Optimization of antibacterial and antifungal activities in Moroccan saffron by-products using mixture design and simplex centroid methodology

The health risks associated with synthetic preservatives have intensified the search for natural antimicrobial alternatives. Crocus sativus L. (saffron) generates abundant by-products, such as tepals and leaves, which are rich in bioactive compounds with demonstrated antimicrobial potential. Compared to other natural alternatives, saffron by-products offer distinct advantages, including a unique combination of phenolic compounds (e.g., ellagic acid, rutin) and carotenoids (e.g., crocin) that act synergistically against both Gram-positive and Gram-negative bacteria, as well as fungi. Additionally, these by-products represent a sustainable solution, with approximately 63 kg of agricultural waste generated per kg of saffron spice. This study optimized the antibacterial and antifungal efficacy of saffron extracts using a simplex centroid mixture design. Phytochemical analysis using high-performance liquid chromatography with diode-array detection (HPLC-DAD) identified key antimicrobial compounds, including ellagic acid (68.43% in leaves, 50.31% in tepals) and crocin (9.59% in stigmas). Antimicrobial assays against Staphylococcus aureus, Escherichia coli, Candida albicans, and Geotrichum candidum revealed that stigma extracts exhibited superior antibacterial activity (MIC = 25 mg/mL for S. aureus and E. coli), while tepal and leaf extracts showed promising antifungal effects (MIC = 12.5 mg/mL for G. candidum). The mixture design approach uncovered synergistic interactions, with an equimolar combination of stigma, tepal, and leaf extracts (33:33:33) demonstrating the strongest antibacterial activity (MIC = 25 mg/mL) and a ternary mixture (34% stigma, 30% leaf, 36% tepal) achieving the lowest antifungal MIC (6.25 mg/mL). These findings highlight saffron by-products as highly effective and sustainable natural antimicrobials, providing a cost-efficient (40-60% reduction compared to conventional extracts) and multi-functional alternative to synthetic preservatives. Their dual functionality (antimicrobial + natural coloring) and agricultural waste origin make them particularly valuable for industrial applications in food preservation, pharmaceuticals, and biopharmaceuticals. The integration of statistical modeling maximizes their potential, meeting the growing demand for safe, natural antimicrobial solutions with clear competitive advantages.