Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria, after incorporation into biopolymer edible films

The antimicrobial activity of twelve natural extracts was tested against two fish spoilage bacteria (Pseudomonas fluorescens and Aeromonas hydrophila/caviae) and Listeria innocua, in order to assess their potential utilization in the preservation and safety of minimally processed fish products. After a screening of the active extracts by agar diffusion and vapour diffusion methods, oregano and thyme essential oils and citrus extract were selected. The minimum inhibitory concentration (MIC) of the selected extracts was determined by disc diffusion method against target bacteria and at two temperatures: bacteria's optimal growth temperature (30 °C or 37 °C) and refrigeration temperature (4 °C). Due to its better solubility, lack of odour and greater inhibitory effect obtained against L. innocua at refrigerated temperature, citrus extract was selected and incorporated at 1% (v/v) into different biopolymer film forming solutions (gelatin, methyl cellulose and their blend 50:50 w/w). The antimicrobial activity of the developed films was then evaluated, just after preparation of the films and after one month of storage at 43 ± 3% relative humidity and 24 ± 3 °C. Regardless of the biopolymer matrix, all the developed films showed antimicrobial activity against the target bacteria. The most sensitive bacterium towards active films was L. innocua while P. fluorescens appeared as the most resistant one, in accordance with the previously performed antimicrobial tests for pure extracts. The differences in activity of the films between the tested two temperatures were not significant except for L. innocua, for which three times higher inhibition diameters were observed at refrigerated temperature. The inhibitory effectiveness of the films against the tested strains was maintained regardless of the biopolymer matrix for at least one month. Therefore, these edible films show potential for their future use in fresh fish fillets preservation.

► Citrus extract has higher activity against L. innocua at 4 °C than at 37 °C.
► Citrus extract's activity is maintained in films regardless of biopolymer matrix.
► The films’ antimicrobial activity was sustained after one month of storage (24 °C).