Potential Applications of Eucalyptus camaldulensis in Food Preservation and Biofilm Inhibition on Food Contact Surfaces

Abstract

Plants are excellent source of bioactive compounds that could be applied in medicine, food, and cosmetics. In this study, the applications of Eucalyptus camaldulensis ethanolic extract in food production were explored. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract against the foodborne pathogen Listeria monocytogenes were 64 to 128 ug/mL and 256 to 512 g/mL, respectively. Time-kill assay revealed growth inhibitory effects after 4 h treatment of the bacteria with the extract. A reduction of ~2 to 3 log CFU/mL was observed against the tested food and environmental isolates after challenging the pathogens with the extract at MIC for 6 h. Sub-MICs of the extract significantly inhibited listerial attachment to food contact surfacé materials, motility and listeriolysin O production up to 80%, with a 60% inhibition of biofilm formation (p<0.05). Antioxidant assay revealed free radical scavenging activity with ICso of 57.07 ug/mL for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 29.01 pg/mL for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Ferric Reducing Antioxidant Power (FRAP) assay further showed a total antioxidant power equivalent to 92.93 uM ascorbic acid equivalent/mg extract. Encapsulation of the extract in sodium alginate - sodium CMC matrix revealed an irregular shaped microparticles with mean diameter ranging from 6.7 to 26.6 pm. Zeta potential and polydispersity index ranged from -17.01 to 2.23 mV and 0.344 to 0.489, respectively. Percentage yield and encapsulation efficiency ranged between 70.4 to 81.5 and 74.2÷0.011 to 82.43t0.772%, respectively. In addition, the microcapsules exhibited high swelling index with poor solubility. DPPH and ABTS assays revealed that the radical scavenging activities varied directly with the concentration of the extract. Minimum inhibitory and minimum bactericidal concentrations of the microcapsules against Gram-positive foodborne pathogens ranged from 0.19 to 3.12 mg/mL and 0.19 to 12.25 mg/mL, respectively. Moreover, the microcapsules at concentration of 1 mg/mL did not show cytotoxic effects on human colon cell Caco-2. In addition, synthesis of silver nanoparticles with the extract as reducing and capping agent yielded spherical nano-sized particle with mean diameters of 13.11 nm, zeta potential of -35.85 mV, and effective diameter of 138.6 nm. MIC and MBC of synthesized silver nanoparticles on pathogenic foodborne, bacteria ranged from 0.99 to 1.99 pg/mL and 3.98 to 15.91 ug/mL, respectively. Fabricated active PVA- Chitosan nanocomposite packaging functionalized by incorporation of the synthesized silver nanoparticles showed good physical and mechanical properties without significant alterations when compared with control PVA-Chitosan packaging (p<0.05). Antimicrobial activity of PVA-CH/silver nanoparticles (1:1) films showed bactericidal effects against important foodborne pathogenic bacteria (Escherichia coli O157:H7, L. monocytogenes, Staphylococcus aureus, and Bacillus cereus), with greater than 99.9% reduction. Antioxidant activities suggested a concentration dependent free radical scavenging activity. In addition, PVA-CH/silver nanoparticles showed first order release with fickian diffusion and were non-toxic to Caco-2 cells at 1 mg/mL. Similarly, UV-vis spectra of silver nanoparticles functionalize packaging nanocomposite fabricated from recycled waste papers presented a peak at 427 mm, indicating the surface plasmon resonance band of silver in the paper. SEM and EDS analysis showed uniform distribution of silver nanoparticles, whereas EDS and ICP-OES results indicated Ag concentration of 0.1 %Wt and 0.0112 ug/cm', respectively. Antimicrobial efficacy of the paper, tested against selected important foodborne pathogens, demonstrated a bactericidal effect against B. cereus and E. coli 0157:H7 with > 3log reduction in CFU/mL after 3 h of treatment and a bacteriostatic effect against L. monocytogenes and S. aureus. Cytotoxicity against HEK293T and Caco-2 cells revealed >80% cell viability. The results of the study suggested that E. camaldulensis leaf could be a source of bio-friendly antimicrobial agent that could be used in food as antimicrobial and antioxidant preservative and/or antimicrobial food-grade sanitizer for the inhibition of microbial attachment and biofilm formation on food contact surfaces. The results also revealed that encapsulation of the extract could be employed to preserve its stability and activity in food system. Moreover, the results further showed that the extract could be used in the synthesis of silver nanoparticles for the fabrication of active nanocomposite packaging material.