Mechanism of Action of Electrospun Chitosan-Based Nanofibers against Meat Spoilage and Pathogenic Bacteria

Abstract

This study investigates the antibacterial mechanism of action of electrospun chitosan-based nanofibers (CNFs), against Escherichia coli, Salmonella enterica serovar Typhimurium, Staphylococcus aureus and Listeria innocua, bacteria frequently involved in food contamination and spoilage. CNFs were prepared by electrospinning of chitosan and poly(ethylene oxide) (PEO) blends. The in vitro antibacterial activity of CNFs was evaluated and the susceptibility/resistance of the selected bacteria toward CNFs was examined. Strain susceptibility was evaluated in terms of bacterial type, cell surface hydrophobicity, and charge density, as well as pathogenicity. The efficiency of CNFs on the preservation and shelf life extension of fresh red meat was also assessed. Our results demonstrate that the antibacterial action of CNFs depends on the protonation of their amino groups, regardless of bacterial type and their mechanism of action was bactericidal rather than bacteriostatic. Results also indicate that bacterial susceptibility was not Gram-dependent but strain-dependent, with non-virulent bacteria showing higher susceptibility at a reduction rate of 99.9%. The susceptibility order was: E. coli > L. innocua > S. aureus > S. Typhimurium. Finally, an extension of one week of the shelf life of fresh meat was successfully achieved. These results are promising and of great utility for the potential use of CNFs as bioactive food packaging materials in the food industry, and more specifically in meat quality preservation.

Keywords: chitosan-based nanofibers; gram-negative; gram-positive; meat packaging; mechanism of action.

Figure 1

Morphology of electrosprayed and electrospun V₁, V₂, and V₃ chitosan nanofibers (CNFs) and fiber diameter distribution of V₃ CNFs. Chitosan’s concentrations: 3, 5, and 7 wt % in 50% (v/v) acetic acid (AcOH); Chitosan/poly(ethylene oxide) (CS/PEO) weight ratio: 80/20. All scale bars represent 1 µm.

Figure 2

Growth curves of (a): E. coli and (b): S. Typhimurium in the absence (black circles) and in the presence (red triangles) of CNFs (2.5 cm², V₃-95/50, rich Luria-Bertani (LB) medium). Filled and empty blue squares refer to bacterial growth in contact with NaCl and sodium dodecyl-sulfate (SDS)-pretreated CNFs, respectively. The shown data are the mean values of the three replicates method.

Figure 2

Growth curves of (a): E. coli and (b): S. Typhimurium in the absence (black circles) and in the presence (red triangles) of CNFs (2.5 cm², V₃-95/50, rich Luria-Bertani (LB) medium). Filled and empty blue squares refer to bacterial growth in contact with NaCl and sodium dodecyl-sulfate (SDS)-pretreated CNFs, respectively. The shown data are the mean values of the three replicates method.

Figure 3

Antibacterial activity of electrospun chitosan/PEO (80/20) nanofibers with different M_W against E. coli, S. Typhimurium, L. innocua, and S. aureus after 4 h incubation in contact with CNFs.

Figure 4

Kinetics of bacterial cell death induced by CNF (V₃ 95/50) on Gram-negative (E. coli and S. Typhimurium) versus Gram-positive (S. aureus and L. innocua) bacteria in PBS (1×, pH 5.8) at 37 °C. Filled symbols refer to controls of bacterial suspension without treatment and empty symbols refer to the same samples after contact with CNFs.

Figure 5

Cell surface hydrophobicity of E. coli, S. Typhimurium, L. innocua, and S. aureus bacteria, as estimated by the bacterial adhesion to a hydrocarbon (BATH) method.

Figure 6

Antibiogram (Inhibition effect) of CNFs compared to kanamycin (Kan) and ampicillin (Amp) antibiotics, against E. coli, S. aureus, L. innocua, and S. Typhimurium. The arrows indicate the inhibition zone caused by chitosan.

Figure 7

Schematic representation of the home-made electrospinning set-up.