**2. Materials and Methods**

#### *2.1. Reactive Agents*

Water-soluble sodium magnesium chlorophyllin, E-140, and water-soluble sodium copper chlorophyllin, E-141, were obtained from Natracol (ROHA Europe S.L.U., Torrente, Spain). Gelatin from porcine skin (G) and (hydroxypropyl) methyl cellulose (HPMC) were supplied by Sigma (Barcelona, Spain). Low-density polyethylene (LDPE) emulsion (50% *w*/*w*) Aquaseal® 2200 (PE) was kindly provided by Paramelt B.V. (Heerhugowaard, The Netherlands), and Gohsenol AH17 polyvinyl alcohol was kindly provided by the Nippon Synthetic Chemical Company (Osaka, Japan).

#### *2.2. Bacterial Strains*

Bacterial strains were obtained from the Spanish Type Culture Collection (Valencia, Spain): *L. monocytogenes* CECT 911 (ATCC 19112) was used as a model of a Gram-positive bacterium and *Escherichia coli* CECT 434 (ATCC 25922) as a model of a Gram-negative bacterium. The strains were stored in Tryptone soy broth (TSB) with glycerol at −80 ◦C. For experimental use, the stock cultures were maintained by regular subculture on agar medium slants at 4 ◦C and transferred every month. A loopful of each strain was transferred to 10 mL of TSB and incubated at 37 ◦C overnight to obtain early stationary phase cells.

Bacteria were spread on selective media, PALCAM agar and Mueller Hinton agar, for *L. monocytogenes* and *E. coli*, respectively.

#### *2.3. Methods*

#### 2.3.1. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of porphyrin were determined for *E. coli* and *L. monocytogenes*. The MIC is defined as the amount or concentration of active compound in which growth inhibition is observed. The MBC is the concentration in which there is no growth of microorganisms [10]. The procedure, which we have called the "drops method", consisted of adding 100 μL of microorganism (107 CFU/mL) and 10 μL of chlorophyllin prepared at different concentrations ranging between 0.0016 mg/mL and 15 mg/mL. Chlorophyllins were activated by two lighting systems: three 500 W Haloline 64702 halogen lamps (Osram) or five 50 W 81.575/Dia LED lights (Electro DH) for 15 min. Their light spectra differ greatly: LED has its maximum radiation in a thin band close to 450 nm and a medium-intensity wide band with a maximum at 550 nm and an unimportant contribution in the infrared region, while halogen light increases in intensity with wavelength, having its maximum in the infrared region [11]. As control samples, identical experiments were also carried out with (a) samples with chlorophyllins stored in dark conditions and (b) an illuminated sample without chlorophyllins. After photosensitization, the suspension of microorganisms was recovered, plated in agar medium, and incubated overnight at 37 ◦C. Cytotoxicity was calculated as the difference between colony forming units (CFU) counted with (photosensitizer and illumination) and without photodynamic treatment. Experiments were carried out in triplicate.

#### 2.3.2. Antimicrobial Coating Preparation

Various matrices were employed as chlorophyllin carriers: gelatin (G), polyethylene (PE), polyvinyl alcohol (PVOH), and (hydroxypropyl) methyl cellulose (HPMC).

G coating-forming solution was prepared by dissolving 10 g of gelatin in 100 mL of 50% (v/v) aqueous ethanol for 2 h at 75 ◦C and adding 25% of glycerol (with respect to polymer dry mass) as plasticizer.

PE coating emulsion was prepared from a commercial emulsion, previously homogenized for 10 min in an ultrasonic bath. Then, 5% of 2-propanol was added to improve wettability, and the mixture was stirred for 10 min using a magnetic stirrer at room temperature.

PVOH coating-forming solution was prepared by dissolving 4 g in 100 mL of deionized water with 10% of glycerol as plasticizer and stirring for 2 h at 75 ◦C.

HPMC coating-forming solution was prepared by dissolving 2.5 g of HPMC in 100 mL of 50% (v/v) aqueous ethanol with 20% of glycerol as plasticizer and stirring for 2 h at 75 ◦C.

G, PE, PVOH, and HPMC coatings were prepared on a 30 μm poly (ethylene terephthalate) (PET) film. The PET film was set on a glass surface and corona-treated (Model BD-20V corona treater, Electro-Technic Products, Chicago, IL, USA) to improve coating adherence. Ten grams of the film-forming solutions was spread using a 50 μm extension bar (Linlab, Logroño, Spain), and the samples were placed in a homemade drying tunnel equipped with a 2500 W heat source and a 30 W fan for 3 min until they were completely dry. Coating thickness was calculated as the difference between the film thicknesses measured with a micrometer before and after the coating process. Finally, the coatings were stored in glass desiccators containing silica gel at 22 ± 2 ◦C prior to use.

#### 2.3.3. Color Measurements

The color of the coated films was measured using a Konica Minolta CM-3500d spectrophotometer (Konica Minolta Business Technologies, Inc., Tokyo, Japan) set to D65 illuminant/10◦ observer. Film samples were measured against the surface of a standard white plate, and the CIELAB color space

was used to obtain the color coordinates *L*\* (lightness) [black (0) to white (100)], *a*\* [green (−) to red (+)], and *b*\* [blue (−) to yellow (+)]. The color was expressed using the polar coordinates *L*\*, *C*\*, *h*◦, and Δ*E*\*, where *L*\* is the same as above, *C*\* is chroma, *h*◦ is hue angle, and Δ*E*\* = [(Δ*L*\*)<sup>2</sup> + (Δ*a*\*)<sup>2</sup> + (Δ*b*\*)2] 1/2. Ten measurements were taken of each sample, and three samples of each film were measured.

#### 2.3.4. Antimicrobial Activity of Coatings

#### Agar Diffusion

First, 100 μL of a bacterial suspension containing approximately 103 CFU/mL was spread on agar medium, and then a disk of film (2.5 cm diameter) was put over the agar with the coating surface facing the agar. Samples with and without chlorophyllins were irradiated with five 50 W LED lights for 15 min. At the same time, samples with and without active agent were treated in dark conditions by covering the Petri dishes with aluminum foil.

All the plates were incubated at 37 ◦C for 24 h, and the diameter of the resulting inhibition zone in the bacterial lawn was measured. The experiment was carried out in triplicate.
