Surface Disinfection

The microorganisms to be tested were spread directly on the G, HPMC, PVOH, and PE coatings (with 1% E-140, and some without antimicrobial agent as controls). For this purpose, the coated films were placed on Petri dishes with the coating facing up and 100 μL of 10<sup>5</sup> CFU/mL of *L. monocytogenes* or *E. coli* was spread over them, ensuring direct contact between the bacteria and the coating. In the case of the *E. coli* bacteria, 20 or 40 mM of ethylenediaminetetraacetic acid (EDTA) was previously added to ease mass transport through the outer membrane. They were then irradiated with 5 LED lights for 15 min. The bacteria were collected with 10 mL of peptone water, and serial dilutions were made and plated on solid agar medium. The incubation was carried out at 37 ◦C for 24 h and CFU/mL was counted. The experiments were carried out in triplicate.

#### 2.3.5. Release of Chlorophyllins

According to Council Directive 85/572/EEC, which determines the list of simulants that should be used to control the migration of components from materials and objects of plastic material intended to come into contact with food products, the simulant that best represents a fatty food such as bologna is vegetable oil. However, since the two chlorophyllins selected are water soluble, we decided to test the films with 50% ethanol, which is a valid simulant for oil-in-water emulsions. In this case, the exposure conditions were 10 mL of simulant and an exposure area of 12.5 cm<sup>2</sup> (5 cm × 2.5 cm rectangles) at a refrigeration temperature of 5 ◦C for 16 days. At 1 h, 2 h, 4 h, 6 h, 24 h, 4 days, 8 days, and 16 days, samples were analyzed at 655 nm by UV-vis spectrometry (Agilent UV-Visible 8453 Spectroscopy System, Agilent Technologies, Wilmington, DE, USA). To quantify the concentrations of migrated porphyrin, a previous calibration with known concentrations in 50% ethanol was prepared.

#### 2.3.6. Application to Bologna Slices

Coatings on PET were used as separators of slices of a real food-bologna. In this test, the antibacterial activity of PVOH, G, PE, and HPMC with 1% porphyrin E-140 coatings was tested against the usual microbial load and against *L. monocytogenes*, which had been previously inoculated. An 800 g piece of classic bologna containing 50% pork and 15% turkey was purchased and sliced. Slices that were 3 mm thick and 6.8 cm in diameter were prepared, with an average weight per slice of 10.12 ± 0.13 g. Slices of bologna were inoculated with 100 μL of 10<sup>5</sup> CFU/mL of *L. monocytogenes* spread over their surface. Then, the active- or control-coated film (with/without porphyrin) was placed on the bologna slices and irradiated for 15 min with 5 LED lamps on a tray with ice to avoid sample overheating (Figure 1). In parallel, control samples with films with and without antimicrobial were kept in darkness for 15 min.

**Figure 1.** Example of test: (**a**) from left to right: bologna slice without coating, sample with a control gelatin (G) coating on poly(ethylene terephthalate) (PET), and sample of G-coated PET containing E-140 porphyrin; (**b**) image of an experiment of treatment during the photoactivation process.

After the treatment, bologna slices were placed in a Stomacher bag together with 10 mL of peptone water and homogenized in a Stomacher (Bagmix, Interscience, St. Nom, France) for 4 min. Serial dilutions were carried out, and the enumeration of particular microbial groups was performed using the following culture conditions and media (from Scharlab, Barcelona, Spain): (a) PALCAM Listeria selective agar for *L. monocytogenes* incubated at 37 ◦C for 48 h; (b) plate count agar for total aerobic plate count, pour-plated and incubated at 30 ◦C for 48 h and plate count agar for total aerobic psychrotrophic count, pour-plated and incubated at 10 ◦C for 10 days; (c) Violet Red Bile Glucose agar for total enterobacteria, pour-plated and incubated at 37 ◦C for 48 h; (d) Eosin Methylene Blue agar for coliform bacteria, pour-plated and incubated at 37 ◦C for 48 h; (e) Brilliant Green agar for *Salmonella* isolation, pour-plated and incubated at 37 ◦C for 48 h; (f) King agar for *Pseudomonas* spp. count, pour-plated and incubated at 25 ◦C for 4 days; (g) De Man, Rogosa and Sharpe agar for lactic bacteria count, pour-plated and incubated at 25 ◦C for 4 days. The counts were performed in triplicate.

Once the antimicrobial activity had been evaluated, the possible impact on the color characteristics of the food due to active agent release was studied. The color of bologna samples before and after irradiation was determined using a Konica Minolta CM-3500d spectrophotometer and the same procedure as described in Section 2.3.3. Eight measurements were taken of each sample, and three samples of each film were measured.
