3.3.1. Agar Diffusion Method

The antimicrobial capacity of G, PVOH, PE, and HPMC films incorporating 1% of chlorophyllin E-140 was studied against *L. monocytogenes* and *E. coli* using the agar diffusion method. The results obtained showed that the G, PVOH, and HPMC films had a bactericidal effect against both microorganisms, while the PE film had a certain degree of inhibition, as can be seen in Table 5.


**Table 5.** Microbiological counts (log CFU/mL) on the surface of the agar in the area under the coated films and antimicrobial effect expressed as log reduction values (LRV).

The results showed that the most hydrophilic films, i.e., with a higher degree of affinity for water, presented a greater antibacterial capacity. It is possible that the excited oxygen reacts with water molecules to produce hydroxyl radicals that have a longer lifetime and are more active against bacteria [16]. Accordingly, it was observed that photoexcited G, PVOH, and HPMC films with 1% chlorophyllin E-140 had a lethal effect on *L. monocytogenes* or *E. coli* in the area covered by the films. In the case of PE-coated films, a certain degree of inhibition was seen, but it was less than that with the other films, despite the higher amount of porphyrin present owing to the greater thickness of the coating. The absence of water in this hydrophobic polymer may limit the photoactivation to the formation of singlet oxygen, which rapidly reduces to triplet oxygen without interacting with the cells. Another explanation is that the low affinity between chlorophyllin and PE causes itsaggregation, and this results in a lower production of singlet oxygen. The films that developed acted mainly by contact, although in the case of the PVOH, G, and HPMC matrices, an inhibition halo of ca. 5 mm was observed around the films.

Bozja et al. (2003) tested the antimicrobial activity of porphyrins grafted onto nylon-6 fibers with polyacrylic acid. They found that the antimicrobial system developed was very effective against *S. aureus* but had no effect on *E. coli* bacteria at any light intensity [7]. There have been some similar studies, such as the one carried out in 2008 in which the effectiveness of E-140 porphyrin-bearing gelatin films as antibacterial agents [2] was demonstrated against *S. aureus* and *L. monocytogenes*, but was found to have no effect on *Salmonella* spp. and *E. coli* bacteria after photoactivation with 30,000 luxes for five minutes.
