Bioautographic method for detecting natural preservatives against Mycobacterium in edible essential oils

Abstract

The interest in essential oils and their use in antimicrobial food preservation has increased in recent years, driven by a growing consumer preference for natural alternatives over synthetic preservatives. Since essential oils are compound mixtures, their study can benefit from effective, simple, and low-cost tools that enable the linking of an observed biological effect with their actual bioactive constituents. In this study, thin-layer chromatography was coupled with an agar-overlay Mycobacterium assay to detect antimycobacterial compounds in essential oils. The optimization of key parameters, including the culture optical density, addition of tyloxapol, incubation time, and final gel volume on the chromatographic plate was carried out through factorial analysis. The results demonstrated acceptable linearity within a range of 0.3–5.0 μg for isoniazid, with a coefficient of determination (r²) = 0.96 and a limit of detection equal to 0.20 μg. A set of 36 essential oils was studied with the developed bioautography. Two active essential oils, Origanum vulgare L. and Allium sativum L., were selected for further analysis by gas chromatography coupled to mass analysis (BioMSId strategy) identifying the compounds responsible for the observed inhibition. The 1,2-diallyl disulfide and 5-isopropyl-2-methylphenol were identified in A. sativum and O. vulgare respectively. The current assay can be applied to the study of mixtures with varying levels of complexity, facilitating dereplication.