Optimization of a Functional Food Formulation Containing Moringa oleifera Leaf, Tualang Honey, and Kefir Milk for Enhanced Antidiabetic, Antioxidant, and Anti-Escherichia coli Activities using Response Surface Methodology

Diabetes increases the risk of Escherichia coli infections and systemic oxidative stress. While Moringa oleifera, Tualang honey, and kefir milk individually possess therapeutic properties, their combined synergistic potential to provide multi-target protection has not been investigated. This study optimized a functional formulation containing M. oleifera, Tualang honey, and kefir milk (MTK) using Response Surface Methodology (RSM) combined with a Box-Behnken Design (3 factors-3 levels) to enhance the complementary bioactivities of these ingredients. Independent variables included M. oleifera (X₁; 1-3 g), Tualang honey (X₂; 20-40 g), and kefir milk (X₃; 30-50 mL), while the biological activities were considered as responses. The antidiabetic activity of MTK formulations was assessed using the α-glucosidase assay, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was performed to evaluate their antioxidant activity. Anti-E. coli activity of MTK formulations was assessed using disc diffusion assay (DDA), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Results indicated that all 15 MTK formulations, including three replicated central points, designed by the RSM exhibited antidiabetic (% inhibition: 48.85-76.79), antioxidant (% inhibition: 72.42-93.21), and anti-E. coli (inhibition size: 1.10-1.73 cm) activities. However, mathematical models showed that the synergistic effect of all three independent ingredients was most prominent in inhibiting E. coli growth. Meanwhile, the interaction between Tualang honey and kefir milk is limited to a narrow range in α-glucosidase inhibitory and DPPH scavenging activities. Our study confirmed that the optimized formulation consisting of 1 g of M. oleifera, 33.85 g of Tualang honey, and 37.27 mL of kefir milk achieved simultaneous antidiabetic (74.68%), antioxidant (84.18%), and anti-E. coli (1.50 cm) activities. These findings support the application of RSM to optimize a multifunctional therapeutic blend and highlight the potential of MTK in managing health issues related to hyperglycemia, free radicals, and E. coli infection.