Inhibitory Effect against Listeria monocytogenes of Carbon Nanoparticles Loaded with Copper as Precursors of Food Active Packaging

Abstract

Human listeriosis is a serious foodborne disease of which outbreaks are occurring increasingly frequently in Europe. Around the world, different legal requirements exist to guarantee food safety. Nanomaterials are increasingly used in the food industry as inhibitors of pathogens, and carbon nanomaterials are among the most promising. In the present study, novel carbon nanoparticles loaded with copper (CNP-Cu) were prepared, and their antimicrobial activity against Listeria monocytogenes was assessed. CNPs of two sizes were synthesized and characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS) and electron microscopy (EM). The minimum inhibitory concentration (MIC) of CNP-Cu was determined in accordance with the available standard. To get insights into its mechanism of action, the release of copper ions into a cell media was assessed by inductively coupled plasma optical emission (ICP-OE), and the ability of loaded CNPs to generate cytotoxic reactive oxygen species (ROS) was evaluated by EPR spectroscopy. Finally, the extent of release of copper in a food simulant was assessed. The results demonstrated the antimicrobial effectiveness of CNP-Cu, with growth inhibition up to 85% and a release of copper that was more pronounced in an acidic food simulant. Overall, the results indicate CNP-Cu as a promising agent for the design of active food packaging which is able to improve food shelf-life.

Keywords: Listeria monocytogenes; action mechanism; active food packaging; antimicrobial activity; carbon nanoparticles; copper-loaded nanoparticles; minimum inhibitory concentration (MIC); shelf-life of food.

Figure 1

Size distribution of CNPs. (A) Hydrodynamic diameter distribution, as evaluated by DLS of pristine and Cu-loaded CNPs suspended in water. Each line is the mean of five measurements of the same suspension. Error bars represent the SD. (B,C) FE-SEM images of (B) SCNP and (C) LCNP.

Figure 2

The antimicrobial activity of CuSO₄, SCNP-Cu e LCNP-Cu (low, medium, high copper concentration) was evaluated against Listeria monocytogenes. The optimal growth condition of Listeria monocytogenes as a negative control (10⁹ CFU/mL) during the MIC experiments is indicated as 100% on the y-axis (dashed gray line). The x-axis represents the copper concentration (low, medium and high) for each antimicrobial. Average values are used for the histogram representation, and values with different superscript letters (a, b, c) are significantly different (p < 0.05).

Figure 3

Release of copper in the BHI broth and ROS generation by CNPs. (A) Concentration of copper in BHI broth following incubation with three different concentrations of SCNP-Cu (S1–S3) or LCNP-Cu (L1-L3) for 24 h (red columns) detected by ICP-OES in comparison with the theoretical concentration of copper relative to total release (black columns). (B,C) EPR spectra generated by (B) SCNP and SCNP-Cu and (C) LCNP and LCNP-Cu after 60 min of incubation in the presence of 88 mM DMPO and 40 mM H₂O₂ in 4 mM PBS at pH 7.4.

Figure 4

Copper release from SCNP-Cu into each food simulant after 10 days of incubation in the dark at 40 °C. The dotted line represents the total amount of copper loaded on the SCNP-Cu during incubation with food simulants, corresponding to the maximum concentration. Values are expressed as % of CU release.