*3.1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Chlorophyllins*

Antimicrobial activity of E-140 and E-141 chlorophyllins was tested against *L. monocytogenes* and *E. coli* using two types of irradiation: halogen and LED lights. The results obtained are shown in Table 1 (halogen light) and Table 2 (LED light).


**Table 1.** Minimum inhibitory concentration (MIC in mg/mL) and minimum bactericidal concentration (MBC in mg/mL) of chlorophyllins E-140 and E-141 against *L. monocytogenes* and *E. coli* activated by halogen light (15 min).

**Table 2.** Minimum inhibitory concentration (MIC in mg/mL) and minimum bactericidal concentration (MBC in mg/mL) of chlorophyllins E-140 and E-141 against *L. monocytogenes* and *E. coli* activated by LED light (15 min).


The first important data were that the MIC values were higher against *E. coli* than against *L. monocytogenes*. In general, Gram-positive bacteria are more sensitive than Gram-negative bacteria to chemical compounds owing to the protection of the outer membrane surrounding the Gram-negative cell wall, which restricts the diffusion of antimicrobial substances [12,13]. Caires et al. [14] reported on the antimicrobial activity of two photosensitizers—eosin methylene blue and chlorophyllin sodium copper salt (E-141)—and showed that eosin methylene blue was able to photoinactivate *E. coli* and *S. aureus*, while E-141 was only active against *S. aureus*. In that test, the maximum concentration tested was 50 μM, which was much lower than the 4.15 mM obtained in this study. However, it is difficult to compare the published results because the important parameters of the photoactivation process—intensity, time, and type of radiation—were either different or were not provided. In this test, 15 min was enough to induce photoactivation, much less than the 120 min employed elsewhere [14].

It is also important to note that although both chlorophyllins presented high antimicrobial activity, porphyrin E-140 was much more effective (with much lower MIC and MBC values) than E-141. With regard to the type of photoactivation, halogen lights were more effective than LED lights. The use of LEDs slightly reduced the efficacy of E-140 against *L. monocytogenes* and *E. coli*. Other authors have reported the antimicrobial effectiveness of zinc complexes of tetrakis (*N*-methylpyridinium-4-yl) tetraiodide porphyrin and tetrakis (*N*-methylpyridinium-4-yl) tetraiodide phthalocyanine impregnated in paper using an inexpensive consumer LED lamp as activation mechanism [9].

In the case of E-141, porphyrin appeared to be inactive when exposed to the radiation of LED lamps, and neither the MBC nor the MIC of the two tested bacteria was found. Our hypothesis is that the LED lamps employed did not emit radiation of sufficient intensity in the wavelength range necessary for E-141 activation. Moreover, copper chlorophyillins have been reported to present low yield of singlet oxygen [15].

Finally, two control tests were carried out. In one, the two microorganisms were exposed to the chlorophyllins in darkness; in the other, they were exposed to radiation without porphyrin. The two microorganisms tested reflected the absence of antimicrobial effect, indicating that chlorophyllins were nontoxic for the two model bacteria, and light radiation alone did not produce antimicrobial activity.

### *3.2. Development of Coated Films*

G, PE, PVOH, and HPMC coatings on PET film with and without 1% of E-140 or E-141 were successfully obtained. They were homogeneous, without discontinuities, flexible, transparent, had a light green color when the chlorophyllins were added, and with thicknesses that are shown

in Table 3. As can be seen, the coating thicknesses varied according to the polymer material as a consequence of the different solid content of the film-forming solution and the polymer density; the PE-based materials were the thickest, while PVOH and HPMC were the thinnest. The incorporation of chlorophyllins in the coatings did not significantly affect thickness.


**Table 3.** Thicknesses (μm) of films and coatings with and without chlorophyllin.

The most significant effect of the addition of the chlorophyllins to the coatings was the yellowish (E-140) or greenish (E-141) color induced. Table 4 shows the color coordinates in the CIELAB system for different coatings, including chromaticity (*C*\*) and tone (*h*). As can be seen, in general, the coated films presented high luminosity as revealed by the *L*\* values ranging between 83 and 90. The only exceptions were the coatings based on PE-incorporated chlorophyllins, which presented a considerable reduction in luminosity. The addition of E-140 provided coatings with a yellowish color, which is characterized in CIELAB coordinates by positive *b*\* values and low negative *a*\* values. As a consequence of this, the tone of the coatings was characterized by values ranging between 95 and 110◦. The incorporation of E-141 provided a greener color, with higher negative *a*\* values and positive *b*\* and tone values ranging between 107 and 135◦. With respect to saturation or chromaticity, the values were greater for E-141 samples than for E-140. Comparing the polymeric materials, *C*\* values were greatest for the thickest material (PE).

**Table 4.** Color coordinates *L*\*, *a*\*, *b*\*, chromaticity (*C*\*) and tone (*h* in ◦) of films coated with gelatin (G), polyvinyl alcohol (PVOH), polyethylene (PE), (hydroxypropyl)methyl cellulose (HPMC) without (control) and with active agents (E-140 and E-141).


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

Figure 2 shows, as examples, photos of the different coatings. As can be seen, in the PE coatings there was a clearly visible alteration of the color of the material after incorporation of both chlorophyllins. On the other hand, the color of the HPMC coatings with E-140 or E-141 was hardly distinguishable from the control.

**Figure 2.** Images of the PET films coated with G, PVOH, HPMC, and PE for the control and the films containing chlorophyllins (E-140 and E-141).
