P26 | Evaluation of the in vitro and in situ anti-Listeria activity of an ethanolic extract of propolis from Tunisia

L. Nalbone¹, S. Forgia¹, G. Ziino^1|2, N. Nefzi³, A. Giuffrida^1|2, W. Megdiche-Ksouri³, F. Giarratana^1|2 | ¹Dipartimento di Scienze Veterinarie, Università di Messina, Polo Universitario dell’Annunziata, Messina, Italia; ² Riconnexia srls, Department of Veterinary Science, University of Messina, Polo Universitario dell'Annunziata, Messina, Italy; ³ Laboratory of Aromatic and Medicinal Plants, Biotechnology Center of Borj-Cédria, Tunisia

The aim of this study was to evaluate the anti-Listeria activity of an ethanolic extract of propolis (EEP) from Tunisia, both in vitro and in situ in sweetened ricotta cheese using a challenge test, while also investigating whether any antimicrobial activity could extend its shelf life without altering its sensory characteristics. The ethanolic extract, obtained from raw propolis collected from Apis mellifera hives, was prepared at a ratio of 20% propolis and 80% solvent (80% Ethanol) (w/v). The antibacterial activity of EEP was analyzed in vitro using the Disk Diffusion Assay method (1 g/ml; 250 mg/ml; 150 mg/ml), and the minimum bactericidal (MBC) and inhibitory (MIC) concentrations were also evaluated from 25 to 0.0125 mg/ml against 6 strains of Listeria monocytogenes (ATCC 19111; 19112; 7644 + three strains from the EURL guidelines for dairy products 12MOB098LM, 12MOB096LM, and 12MOB079LM). The in situ evaluation was conducted on 2 types of sweetened ricotta: an industrial vaccine (RZIV) and the second semi-artisan sheep vaccine (RZSP) on the market, both supplemented in the laboratory with 40% sucrose. Each type of sweetened ricotta was divided into 2 portions: From the first, intended for the challenge test, multiple sub-samples were obtained, experimentally contaminated with a mix of 3 strains of L. monocytogenes (12MOB098LM, 12MOB096LM, and 12MOB079LM) and treated with different concentrations of EEP (0, 0.1, and 1%). The second batch, which was further divided into sub-batches, was supplemented with 0 and 0.1% EEP for RZIV and 0 and 1% EEP for RZSP, and analyzed at regular intervals for sensory evaluation and shelf life by analyzing the following microbiological parameters: Total bacterial count (TBC), Enterobacteriaceae, Pseudomonas spp., and lactic acid bacteria. The results of the disk diffusion assay showed good antibacterial activity of the EEP against all L. monocytogenes strains at all concentrations tested. The MIC obtained for L. monocytogenes ranged from 0.25 for the ATCC strains to 0.625 mg/ml for the three EURL strains, while the MBC was the same as the MIC for two of the three EURL strains and 1.25 mg/ml for all other strains. Regarding the challenge test, after 14 days in the RZIV, a significant difference in the growth of L. monocytogenes was observed between the control sample (Δ +6.10 log CFU/g) and the sample treated with 1% EEP (+3.84 log CFU/g). In the RZSP, slower pathogen growth was observed, with a Δ of the control sample (+2.78 log CFU/g) and the samples treated with 1% EEP (+1.04 log CFU/g). Regarding the influence on shelf life, differences were observed for both CBT (-1.25 log CFU/g) and lactic acid bacteria (-0.81 log CFU/g) only in the RZSP samples treated with 1% EEP. Finally, in the samples treated with 1%, a decrease in sensory impact over time was observed. The results of this study highlighted that EEP possesses moderate anti-L. monocytogenes activity, showing promising results even in sweetened ricotta treated with 1% EEP. Therefore, plant extracts could represent a valid natural alternative to the common additives and treatments used industrially for managing the risk of this pathogen.