3.3.2. Surface Disinfection

The coatings were inoculated on the surface and then illuminated in order to verify whether they could be self-sanitized by photoactivation. As an example, Figure 3 shows the results obtained with gelatin films contaminated with *Listeria*. As can be seen, there is growth both in the control plate that was exposed to light and in the plate with porphyrin films that was kept in darkness. By contrast, irradiated samples containing porphyrin E-140 showed no microbial growth.

**Figure 3.** Microbial growth of *L. monocytogenes*. Left to right: control G film which had been irradiated, G film with E-140 which had been kept in darkness, and G film with E-140 which had been irradiated.

Similar results were obtained for the other films: HPMC, PVOH, and PE. This shows that the films developed can be self-sanitized in case of possible contamination simply by subjecting them to visible light irradiation for 15 min.

However, no clear bactericidal effect against *E. coli* was observed with any of the coated films that contained E-140. These results are in agreement with other authors who obtained an antimicrobial effect against Gram-positive bacteria using porphyrins grafted onto nylon fibers but found no effect on *E. coli* bacteria at any light intensity [7]. As explained previously, this may be due to the extra protection of the outer membrane in Gram-negative bacteria. The outer membrane of Gram-negative bacteria plays an important role, which is related to their resistance to many active agents that are very effective against Gram-positive bacteria [17]. The resistance of Gram-negative bacteria to photosensitization has been widely reported [18,19]. In order to increase the permeability of *E. coli*, we added 40 mM EDTA (ethylenediaminetetraacetic acid) to the 100 μL dilution of *E. coli*. EDTA is a chelating agent that destabilizes the outer membrane and may facilitate access of free radicals generated with light to bacterial cells [20]. However, we did not observe significant differences between the controls and the treatment with light and porphyrin. Other authors have employed dimethyl sulfoxide (5%) to increase the permeability of *E. coli* to porphyrins without achieving the same results as with Gram-positive bacteria [14].

#### *3.4. Migration of Chlorophyllins from Coatings to the Food Simulant*

The release of chlorophyllin E-140 was studied in 50% ethanol as a fatty food simulant in accordance with Directive 85/572/EEC prior to application on bologna slices. The regulation stipulates that the results of overall migration obtained must be in accordance with the overall migration limit of 10 mg/dm2 established in Directive 2002/72/EC and in Royal Decree 866/2008, which are both related to materials and plastic objects in contact with food products. The results are shown in Figure 4.

**Figure 4.** Concentration of E-140 released from G, PVOH, HPMC, and PE coatings on PET in fatty simulant as a function of time.

The release of chlorophyllins from G, PVOH, and HPMC coatings on PET was very low—practically zero—indicating that there was no substantial migration. This means the antimicrobial effect was produced by the generation of free radicals and it was not necessary for the chlorophyllins to migrate to the simulating medium. On the other hand, the PE coatings presented a maximum release of 6.4 mg/dm<sup>2</sup> (according to the surface of each coating, which was 0.125 dm2), which was less than the overall migration limit set by legislation (10 mg/dm2).

In the case of the other coatings, the low migration obtained makes it unnecessary to calculate migration limits, and therefore they can be used as packaging systems for fatty food. In this case, they were tested with bologna slices. The fact that the greatest release was observed in PE could be due to several factors. First, the thickness of the PE coating—well above 25 μm—results in a large amount of porphyrin in absolute values. Second, the poor affinity of LDPE for chlorophyllins might result in their separation, forming a two-phase matrix in which the agent is isolated in small regions dispersed in the pure LDPE matrix. When the material comes into contact with a solvent medium, the porphyrin located close to the surface is immediately released, as observed in Figure 4. In the other matrices there would be specific interactions between E-140 and the polymers, keeping the agent in the package where porphyrin molecules have a compatible chemical environment. Similar release results were observed for films containing E-141 (data not shown).

#### *3.5. Application to Food*

Once the antimicrobial and self-sanitizing capacity of the films had been determined, their effectiveness when applied to a real food was studied. The experiment was carried out with G, HPMC, PE, and PVOH coatings on PET films used as bologna slice separators.

This food product was chosen to test the coatings developed because it has a high water activity (*a*w = 0.970) and is therefore very susceptible to spoilage by microorganisms. The antimicrobial effect of the films was studied against *L. monocytogenes*, which was inoculated on the bologna surface and against the usual microbial load of this meat product. The results are shown in Figure 5.

**Figure 5.** Antimicrobial activity of (**a**) G, (**b**) PVOH, (**c**) PE, and (**d**) HPMC coatings on PET films applied to bologna slices.

There was no microbial growth of *Salmonella* spp., enterobacteria, or coliform bacteria on any sample. Control coatings and coatings kept in darkness showed no antimicrobial effect, confirming the nontoxicity of the active agents without photoactivation. However, coatings with E-140 exposed to LED lights inhibited microbial growth of *L. monocytogenes* successfully. The greatest antimicrobial effect was observed with the G coating and the least with the PE coating. The HPMC and PVOH coatings were slightly less effective than G (Figure 5).

Growth of the microbial load was also inhibited in irradiated samples with chlorophyllin. Lactic, mesophilic, and psychrophilic bacteria and *Pseudomonas* were significantly reduced. Once again, the G coatings presented the greatest antimicrobial effect against all the microorganisms tested. The highest efficiency was observed against psychrophiles and mesophiles, with reductions of 2.87 and 2.30 log, respectively. In the case of lactic acid bacteria and *Pseudomonas*, although the degree of inhibition was lower, it was also considerable. The HPMC and PVOH coatings with chlorophyllin

presented similar degrees of inhibition against all the microorganisms studied, ranging between 0.76 and 1 log. Finally, the antimicrobial effect of the PE coatings was lower, ranging between approximately 0.24 and 0.69 log.

Results showed that all the coatings developed in this study had antimicrobial activity, with gelatin being the most effective matrix. This is possibly due to its morphology that facilitates the release of free radicals produced by the porphyrin molecule, which is responsible for the antimicrobial action. It should also be taken into account that the G coatings were thicker, and therefore there was a greater amount of agent in absolute values than in the HPMC and PVOH coatings. The application of the gelatin coating would be very useful given gelatin is a component that is usually present in many foods and does not constitute a risk when it forms part of the surface of a package intended for food use.

To confirm the absence of effects on the organoleptic properties of the product and because the release of chlorophyllin could produce a green color, the color of the bologna slices was measured before and after the photoactivation treatment. The parameters *L*\* [black (0) to white (100)], *a*\* [green (−) to red (+)], and *b*\* [blue (−) to yellow (+)] were obtained, and the polar coordinates, chroma *C*\*, and hue angle *h* that were calculated are shown in Table 6.

**Table 6.** The color coordinates *L*\*, *a*\*, *b*\*, the chromaticity (*C*\*), and the tone (*h in* ◦) of bologna slices covered with G, PVOH, PE, and HPMC coatings on PET before and after photoactivation.


a–c Different letters in the same column indicate significant differences (Tukey's adjusted analysis of variance, *p* < 0.05).

The bologna slices were displaced towards the + *a* coordinate, i.e., towards red and slightly towards the + *b* coordinate (yellow), which is logical given their rosy hue.

In general, irradiation produced a slight decrease in the luminosity of the bologna slices and a slight shift to the left of the *b*\* coordinates. However, these color modifications were not distinguishable to the naked eye. On the other hand, no color difference was observed between samples with and without porphyrin submitted to the same treatment (darkness or light). Therefore, from the data shown above, it can be confirmed that no substantial release of porphyrin to the bologna took place. Similar results were reported in a study of chlorophyllin gelatin films applied as wraps to frankfurter sausages, which found no substantial differences between uncoated and coated products [2].

Finally, it can be concluded that chlorophyllin-based photosensitization of coated films is an effective way of reducing the population of microorganisms naturally present in meat and poultry products and as a way of improving asepsis of packaging materials through their self-sanitizing

function. Moreover, this efficiency has also been demonstrated when light is applied through the supporting material (in this case, PET) if the material is transparent to visible light. This aspect is of great importance because it would greatly facilitate carrying out its antimicrobial or self-sanitizing function in food products that have already been packaged. The process that has been developed in this study, which requires only white light, could become an alternative food process that is nonchemical, nonthermal, inexpensive, and environmentally friendly.

The films developed could be applied as part of a package intended for meat derivatives—either as an external protective cover or as separators of cold cuts—or in some dairy products, such as fresh cheese. In addition, the films developed could be used to wrap food that are sensitive to microbial contamination in daily use, such as pieces of cooked ham that are not immediately consumed completely but are rather manipulated (cut) on a number of occasions before being completely consumed, therefore making them potential vectors for the transmission of microorganisms. These films could be used to cover the cut surface and protect it from microbial spoilage thanks to the photoactivation induced by the white lights of refrigerated displays in grocery stores. Furthermore, this active packaging does not require any agent release, thereby increasing food shelf life without substantial changes in aroma, flavor, or color.

**Author Contributions:** Conceptualization, P.H.-M. and R.G.; Methodology, G.L.-C.; Validation, P.H.-M., R.G. and G.L.-C.; Formal Analysis, G.L.-C.; Investigation, P.H.-M., R.G. and G.L.-C.; Resources, P.H.-M. and R.G.; Writing, Original Draft Preparation, G.L.-C., R.G. and P.H.-M.

**Funding:** This research was funded by the Ministry of Economy and Competitiveness (No. AGL2015-64595-R).

**Acknowledgments:** The authors acknowledge the assistance of Karel Clapshaw (translation services) and the supply of materials by Paramelt B.V. (Heerhugowaard, The Netherlands) and the Nippon Synthetic Chemical Company (Osaka, Japan).

**Conflicts of Interest:** The authors declare no conflict of interest.
