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The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 μg protein/cm 2 and 5.74 μg protein/cm 2 for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH−lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29−lysozyme, 0.95 log for EVOH 44−lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

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with those of standard PP films. Antimicrobial tests were carried out for enterobacteria, total 20 aerobic counts, yeasts and moulds, and lactic acid bacteria and psychrotrophic bacteria, and the 21 effect of the release of the antimicrobial agent on the sensory characteristics of the salads was 22 also studied. The application of the EVOH coating results in an increase in the tensile resistance 23 of the PP films and a reduction in the elongation at break. The results showed that 24 microorganism counts bacteria decreased especially at the beginning of the storage period. OEO 25 and CITRAL samples had reductions of 1.38 log and 2.13 log respectively against 26 enterobacterias, about 2 log against yeasts and moulds. The total aerobic counts reduced 1.08 27 log with OEO and 1.23 log with CITRAL and the reduction of lactic acid bacteria and 28 psychrotrophic was about 2 log.. Citral-based films appeared to be more effective than materials 29 containing oregano essential oil in reducing spoilage flora during storage time. Sensory studies 30 also showed that the package with citral was the most accepted by customers at the end of the 31 shelf life 32 33 Keywords: minimally processed salads, citral, oregano essential oil, antimicrobial packaging, 34 mechanical properties, EVOH.

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13The aim of the present work was the development of antimicrobial films containing garlic 14 extract to be applied as active packaging for preservative-free sliced pan loaf, with the goal of 15 extending its shelf-life. First, the antimicrobial capacity of garlic extract, a compound used as 16 active agent, was tested against Penicillium expansum by the disc diffusion method. The 17 extract showed high antimicrobial activity, 0.1 µL per Petri dish being the minimum inhibitory 18 amount, and 0.25 µL the minimum fungicidal amount. Bread aroma was also used to mask the 19 pungent odour of garlic and it was confirmed to have no antimicrobial activity. Subsequently, 20 polyethylene (PE) aqueous emulsion and ethylene-vinyl alcohol copolymer (EVOH) and zein 21 hydroalcoholic solutions containing 0.25 and 0.5% (w/w per dry polymer) of garlic extract and 22 34 INTRODUCTION 35Bread is an essential food product in the traditional diet in Europe, the Middle East, India, 36America and Oceania. According to the Codex Alimentarius, bread is the product resulting from 37 baking dough obtained by mixing flour and water, with or without addition of edible salt, 38 fermented by baker's yeast, Saccharomyces cerevisiae. Bread slowly deteriorates after baking, 39owing to a combination of chemical and physical processes called staling and owing to 40 microbiological spoilage. 41Pan loaf is a type of bread baked in a pan, with a soft texture and high water activity 42 (0.94

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