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This study investigates the antibacterial action of chitosan‐based nanofibers (CNFs) obtained by the electrospinning process on the permeability of bacterial membranes. The bactericidal efficiency of CNFs was first determined against Gram‐negative Escherichia coli and Salmonella Typhimurium, and Gram‐positive Staphylococcus aureus and Listeria innocua bacteria as a baseline. The results strongly suggest that CNFs interact with the negatively charged bacterial cell wall causing membrane rupture and inducing leakage of intracellular components among which are proteins and DNA. Results clearly indicate that the release of such components after contact with CNFs is an indication of membrane permeabilization and perforation, as pore formation was observed in transmission electron microscopy (TEM). This work suggests a plausible antibacterial mechanism of action of CNFs and also provides clear evidence in favor of chitosan as a bacterial membrane disruptor and perforator. As a result, CNFs can find promising applications as bioactive food packaging materials capable to extend shelf life of food products while inhibiting the spread of alteration flora and foodborne pathogens.

The fabrication of nonwoven mats containing chitosan and bacterial nanocellulose by electrospinning were considered using two different approaches: (1) simultaneous spinning of chitosan and bacterial nanocellulose solutions using two separate syringes towards the same target and (2) coaxial electrospinning, where chitosan and bacterial nanocellulose were simultaneously electrospun through a spinneret composed of two concentric needles to produce core–shell structures. Co-spinning agents were required in both approaches. A direct blend of chitosan and bacterial nanocellulose and subsequent electrospinning was not feasible due to the incompatibility of their respective solvents. The first approach led to the production of mats containing both chitosan and bacterial nanocellulose nanofibers. However, few bacterial nanocellulose fibers were deposited on the collector. Addition of polylactide as a co-spinning agent and an increase in solution temperature (from 22 to 60 °C) during electrospinning was required to improve both fiber formation and collection. On the other hand, coaxial electrospinning showed the best results for the production of nanofibers containing both chitosan and bacterial nanocellulose. Nanofibers with a good yield were obtained by using a chitosan/poly(ethylene oxide) (2.4/0.6 wt/v%) aqueous solution as the inner layer, and a bacterial nanocellulose solution (0.6 wt/v%) as the outer layer. Co-electrospun nanofibers had a diameter of 85 nm in average, and a narrow size distribution. The core/shell nanostructure was validated by transmission electron microscopy whilst energy-dispersive X-ray spectroscopy analysis showed that the nanofibers contained both chitosan and bacterial nanocellulose along their structure. Finally, the mats obtained by the coaxial approach exhibited strong antimicrobial activity with a decrease of 99.9 % of an Escherichia coli population.