temperature, time, stationary or shaking conditions, carbon or nitrogen source, etc.) were determined through step-by-step optimization. The two strains (K10:K06 = 7:3), cultured together at 30°C for 48 h in stationary conditions, resulted in enhanced antibacterial effectiveness. This was evident against B. subtilis ATCC 9372 (clear zone size of 22.6 ± 2.3 mm), E. coli S-99 (14.8 ± 1.8 mm), Salmonella Gallinarum ATCC 9184 (27.6 ± 2.4 mm), V. parahaemolyticus KCTC2471 (13.2 ± 3.2 mm), and S. aureus KACC 10768 (12.0 ± 2.1 mm). These results surpassed the clear zone sizes of 13.2 ± 1.4 mm, 12.0 ± 2.2 mm, 19.3 ± 1.0 mm, 9 ± 0.5 mm, and 9.8 ± 0.7 for each corresponding bacterium, respectively, in K10-alone control groups (37°C for 48 h in conventional medium). We further confirmed the production of these cyclodipeptides within cultures. When LBP-K10 and LBP-K06 were co-cultured at a 7:3 ratio under stationary conditions at 30°C for 48 h with 4% sucrose and 1.5% mixed amino acids supplementation, the total production of cyclo(Leu-Pro) and cyclo(Phe-Pro) exceeded that of the LBP-K10 mono-culture by more than 35%, reaching 6.65 ppm compared to 4.91 ppm. These findings hold significant potential for industrial-scale production of CDPs and related products.