Investigating the synergistic action of three essential oils for antibacterial and antioxidant applications: A comprehensive framework using mixture design, artificial neural networks, and in silico methods

Essential oils (OEs), recognized for their antibacterial and antioxidant characteristics, have gained extensive application in the food sector as preservative agents. Moreover, mixtures of OEs may have more potent antibacterial and antioxidant effects than a single essential oil. Expanding on this foundation, the objective of this work was to highlight the combined antibacterial and antioxidant action of blending essential oils (EOs) derived from Moroccan Cladanthus mixtus (C. mixtus), Myrtus communis (M. communis), and Helichrysum italicum subsp. Italicum (H. italicum). Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detection (GC-FID) analyses have revealed that C. mixtus EO was marked by α-pinene (19.7%), santolina alcohol (11.53%) and germacrene D (10.59%), M. communis was characterized by 1,8-cineole (30.88%), α-pinene (25.65%), myrtenyl acetate (14.69%) and limonene (10.99%), while neryl acetate (16.27%), α-pinene (13.64 %), β-selinene (8.79%), γ-curcumene (7,7%) were major compounds in H. italicum EO. C. mixtus showed the strongest efficacy against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), while H. italicum demonstrated the strongest antioxidant effect. Subsequently, the methodology of mixture design (MDM) combined with artificial neural networks (ANNs) were used to improve the formulations of the EOs. The results obtained from the predictive modeling revealed that the inhibition of E. coli and S. aureus was impacted by the ternary mixtures of C. mixtus, M. communis, and H. italicum, whereas the binary mixture of C. mixtus and H. italicum displayed the highest antioxidant activity. Statistical analysis indicated that both optimization approaches accurately predicted the biological activities. However, the accuracy of the ANNs models outperformed that of MDM.