International Journal of Food Microbiology 126 (2008) 6570

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Photoactivated chlorophyllin-based gelatin lms and coatings to prevent microbial contamination of food products
G. Lpez-Carballo a, P. Hernndez-Muoz a, R. Gavara a, M.J. Ocio a,b,
a b

Institute of Agrochemistry and Food Technology (IATA), CSIC, Apartado Correos 73, 46100 Burjassot Valencia, Spain Department of Preventive Medicine, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain

A R T I C L E

I N F O

A B S T R A C T
The aim of this work was to develop antimicrobial photosensitizer-containing edible lms and coatings based on gelatin as the polymer matrix, incorporating sodium magnesium chlorophyllin (E-140) and sodium copper chlorophyllin (E-141). Chlorophyllins were incorporated into the gelatin lm-forming solution and the inhibiting effect of the cast lms was tested against Staphylococcus aureus and Listeria monocytogenes. The results demonstrated that water soluble sodium magnesium chlorophyllin and water soluble sodium copper chlorophyllin reduced the growth of S. aureus and L. monocytogenes by 5 log and 4 log respectively. Subsequently, the activity of self-standing lms and coatings containing E-140 was assessed on cooked frankfurters inoculated with S. aureus and L. monocytogenes. These tests showed that it was possible to reduce microorganism growth in cooked frankfurters inoculated with S. aureus and L. monocytogenes by covering them with sodium magnesium chlorophyllin-gelatin lms and coatings. 2008 Elsevier B.V. All rights reserved.

Article history: Received 10 March 2008 Received in revised form 29 April 2008 Accepted 1 May 2008 Keywords: Active packaging Antimicrobial packaging Food safety Porphyrins Foodborne pathogens

1. Introduction The incidence of foodborne diseases associated with microbial pathogens is widespread and represents a threat to public health, and a challenge for the food industry. Foodborne pathogens are generally eliminated by standard thermal treatments during processing; hence, microbial contamination of processed food is mostly limited to the food surface as a result of the post-processing steps, where the risk of cross-contamination involving food, personnel, equipment and the processing environment greatly increases. Considerable efforts are underway to nd effective treatments to control this recontamination of meat and poultry products in order to enhance their safety without compromising their quality. Applications of antimicrobial lms and coatings to food have received considerable attention in recent years because they can act as protective barriers against microbiological contamination of food products (Suppakul et al., 2003; Cagri et al., 2004). Besides, edible coatings and lms can be composed of polysaccharides, proteins, and lipids, which extend the shelf-life of foods by functioning as solute, gas, and vapor barriers (Gennadios et al., 1997). Edible lms and coatings can serve as carriers for a wide range of food additives, including various antimicrobials that can extend product shelf-life and reduce the risk of pathogen growth on food surfaces (Cagri et al., 2004). The use of generally recognized as safe (GRAS) chemical

Corresponding author. Department of Preventive Medicine, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain. E-mail address: ajo@iata.csic.es (M.J. Ocio). 0168-1605/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.05.002

antimicrobials incorporated into processed meat formulations, applied as dipping solutions or sprayed on the surface of the nished product has become a common practice to control foodborne pathogens. In recent years, much attention has been paid to the use of bacterial starter cultures, biopreservatives and plant extracts as antimicrobial hurdles (Zhu et al., 2005). Porphyrin molecules are of interest due to their antimicrobial ability. When the molecules are activated by visible light in the presence of air, they generate singlet oxygen and free radicals that are cytotoxic to most live cells (Romanova et al., 2003). When these shortlife free radicals are in close proximity to the cell surface, they can trigger extensive cell damage, resulting in cell death. Chlorophyllins are semi-synthetic porphyrins obtained from chlorophyll. They are used as food colorants, in dietary supplements, in cosmetics, as an internal deodorant and as an accelerant in wound healing (Kephart, 1955). Anti-oxidative, anti-mutagenic and anti-carcinogenic properties have been reported for chlorophyllin (Dashwood, 1997; Ferruzzi et al., 2002; Kapiotis et al., 2005). Although different classes of porphyrins have been tested against gram-positive and gram-negative bacteria (Nitzan et al., 1983; Nitzan et al., 1987; Merchat et al., 1996; Kreitner et al., 2001; Reddi et al., 2002), the photodynamic effect of chlorophyllins does not appear to have been applied to inactivating food-contaminating microorganisms. Several authors have successfully developed porphyrin lms with nylon or cellulose for industrial, household, and medical environment uses (Bozja et al., 2003; Krouit et al., 2006). Nevertheless, the application of polymeric lms and coatings containing these antimicrobial compounds to improving food preservation has not yet been reported.

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The objective of this work was to develop and apply novel antimicrobial photosensitizer-containing edible lms and coatings based on gelatin as the polymer matrix. The inhibiting effect of these lms and coatings was tested against Staphylococcus aureus and Listeria monocytogenes in laboratory conditions and in cooked frankfurters. 2. Materials and methods 2.1. Bacterial strains and growth conditions S. aureus CECT 86, L. monocytogenes CECT 934, E. coli CECT 515 and Salmonella spp. CECT 409 were obtained from the Spanish Type Culture Collection (Valencia, Spain). Strains were stored in Tryptone Soy Broth (TSB) with 20% glycerol at 80 C until needed. For experimental use, the stock cultures were maintained by regular subculture on agar Tryptone Soy Agar (TSA) slants at 4 C and transferred monthly. Prior to inoculation of plates or food systems, a loopful of each strain was transferred to 10 mL of TSB and incubated at 37 C for 18 h to obtain early-stationary phase cells. 2.2. Preparation of the porphyrin solutions Water soluble sodium magnesium chlorophyllin, E-140, and water soluble sodium copper chlorophyllin, E-141, were obtained from ROHA EPSA S. L. Porphyrin stock solutions were prepared with sterile deionized water and stored at 4 C in dark conditions until used. 2.3. Preparation of cooked frankfurters for sampling Cooked frankfurters were purchased from a local supermarket. Cooked frankfurter slice surfaces of about 2 cm2 were wiped with 70% ethanol-sterile deionized water solution and exposed to UV light for 1 h. 2.4. Antimicrobial edible lm and coating preparation Edible lm and coating formulations were prepared by dissolving 10 g of gelatin (Sigma) in 100 mL of 50% (v/v) aqueous ethanol. Glycerol (2.5 g) was gently stirred into the formulation as a plasticizer. Chlorophyllin E-140 or E-141 was added to the solution at 80 g/mL. For lm formation, 10 g of lm-forming solution was poured onto Petri dishes and water and ethanol were allowed to evaporate at 23 2 C and 50 5% relative humidity for 5 h. The dry lms were peeled from the casting surface and kept in a desiccator at 23 2 C and 50 5% relative humidity prior to use. Edible coatings were applied by immersing sterilized cooked frankfurters for 2 min in the coating formulation. The frankfurters were then allowed to dry for 30 min at room temperature and 50% relative humidity. After drying, they were stored at 23 C and 50% relative humidity prior to the experiments. Control lms and coatings were obtained by omitting the addition of chlorophyllins to the formulation. 2.5. Antimicrobial assay

tion treatment, the TSA plates were incubated at 37 C for 24 h and bacterial colonies were counted. The results were expressed as the number of colony forming units per mL. A further control experiment was carried out in dark conditions by covering samples of all the lm treatments with aluminium foil for the same lengths of time before incubation as the irradiated samples. Additionally, the inhibitory effect of 80 g/mL E-140 chlorophyllin-gelatin lms on early-stationary phase cells of S. aureus and L. monocytogenes inoculated on slices of cooked frankfurters was tested. Using a sterile L-shaped glass rod, 50 L samples containing 106 cells were spread across the surface of cooked frankfurter slices. After inoculation, samples were held for 10 min to allow sorption of the microorganisms tested. The sample surfaces were then wrapped in E-140 chlorophyllin edible lms and exposed to 30,000 lux for 15 min. After photosensitization, the cooked frankfurters were homogenized in a stomacher (Masticator Delabo IUL instruments) with 10 mL of peptone water for 5 min. Serial dilutions in 0.1% peptone water were made and the microorganism suspensions were plated on TSA. Colonies were counted after incubation at 37 C for 24 h. Finally, the inhibitory effect of E-140 chlorophyllin-gelatin coatings on the viability of S. aureus and L. monocytogenes inoculated on meat products was studied. Cooked frankfurter slice surfaces were immersed for 2 min in an edible coating solution and dried at room temperature for 30 min. They were then supercially inoculated with 50 L of the microorganism suspension (106 CFU/mL), and exposed to 30,000 lux for 15 min. After photosensitization, the cooked frankfurters were homogenized as described above. A control experiment was carried out in dark conditions by covering samples of with aluminium foil. 2.6. Color measurements Film color was determined with a hand-held Minolta Chroma Meter CR300 (Minolta Camera Co., Ltd., Osaka, Japan) set to D65 illuminant/10 observer. The lm specimens were placed on the surface of a white standard plate and the CIELAB color space was used to determine the parameters: L (0 black to 100 white), a ( greenness to +redness; b( blueness to +yellowness). Hue angle (H) was calculated as tan 1 (b/a), and chroma (C) as (a2 + b2)1 / 2. Five measurements were taken of each sample, using three samples of each lm. The lms were conditioned at 50% relative humidity for 48 h prior to testing. Sausage surface color was measured at ve different locations on a total of ten samples for each treatment. The total color difference (E) between uncoated and coated frankfurters was determined by the formula (L2 + a2 + b2)1 / 2. 2.7. Statistical analysis All the experiments were conducted in triplicate. The data were subjected to an analysis of main effects and interaction factors by multifactorial ANOVA using the StatGraphics plus 5.1 program. Differences between means were considered signicant when p 0.05. 3. Results and discussion

The antimicrobial effect of photoactivated chlorophyllin-gelatin lms against S. aureus, L. monocytogenes, E. coli and Salmonella spp. was studied as follows. Cell cultures of each microorganism in the early-stationary phase, with an optical density of 0.9 at 600 nm, were diluted in 10 mM phosphate-buffered saline (PBS) to obtain a suspension of 108 cells/mL. 100 L of the previous suspension cells were spread on the surface of TSA plates. Control and E-140- and E141-containing gelatin lms were placed on the surface of the inoculated TSA plates and irradiated for 5 or 15 min at several uence rates: 10,000, 30,000 and 50,000 lux. The irradiation was performed with white light using a system of quartz/halogen lamps equipped with UV and infrared lters and a fan near the lamps to keep the temperature below 30 C during the process. After the photosensitiza-

3.1. Antimicrobial activity of chlorophyllins immobilized in gelatin lms under laboratory conditions The phototoxic effect of chlorophyllin lms against S. aureus, L. monocytogenes, E. coli and Salmonella spp. is shown in Table 1. The results indicate that photoactivated chlorophyllins immobilized in gelatin lm have a strong bactericidal effect on the viability of Grampositive bacteria. S. aureus growth was reduced by 5 log and 4 log by chlorophyllin E-140 and E-141 respectively. Similar results were obtained when stationary phase cultures of L. monocytogenes were exposed to the two chlorophyllins. When the microorganisms that had not been exposed to light were incubated, there was no loss of viability

G. Lpez-Carballo et al. / International Journal of Food Microbiology 126 (2008) 6570 Table 1 Susceptibilities of several foodborne pathogens to chlorophyllins E-140 and E-141 incorporated into gelatin lms; gelatin lms without chlorophyllins were used as controls Microorganism CFU/mL Dark Gelatin lm S. aureus L. monocytogenes E. coli Salmonella spp. 3.4 107 0.40a 3.8 107 0.19 5 107 0.43 6.3 107 0.31 Gelatin + E-140 lm 3.8 107 0.34 3.0 107 0.11 3.5 107 0.87 5.3 107 0.52 Gelatin + E-141 lm 3.2 107 0.33 4.5 107 0.24 3.9 107 0.67 4.6 107 0.38 Light Gelatin lm 2.3 107 0.87 3.9 107 0.79 4.5 107 0.26 4.9 107 0.17 Gelatin + E-140 lm 1.3 102 0.70 1.4 102 0.79 3.5 107 0.44 4.5 107 0.66

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Gelatin + E-141 lm 4.0 103 0.13 2.8 103 0.82 4.9 107 0.75 4.2 107 0.93

Light treatment was 30,000 lux for 5 min. CFU: unit forming colonies. a Data are presented as mean of 3 values standard deviation.

with any treatment tested. When cells were irradiated in contact with the gelatin lm without photosensitizers no loss of viability was found. These two control tests indicated, rstly, that the porphyrins used were not in themselves toxic to the microorganisms and secondly, that the visible light was not responsible for the observed bactericidal effect. While L monocytogenes and S. aureus cells treated with photosensitizers were shown to be successfully inactivated, no photosensitizer treatment showed any effect on the viability of E. coli and Salmonella cells (Table 1). These discrepancies could be explained by differences in cell wall structure between Gram-positive and Gram-negative bacteria. Gram-positive bacteria lack an outer membrane, allowing singlet

oxygen to come into contact with sensitive bacterial targets located in the inner membrane, but Gram-negative bacteria are mainly protected from damage by their outer membrane (Romanova et al., 2007; Bozja et al., 2003).These results are in agreement with Bozja et al. (2003) study of antimicrobial activity against S. aureus and E. coli using specic porphyrins grafted to nylon bers. The authors found that the antimicrobial system they had developed was very effective for killing S. aureus but had no effect on the E. coli bacteria at any light intensity. Fig. 1a shows a photograph of TSA plates inoculated with S. aureus, covered with E-141 chlorophyllin-gelatin lm (left) and gelatin lm (right), exposed to light and incubated at 37 C for 24 h. As can be seen

Fig. 1. Example of photoinactivation of S. aureus when exposed to chlorophyllin E-141 incorporated in (a) gelatin lm (triangle area 4.5 cm2) and (b) polyethylene lm (circle area of 1.75 cm2). Petri dishes with E-141 lms (left), and lms without photosensitizer (right). Cells irradiated at 30,000 lux for 15 min and incubated in darkness.

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Table 2 Cytotoxicity of gelatin lms with E-140 and E-141 against S. aureus and L. monocytogenes at several illumination intensities and different exposure times Bacteria S .aureus Time Illumination Gelatin lm (min) (lux) (CFU/mL) 5 Control 10,000 30,000 50,000 Control 10,000 30,000 50,000 Control 10,000 30,000 50,000 Control 10,000 30,000 50,000 3.1 107 0.80a 2.7 107 0.48 2.3 107 0.87 1.8 107 0.21 3.1 107 0.41 2.0 107 0.22 2.0 107 0.17 1.9 107 0.30 4.1 107 0.26 3.1 107 0.88 3.9 107 0.79 2.4 107 0.27 4.1 107 0.44 3.3 107 0.18 3.1 107 0.40 3.3 107 0.51 Gelatin+E-140 Gelatin+E-141 lm (CFU/mL) lm (CFU/mL) 3.0 107 0.35 6.9 103 0.19 1.3 102 0.70 N.G. 3.0 107 0.52 N.G. N.G. N.G. 3.3 107 0.74 1.5 104 0.48 1.4 102 0.79 N.G. 3.3 107 0.30 4.8 103 0.39 0.3 101 0.55 N.G. 2.4 107 0.93 1.5 106 0.24 4.0 103 0.13 2.0 103 0.36 2.4 107 0.15 2.8 104 0.70 1.4 103 0.81 N.G. 3.5 107 0.64 2.2 106 0.77 2.8 103 0.82 N.G. 3.5 107 0.87 3.5 104 0.60 2.8 103 0.33 N.G.

15

L. monocytogenes 5

15

CFU: unit forming colonies. N.G.: no growth observed. a Data are presented as mean of 3 values standard deviation.

from the gure, in the gelatin lm samples there were innumerable colonies after incubation, ruling out any suffocation of the microorganisms caused by the presence of the lm. However, practically no growth was observed in the case of E-141 chlorophyllin-gelatin lms, which would suggest that the chlorophyllin was uniformly distributed in the lm and that singlet oxygen molecules diffused regularly into the inoculated agar. Taking into account that the free radicals and singlet oxygen generated by the chlorophyllins during light exposure can only travel a very small distance because of their short lifetime, only cell molecules close to the photosensitizer would be affected. Therefore the possible explanation of this strong bactericidal effect could be that gelating lm is not longer visible after incubation allowing the interactions between the single oxygen and the bacteria on the agar surface. To check this hypothesis, an independent experiment was carried out using polyethylene lm, a non-polar hydrophobic polymeric material, as a polymer matrix to contain oil-soluble E-141 as the antimicrobial agent. These lms were obtained by compression molding at 180 C. Even though chlorophyll and chlorophyll derivatives are reasonably heat-stable, a sample of the porphyrin was heated in a vial at 180 C for 5 min and its light-induced antimicrobial activity against S. aureus was conrmed by direct exposure. Fig. 1b shows that microbial growth took place under both the polyethylene lms and the E-141

chlorophyllin-polyethylene lms after light exposure. The great differences in antimicrobial effect observed between the E-141 in gelatin and that in the polyethylene matrix could be caused by the different behavior of these polymer matrixes under the experimental conditions. The gelatin lms with and without chlorophyllin were absorbed onto the culture surface after 24 h in contact with the inoculated plate, (possibly due to the high water activity of the agar medium). In these samples, the lms were no longer discernible (Fig. 1a). The polyethylene lms, on the contrary, did not seem to be affected by the high humidity of the TSA plate (see example in Fig. 1b), as they preserved their original morphology throughout the experiment, indicating minimum interaction with the inoculated surface. Apparently, only in the case of the gelatin lm could the singlet oxygen reach the cell membrane and interact with different components, leading to cell death. In order to study the optimal efciency of chlorophyllin-gelatin lms in terms of food safety, the antimicrobial effect on S. aureus and L. monocytogenes of E-140 and E-141 incorporated into gelatin lms was tested at different setting of light intensities and periods of time by recovering the survival bacteria in the most favorable microbial growth conditions after each treatment. Table 2 summarizes the results obtained when these microorganisms were exposed to different light intensities and exposure times. The cell viability of both microorganisms decreased with greater light intensity at both of the illumination times tested. Greater bactericidal effect was observed with the longer exposure time, as cell viability dropped considerably. Thus, the results demonstrated that after 15 min treatment with the E-140-gelatin lm there was no S. aureus growth, irrespective of the illumination intensity applied. For S. aureus cells treated with E-141-gelatin lms, a longer exposure period and higher light intensity were required to obtain a similar inhibitory effect to that observed with E-140. Similar results were obtained with L. monocytogenes, although the bactericidal effect achieved was weaker. The statistical analysis clearly showed that chlorophyllin E-140 exerted a greater inhibition effect than chlorophyllin E-141 on the cell viability of the microorganisms tested. Differences in the antibacterial activity of the two chlorophyllins could be due to E-140 having a greater ability than E141 to generate singlet oxygen under the experimental conditions tested. This analysis also showed that the exposure time was the most inuential factor on the viability of the cells, from which it was concluded that exposure time plays a key role in the photodynamic effect. 3.2. Antimicrobial activity of chlorophyllins immobilized in gelatin lms and coatings applied on cooked frankfurters For the active system to be effective, an adequate chlorophyllin activation procedure is necessary in order to assure that the antimicrobial

Fig. 2. Antimicrobial activity of E-140 immobilized in gelatin lms and coatings on cooked frankfurters. Microorganism tested: a) S. aureus b) L. monocytogenes.

G. Lpez-Carballo et al. / International Journal of Food Microbiology 126 (2008) 6570 Table 3 Color coordinates and total color difference of gelatin lms and uncoated and coated cooked frankfurters Samples Films Gelatin Gelatin + E-140 Gelatin + E-141 Coatings Frankfurter (uncoated) Gelatin Gelatin + E-140 Gelatin + E-141
a

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L 88.1 0.6a 84.7 1.4 86.6 0.5

a 1.02 0.07 3.2 0.2 4.4 0.5

b 0.5 0.1 7.2 0.5 4.6 1.1

53.2 0.7 53.4 1.0 53.4 1.5 53.4 0.4

14.2 0.5 14.3 0.7 13.4 0.6 12.3 0.6

33.8 1 34.4 0.6 39.3 1.2 36.7 1.1

1.3 0.6 5.8 1.5 2.6 0.4

Data are presented as mean of 3 values standard deviation.

compound can exert its biocide effect on the surface of the food. In this study, two different methods were used to test the efciency of selected porphyrins in decreasing microbial growth on the surface of cooked frankfurters: wrapping or coating the food sample with E-140-containing gelatin lms and coatings. Both procedures were successful, although the results of the antimicrobial studies were slightly different. The E-140 gelatin lm was placed on the surface of a previously inoculated frankfurter sample as described in the Materials and methods section. As can be seen from Fig. 2, the combined treatment of wrapping with E-140 gelatin lm and illumination produced an approximately 1.5 log fall in the cell viability of S. aureus (Fig. 2a) and L. monocytogenes (Fig. 2b). No bactericidal effect was evident in either the gelatin lm without chlorophyllin or the non-irradiated lm with chlorophyllin. The E-140 gelatin coating formulation also yielded successful results (Fig. 2). In this case, a decrease of about 2 log in S. aureus (Fig. 2a) and L. monocytogenes (Fig. 2b) populations were found after the photosensitization treatment. However, the antimicrobial result was less effective in cooked frankfurters than in laboratory conditions. The effectiveness of placing an antimicrobial lm directly over foods

may often be reduced by the presence of substances that interact with it, so inactivating or reducing its antimicrobial effect. Franklin et al. (2004) developed hot dog packaging with 10,000 and 7500 IU/mL nisin and signicantly decreased the L. monocytogenes populations on the surface of the hot dogs, by 2 log CFU per package, throughout the 60-day study (Franklin et al., 2004). Starting from natural products such cellulose, protoporphyrin IX, and lauric acid, Krouit et al. (2006) developed protoporphyrinated plastic lms in which the dye units were covalently bound to the cellulose polymer; they observed inhibition growth of S. aureus and E. coli below the lm (Krouit et al., 2006). The main disadvantage of their system is that the porphyrin they used cannot be applied to food preservation. However, it is safe to use chlorophyllin lms, as chlorophyllin E-140 and E-141 are food additives. Another feasible explanation of the decrease in antimicrobial effect when tested in food matrices could be associated with the distinctive experimental procedure undertaken. A homogenization step in a stomacher was required to process the food samples, serial dilutions were made in 0.1% peptone water and suspensions of microorganisms were plated on TSA. Consequently, the cooked frankfurters, along with the lms or coatings, were no longer in the system during microorganism incubation at 37 C. When the samples were processed under laboratory conditions, however, after their exposure to the light the inoculated plates with the lm covering the bacteria were incubated in the dark, allowing bacteria-bound sensitizers to remain undisturbed. This might suggest that some reactive species could still be being generated on the lm surface during the resting period afforded by this incubation. Indeed, a comparable reduction in the bactericidal effect was obtained when the experiment was carried out with the agar plate simulating the food matrix, as the decrease in viable count registered after the stomacher step was only about 2 log (data not shown). Merzlyak (Merzlyak et al., 1996) suggested that photodegradation of chlorophylls results in opening of the porphyrin ring and formation of a number of intermediate and nal products. Nonetheless, little is known about the chemical structures of these

Fig. 3. Hue and chroma coordinates of edible lms and coated and uncoated sausages.

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products because of the diversity of the pathways of complicated photochemical transformations (Juzenas et al., 2001; Rotomskis et al., 1996). 3.3. Color properties Color plays an important role in the acceptability of food since consumers tend to judge the quality of food by its color. In general, lms intended for use as edible coatings should be transparent and achromatic in order not to cause consumer rejection. Table 3 shows the Lab coordinates of the edible lms and uncoated and coated frankfurters, and the total color differences between coated and uncoated frankfurters. As can be observed, gelatin lms present good transparency, indicated by high lightness values, and are almost colorless regarding the values of the a and b coordinates. The incorporation of chlorophyllin into the polymer matrix gave rise to the development of lms of similar transparency but with a vivid greenyellow color compared with the gelatin lms. Higher hue angles were achieved by lms containing E-141 chlorophyllin, which acquired a greener color, while lms with E-140 developed a yellower color. In addition, chroma values were somewhat higher for the lms with E-140 chlorophyllin (Fig. 3). The lightness of the frankfurters was not modied by the coating applications and no signicant differences were found between the hue angle and chroma of uncoated frankfurters and frankfurters coated with gelatin. However, frankfurters coated with gelatin containing chlorophyllins presented slightly higher values of hue angle and chroma, as shown in Fig. 3. No signicant differences were found between the hue angles of frankfurters coated with the gelatins containing E-140 and E-141 chlorophyllins. Differences in chroma were more pronounced between uncoated frankfurters and frankfurters coated with gelatin containing E-140 chlorophyllin, and the latter also presented higher total color difference values. Therefore, the authors can conclude that the results of this study suggest these photoactivated antimicrobial lms could be applied as wraps or castings for fresh and processed products in order to avoid contamination on the surface of foods and therefore contributing to increase the shelf-life and safety of the food products. In addition, by using low concentrations of chlorophyllin incorporated into gelatin lm as a carrier, minimal impact on the visual sensory properties of the food is observed. Acknowledgements The authors would like to thank I. Galdeano for technical support. They are also grateful for the nancial support of the Generalitat Valenciana (Project GV04B-194) and the Spanish Ministry of Education and Science (project AGL2003-07326-C02-01).

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