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One critical challenge when developing a new antimicrobial packaging material is to demonstrate the mode of action of the antimicrobials incorporated into the packaging. For this task, several analytical techniques as well as microbiology are required. In this work, the antimicrobial properties of benzyl isothiocyanate, allyl isothiocyanate and essential oils of cinnamon and oregano against several moulds and bacteria have been evaluated. Benzyl isothiocyanate showed the highest antimicrobial activity and it was selected for developing the new active packaging material. Scanning electron microscopy and Raman spectroscopy were successfully used to demonstrate the mode of action of benzyl isothiocyanate on Escherichia coli. Bacteria exhibited external modifications such as oval shape and the presence of septum surface, but they did not show any disruption or membrane damage. To provide data on the in vitro action of benzyl isothiocyanate and the presence of inhibition halos, the transfer mechanism to the cells was assessed using solid-phase microextraction-gas chromatography-mass spectrometry. Based on the transfer system, action mechanism and its stronger antimicrobial activity, benzyl isothiocyanate was incorporated to two kinds of antimicrobial labels. The labels were stable and active for 140 days against two mould producers of ochratoxin A; Penicillium verrucosum is more sensitive than Aspergillus ochraceus. Details about the analytical techniques and the results obtained are shown and discussed. [ABSTRACT FROM AUTHOR]

In this work, the effects of cinnamon and mustard essential oils against nine foodborne bacteria were evaluated. Their antimicrobial susceptibility and action mechanism was also assessed. Additionally, the effects of the combination between both were evaluated. To provide data on the in vitro action of essential oils, the transfer mechanism was assessed using gas chromatography–mass spectrometry. The results indicated that the mustard presents 10 times more bactericidal/bacteriostatic effect than cinnamon essential oil. In both cases the action mechanism happened intracellularly, but since mustard induces cell cycle arrest, cinnamon produces protrusions and aggregation. The combinatorial assays demonstrated in most cases an additive effect. In addition, synergies for P. putida and E. coli OI57:H7 were shown. Therefore, to introduce them in combination, could be a great way to make a natural-active packaging. The distinct activity profiles can be explained by their transfer mechanism that involves vapour release and agar diffusion. Industrial relevance The industry is focused on improving food safety by controlling the growth of foodborne and food spoilage microorganisms and by reducing the use of synthetic preservatives. The novelty of this research article lies in the study of antimicrobial activity of two natural active agents (mustard and cinnamon essential oil), separately and in combination, against nine foodborne bacteria; as well as the study of its mechanism of action and transfer. The set of all outcomes give the necessary information to be able to design a novel food active packaging based on the applied microbiology studied. [ABSTRACT FROM AUTHOR]

Raman imaging spectroscopy is a nondestructive analytical method that can be a useful tool to obtain detailed information about the molecular composition and morphology of biological samples. Its high spatial resolution was used to collect spectra of Aspergillus ochraceus, a mold producer of ochratoxin A (OTA), in order to investigate the cell damage caused on it by the action of the antimicrobial benzyl isothiocyanate (BITC). The study was performed in both direct contact and vapor phase, in order to check the use of BITC as active agent in food packaging material. The results showed that there were morphologic alteration and a characteristic Raman spectrum on spore and hyphae exposed to BITC. BITC was accumulated in the mold cells where it caused an enormous amount of alterations in cellular components (lipids, proteins, saccharides, amino acids...) and cellular functions (cell cycle, respiration, metabolism, transcription of genes, fluidity of the cellular wall). All these structural, composition, and metabolic changes will affect the production of OTA. Pattern recognition with chemometrics using principal component analysis (PCA) demonstrated an excellent separation between control and BITC treated samples, both in spores and hyphae. PCA results also showed two different affection levels when samples were exposed to BITC in the vapor phase. [ABSTRACT FROM AUTHOR]