International Journal of Food Microbiology 126 (2008) 65–70

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International Journal of Food Microbiology

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Photoactivated chlorophyllin-based gelatin films and coatings to prevent microbialcontamination of food products

G. López-Carballo a, P. Hernández-Muñoz a, R. Gavara a, M.J. Ocio a,b,⁎a Institute of Agrochemistry and Food Technology (IATA), CSIC, Apartado Correos 73, 46100 Burjassot Valencia, Spainb Department of Preventive Medicine, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain

⁎ Corresponding author. Department of Preventive MUniversity 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. Aldoi:10.1016/j.ijfoodmicro.2008.05.002

A B S T R A C T

A R T I C L E I N F O

Article history:

The aim of this work was to Received 10 March 2008Received in revised form 29 April 2008Accepted 1 May 2008

Keywords:Active packagingAntimicrobial packagingFood safetyPorphyrinsFoodborne pathogens

develop antimicrobial photosensitizer-containing edible films and coatings basedon gelatin as the polymer matrix, incorporating sodium magnesium chlorophyllin (E-140) and sodiumcopper chlorophyllin (E-141). Chlorophyllins were incorporated into the gelatin film-forming solution andthe inhibiting effect of the cast films was tested against Staphylococcus aureus and Listeria monocytogenes.The results demonstrated that water soluble sodium magnesium chlorophyllin and water soluble sodiumcopper chlorophyllin reduced the growth of S. aureus and L. monocytogenes by 5 log and 4 log respectively.Subsequently, the activity of self-standing films and coatings containing E-140 was assessed on cookedfrankfurters inoculated with S. aureus and L. monocytogenes. These tests showed that it was possible toreduce microorganism growth in cooked frankfurters inoculated with S. aureus and L. monocytogenes bycovering them with sodium magnesium chlorophyllin-gelatin films and coatings.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The incidence of foodborne diseases associated with microbialpathogens is widespread and represents a threat to public health, anda challenge for the food industry. Foodborne pathogens are generallyeliminated by standard thermal treatments during processing; hence,microbial contamination of processed food is mostly limited to thefood surface as a result of the post-processing steps, where the risk ofcross-contamination involving food, personnel, equipment and theprocessing environment greatly increases.

Considerable efforts are underway to find effective treatments tocontrol this recontamination of meat and poultry products in order toenhance their safety without compromising their quality. Applicationsof antimicrobial films and coatings to food have received considerableattention in recent years because theycanact asprotectivebarriers againstmicrobiological contamination of food products (Suppakul et al., 2003;Cagri et al., 2004). Besides, edible coatings and films can be composed ofpolysaccharides, proteins, and lipids, which extend the shelf-life of foodsby functioning as solute, gas, and vapor barriers (Gennadios et al., 1997).

Ediblefilmsand coatings can serve as carriers for awide rangeof foodadditives, including various antimicrobials that can extend productshelf-life and reduce the risk of pathogen growth on food surfaces (Cagriet al., 2004). The use of generally recognized as safe (GRAS) chemical

edicine, Faculty of Pharmacy,

l rights reserved.

antimicrobials incorporated into processed meat formulations, appliedas dipping solutions or sprayed on the surface of the finished producthas become a common practice to control foodborne pathogens. Inrecent years,much attention has been paid to the use of bacterial startercultures, biopreservatives and plant extracts as antimicrobial hurdles(Zhu et al., 2005).

Porphyrin molecules are of interest due to their antimicrobialability. When the molecules are activated by visible light in thepresence of air, they generate singlet oxygen and free radicals that arecytotoxic to most live cells (Romanova et al., 2003). When these short-life free radicals are in close proximity to the cell surface, they cantrigger extensive cell damage, resulting in cell death.

Chlorophyllins are semi-synthetic porphyrins obtained fromchlorophyll. They are used as food colorants, in dietary supplements,in cosmetics, as an internal deodorant and as an accelerant inwound healing (Kephart, 1955). Anti-oxidative, anti-mutagenic andanti-carcinogenic properties have been reported for chlorophyllin(Dashwood,1997; Ferruzzi et al., 2002; Kapiotis et al., 2005). Althoughdifferent classes of porphyrins have been tested against gram-positiveand gram-negative bacteria (Nitzan et al., 1983; Nitzan et al., 1987;Merchat et al., 1996; Kreitner et al., 2001; Reddi et al., 2002), thephotodynamic effect of chlorophyllins does not appear to have beenapplied to inactivating food-contaminating microorganisms. Severalauthors have successfully developed porphyrin films with nylon orcellulose for industrial, household, and medical environment uses(Bozja et al., 2003; Krouit et al., 2006). Nevertheless, the application ofpolymeric films and coatings containing these antimicrobial com-pounds to improving food preservation has not yet been reported.

66 G. López-Carballo et al. / International Journal of Food Microbiology 126 (2008) 65–70

The objective of this work was to develop and apply novelantimicrobial photosensitizer-containing edible films and coatingsbased on gelatin as the polymer matrix. The inhibiting effect of thesefilms and coatings was tested against Staphylococcus aureus and Listeriamonocytogenes 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 andSalmonella spp. CECT 409 were obtained from the Spanish TypeCulture Collection (Valencia, Spain). Strains were stored in TryptoneSoy Broth (TSB) with 20% glycerol at −80 °C until needed. Forexperimental use, the stock cultures were maintained by regularsubculture on agar Tryptone Soy Agar (TSA) slants at 4 °C andtransferred monthly. Prior to inoculation of plates or food systems, aloopful of each strainwas transferred to 10mL of TSB and incubated at37 °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 watersoluble sodium copper chlorophyllin, E-141, were obtained fromROHA EPSA S. L. Porphyrin stock solutions were prepared with steriledeionized 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 film and coating preparation

Edible film and coating formulations were prepared by dissolving10 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 μg/mL.For film formation,10 g of film-forming solutionwas poured onto Petridishes and water and ethanol were allowed to evaporate at 23±2 °Cand 50±5% relative humidity for 5 h. The dry films were peeled fromthe casting surface and kept in a desiccator at 23±2 °C and 50±5%relative humidity prior to use. Edible coatings were applied byimmersing sterilized cooked frankfurters for 2 min in the coatingformulation. The frankfurters were then allowed to dry for 30 min atroom temperature and 50% relative humidity. After drying, they werestored at 23 °C and 50% relative humidity prior to the experiments.Control films and coatings were obtained by omitting the addition ofchlorophyllins to the formulation.

2.5. Antimicrobial assay

The antimicrobial effect of photoactivated chlorophyllin-gelatinfilms against S. aureus, L. monocytogenes, E. coli and Salmonella spp.was studied as follows. Cell cultures of each microorganism in theearly-stationary phase, with an optical density of 0.9 at 600 nm, werediluted in 10 mM phosphate-buffered saline (PBS) to obtain asuspension of 108 cells/mL. 100 μL of the previous suspension cellswere spread on the surface of TSA plates. Control and E-140- and E-141-containing gelatin films were placed on the surface of theinoculated TSA plates and irradiated for 5 or 15 min at several fluencerates: 10,000, 30,000 and 50,000 lux. The irradiation was performedwith white light using a system of quartz/halogen lamps equippedwith UV and infrared filters and a fan near the lamps to keep thetemperature below 30 °C during the process. After the photosensitiza-

tion treatment, the TSA plates were incubated at 37 °C for 24 h andbacterial colonies were counted. The results were expressed as thenumber of colony forming units per mL. A further control experimentwas carried out in dark conditions by covering samples of all the filmtreatments with aluminium foil for the same lengths of time beforeincubation as the irradiated samples.

Additionally, the inhibitory effect of 80 μg/mL E-140 chlorophyl-lin-gelatin films on early-stationary phase cells of S. aureus andL. monocytogenes inoculated on slices of cooked frankfurters wastested. Using a sterile L-shaped glass rod, 50 μL samples containing106 cells were spread across the surface of cooked frankfurter slices.After inoculation, samples were held for 10 min to allow sorption ofthe microorganisms tested. The sample surfaces were then wrapped inE-140 chlorophyllin edible films and exposed to 30,000 lux for 15 min.After photosensitization, the cooked frankfurters were homogenized ina stomacher (Masticator Delabo IUL instruments) with 10 mL of peptonewater for 5 min. Serial dilutions in 0.1% peptone water were made andthe microorganism suspensions were plated on TSA. Colonies werecounted after incubation at 37 °C for 24 h.

Finally, the inhibitory effect of E-140 chlorophyllin-gelatin coatingson the viability of S. aureus and L. monocytogenes inoculated on meatproducts was studied. Cooked frankfurter slice surfaces were immersedfor 2min in anedible coating solution anddriedat room temperature for30 min. They were then superficially inoculated with 50 μL of themicroorganism suspension (106 CFU/mL), and exposed to 30,000 lux for15 min. After photosensitization, the cooked frankfurters were homo-genized as described above. A control experiment was carried out indark conditions by covering samples of with aluminium foil.

2.6. Color measurements

Film color was determined with a hand-held Minolta ® ChromaMeter CR300 (Minolta Camera Co., Ltd., Osaka, Japan) set to D65illuminant/10° observer. The film specimenswere placed on the surfaceof a white standard plate and the CIELAB color space was used todetermine the parameters: L⁎ (0 black to 100 white), a⁎ (−greenness to+redness; b⁎(−blueness to +yellowness). Hue angle (H⁎) was calculatedas tan−1 (b⁎/a⁎), and chroma (C⁎) as (a⁎2+b⁎2)1/2. Five measurementswere taken of each sample, using three samples of each film. The filmswere conditioned at 50% relative humidity for 48 h prior to testing.

Sausage surface color was measured at five different locations on atotal of ten samples for each treatment. The total color difference (ΔE)between uncoated and coated frankfurters was determined by theformula (ΔL⁎2+Δa⁎2+Δb⁎2)1/2.

2.7. Statistical analysis

All the experiments were conducted in triplicate. The data weresubjected to an analysis of main effects and interaction factors bymultifactorial ANOVA using the StatGraphics plus 5.1 program.Differences betweenmeanswere considered significant when p≤0.05.

3. Results and discussion

3.1. Antimicrobial activity of chlorophyllins immobilized in gelatin filmsunder laboratory conditions

The phototoxic effect of chlorophyllin films against S. aureus,L. monocytogenes, E. coli and Salmonella spp. is shown in Table 1. Theresults indicate that photoactivated chlorophyllins immobilized ingelatin film have a strong bactericidal effect on the viability of Gram-positive bacteria. S. aureus growth was reduced by 5 log and 4 log bychlorophyllin E-140 and E-141 respectively. Similar results wereobtained when stationary phase cultures of L. monocytogenes wereexposed to the two chlorophyllins. When the microorganisms that hadnot been exposed to light were incubated, there was no loss of viability

Table 1Susceptibilities of several foodborne pathogens to chlorophyllins E-140 and E-141 incorporated into gelatin films; gelatin films without chlorophyllins were used as controls

Microorganism CFU/mL

Dark Light

Gelatin film Gelatin+E-140 film Gelatin+E-141 film Gelatin film Gelatin+E-140 film Gelatin+E-141 film

S. aureus 3.4 107±0.40a 3.8 107±0.34 3.2 107±0.33 2.3 107±0.87 1.3 102±0.70 4.0 103±0.13L. monocytogenes 3.8 107±0.19 3.0 107±0.11 4.5 107±0.24 3.9 107±0.79 1.4 102±0.79 2.8 103±0.82E. coli 5 107±0.43 3.5 107±0.87 3.9 107±0.67 4.5 107±0.26 3.5 107±0.44 4.9 107±0.75Salmonella spp. 6.3 107±0.31 5.3 107±0.52 4.6 107±0.38 4.9 107±0.17 4.5 107±0.66 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.

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with any treatment tested. When cells were irradiated in contact withthe gelatin filmwithout photosensitizers no loss of viability was found.These two control tests indicated, firstly, that the porphyrins used werenot in themselves toxic to the microorganisms and secondly, that thevisible light was not responsible for the observed bactericidal effect.

While L monocytogenes and S. aureus cells treated with photosensi-tizers were shown to be successfully inactivated, no photosensitizertreatment showed any effect on the viability of E. coli and Salmonellacells (Table 1). These discrepancies could be explained by differences incell wall structure between Gram-positive and Gram-negative bacteria.Gram-positive bacteria lack an outer membrane, allowing singlet

Fig. 1. Example of photoinactivation of S. aureuswhen exposed to chlorophyllin E-141 incorp1.75 cm2). Petri dishes with E-141 films (left), and films without photosensitizer (right). Cel

oxygen to come into contact with sensitive bacterial targets located inthe inner membrane, but Gram-negative bacteria are mainly protectedfrom damage by their outer membrane (Romanova et al., 2007; Bozjaet al., 2003).These results are in agreementwithBozja et al. (2003) studyof antimicrobial activity against S. aureus and E. coli using specificporphyrins grafted to nylon fibers. The authors found that the anti-microbial system they had developed was very effective for killingS. 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 film (left) and gelatin film(right), exposed to light and incubated at 37 °C for 24 h. As can be seen

orated in (a) gelatin film (triangle area 4.5 cm2) and (b) polyethylene film (circle area ofls irradiated at 30,000 lux for 15 min and incubated in darkness.

Table 2Cytotoxicity of gelatin filmswith E-140 and E-141 against S. aureus and L. monocytogenesat several illumination intensities and different exposure times

Bacteria Time(min)

Illumination(lux)

Gelatin film(CFU/mL)

Gelatin+E-140film (CFU/mL)

Gelatin+E-141film (CFU/mL)

S .aureus 5 Control 3.1 107±0.80a 3.0 107±0.35 2.4 107±0.9310,000 2.7 107±0.48 6.9 103±0.19 1.5 106±0.2430,000 2.3 107±0.87 1.3 102±0.70 4.0 103±0.1350,000 1.8 107±0.21 N.G. 2.0 103±0.36

15 Control 3.1 107±0.41 3.0 107±0.52 2.4 107±0.1510,000 2.0 107±0.22 N.G. 2.8 104±0.7030,000 2.0 107±0.17 N.G. 1.4 103±0.8150,000 1.9 107±0.30 N.G. N.G.

L. monocytogenes 5 Control 4.1 107±0.26 3.3 107±0.74 3.5 107±0.6410,000 3.1 107±0.88 1.5 104±0.48 2.2 106±0.7730,000 3.9 107±0.79 1.4 102±0.79 2.8 103±0.8250,000 2.4 107±0.27 N.G. N.G.

15 Control 4.1 107±0.44 3.3 107±0.30 3.5 107±0.8710,000 3.3 107±0.18 4.8 103±0.39 3.5 104±0.6030,000 3.1 107±0.40 0.3 101±0.55 2.8 103±0.3350,000 3.3 107±0.51 N.G. N.G.

CFU: unit forming colonies.N.G.: no growth observed.

a Data are presented as mean of 3 values±standard deviation.

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from the figure, in the gelatin film samples there were innumerablecolonies after incubation, ruling out any suffocation of the microorgan-isms caused by the presence of the film. However, practically no growthwas observed in the case of E-141 chlorophyllin-gelatin films, whichwould suggest that the chlorophyllin was uniformly distributed inthe film and that singlet oxygen molecules diffused regularly into theinoculated agar. Taking into account that the free radicals and singletoxygen generated by the chlorophyllins during light exposure can onlytravel a very small distance because of their short lifetime, only cellmolecules close to the photosensitizer would be affected. Therefore thepossible explanation of this strong bactericidal effect could be thatgelating film is not longer visible after incubation allowing theinteractions between the single oxygen and the bacteria on the agarsurface.

To check this hypothesis, an independent experiment was carriedout using polyethylene film, a non-polar hydrophobic polymericmaterial, as a polymer matrix to contain oil-soluble E-141 as theantimicrobial agent. Thesefilmswere obtained by compressionmoldingat 180 °C. Even though chlorophyll and chlorophyll derivatives arereasonably heat-stable, a sample of the porphyrinwas heated in a vial at180 °C for 5 min and its light-induced antimicrobial activity against S.aureus was confirmed by direct exposure. Fig. 1b shows that microbialgrowth took place under both the polyethylene films and the E-141

Fig. 2. Antimicrobial activity of E-140 immobilized in gelatin films and coatings on

chlorophyllin-polyethylene films after light exposure. The great differ-ences in antimicrobial effect observed between the E-141 in gelatin andthat in thepolyethylenematrixcouldbecausedby thedifferentbehaviorof these polymer matrixes under the experimental conditions. Thegelatin films with and without chlorophyllin were absorbed onto theculture surface after 24 h in contact with the inoculated plate, (possiblydue to the highwater activity of the agarmedium). In these samples, thefilmswere no longer discernible (Fig.1a). The polyethylene films, on thecontrary, did not seem to be affected by the high humidity of the TSAplate (see example in Fig. 1b), as they preserved their originalmorphology throughout the experiment, indicating minimum interac-tion with the inoculated surface. Apparently, only in the case of thegelatin film could the singlet oxygen reach the cell membrane andinteract with different components, leading to cell death.

In order to study the optimal efficiency of chlorophyllin-gelatinfilms in terms of food safety, the antimicrobial effect on S. aureus andL. monocytogenes of E-140 and E-141 incorporated into gelatin films wastested at different setting of light intensities and periods of time byrecovering the survival bacteria in the most favorable microbialgrowth conditions after each treatment. Table 2 summarizes the resultsobtained when these microorganisms were exposed to different lightintensities and exposure times. The cell viability of bothmicroorganismsdecreased with greater light intensity at both of the illumination timestested. Greater bactericidal effectwas observedwith the longer exposuretime, as cell viability dropped considerably. Thus, the results demon-strated that after 15min treatmentwith the E-140-gelatin film therewasno S. aureus growth, irrespective of the illumination intensity applied. ForS. aureus cells treated with E-141-gelatin films, a longer exposure periodand higher light intensity were required to obtain a similar inhibitoryeffect to that observed with E-140. Similar results were obtained withL. monocytogenes, although the bactericidal effect achieved was weaker.The statistical analysis clearly showed that chlorophyllin E-140 exerted agreater inhibition effect than chlorophyllin E-141 on the cell viability ofthemicroorganisms tested. Differences in the antibacterial activity of thetwo chlorophyllins could be due to E-140 having a greater ability than E-141 to generate singlet oxygen under the experimental conditions tested.This analysis also showed that the exposure timewas themost influentialfactor on the viability of the cells, from which it was concluded thatexposure time plays a key role in the photodynamic effect.

3.2. Antimicrobial activity of chlorophyllins immobilized in gelatin filmsand coatings applied on cooked frankfurters

For the active system to be effective, an adequate chlorophyllin activ-ation procedure is necessary in order to assure that the antimicrobial

cooked frankfurters. Microorganism tested: a) S. aureus b) L. monocytogenes.

Table 3Color coordinates and total color difference of gelatin films and uncoated and coatedcooked frankfurters

Samples L⁎ a⁎ b⁎ ΔE

FilmsGelatin 88.1±0.6a −1.02±0.07 0.5±0.1 –

Gelatin+E-140 84.7±1.4 −3.2±0.2 7.2±0.5 –

Gelatin+E-141 86.6±0.5 −4.4±0.5 4.6±1.1 –

CoatingsFrankfurter (uncoated) 53.2±0.7 14.2±0.5 33.8±1 –

Gelatin 53.4±1.0 14.3±0.7 34.4±0.6 1.3±0.6Gelatin+E-140 53.4±1.5 13.4±0.6 39.3±1.2 5.8±1.5Gelatin+E-141 53.4±0.4 12.3±0.6 36.7±1.1 2.6±0.4

a Data are presented as mean of 3 values±standard deviation.

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compound can exert its biocide effect on the surface of the food. In thisstudy, two different methods were used to test the efficiency of selectedporphyrins in decreasing microbial growth on the surface of cookedfrankfurters:wrappingor coating the food samplewith E-140-containinggelatin films and coatings. Both procedures were successful, althoughthe results of the antimicrobial studies were slightly different. The E-140gelatin film was placed on the surface of a previously inoculated frank-furter sample as described in the Materials and methods section. As canbe seen from Fig. 2, the combined treatment of wrapping with E-140gelatin film and illumination produced an approximately 1.5 log fallin the cell viability of S. aureus (Fig. 2a) and L. monocytogenes (Fig. 2b).No bactericidal effect was evident in either the gelatin film withoutchlorophyllin or the non-irradiated film with chlorophyllin.

The E-140 gelatin coating formulation also yielded successfulresults (Fig. 2). In this case, a decrease of about 2 log in S. aureus(Fig. 2a) and L. monocytogenes (Fig. 2b) populations were found afterthe photosensitization treatment. However, the antimicrobial resultwas less effective in cooked frankfurters than in laboratory conditions.The effectiveness of placing an antimicrobial film directly over foods

Fig. 3. Hue and chroma coordinates of edible

may often be reduced by the presence of substances that interact withit, so inactivating or reducing its antimicrobial effect. Franklin et al.(2004) developed hot dog packaging with 10,000 and 7500 IU/mLnisin and significantly decreased the L. monocytogenes populations onthe surface of the hot dogs, by 2 log CFU per package, throughout the60-day study (Franklin et al., 2004). Starting from natural productssuch cellulose, protoporphyrin IX, and lauric acid, Krouit et al. (2006)developed protoporphyrinated plastic films in which the dye unitswere covalently bound to the cellulose polymer; they observed inhi-bition growth of S. aureus and E. coli below the film (Krouit et al.,2006). The main disadvantage of their system is that the porphyrinthey used cannot be applied to food preservation. However, it is safe touse chlorophyllin films, as chlorophyllin E-140 and E-141 are foodadditives.

Another feasible explanation of the decrease in antimicrobial effectwhen tested in food matrices could be associated with the distinctiveexperimental procedure undertaken. A homogenization step in a sto-macher was required to process the food samples, serial dilutionswere made in 0.1% peptone water and suspensions of microorganismswere plated on TSA. Consequently, the cooked frankfurters, alongwith the films or coatings, were no longer in the system duringmicroorganism incubation at 37 °C.When the samples were processedunder laboratory conditions, however, after their exposure to the lightthe inoculated plates with the film covering the bacteria wereincubated in the dark, allowing bacteria-bound sensitizers to remainundisturbed. This might suggest that some reactive species could stillbe being generated on the film surface during the resting periodafforded by this incubation. Indeed, a comparable reduction in thebactericidal effect was obtained when the experiment was carried outwith the agar plate simulating the food matrix, as the decrease inviable count registered after the stomacher step was only about 2 log(data not shown). Merzlyak (Merzlyak et al., 1996) suggested thatphotodegradation of chlorophylls results in opening of the porphyrinring and formation of a number of intermediate and final products.Nonetheless, little is known about the chemical structures of these

films and coated and uncoated sausages.

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products because of the diversity of the pathways of complicatedphotochemical transformations (Juzenas et al., 2001; Rotomskis et al.,1996).

3.3. Color properties

Color plays an important role in the acceptability of food sinceconsumers tend to judge the quality of food by its color. In general,films intended for use as edible coatings should be transparent andachromatic in order not to cause consumer rejection. Table 3 showsthe L⁎a⁎b⁎ coordinates of the edible films and uncoated and coatedfrankfurters, and the total color differences between coated anduncoated frankfurters. As can be observed, gelatin films present goodtransparency, indicated by high lightness values, and are almostcolorless regarding the values of the a⁎ and b⁎ coordinates. Theincorporation of chlorophyllin into the polymermatrix gave rise to thedevelopment of films of similar transparency but with a vivid green-yellow color compared with the gelatin films. Higher hue angles wereachieved by films containing E-141 chlorophyllin, which acquired agreener color, while films with E-140 developed a yellower color.In addition, chroma values were somewhat higher for the films withE-140 chlorophyllin (Fig. 3). The lightness of the frankfurters was notmodified by the coating applications and no significant differenceswere found between the hue angle and chroma of uncoatedfrankfurters and frankfurters coated with gelatin. However, frankfur-ters coated with gelatin containing chlorophyllins presented slightlyhigher values of hue angle and chroma, as shown in Fig. 3. Nosignificant differences were found between the hue angles offrankfurters coated with the gelatins containing E-140 and E-141chlorophyllins. Differences in chroma were more pronouncedbetween uncoated frankfurters and frankfurters coated with gelatincontaining E-140 chlorophyllin, and the latter also presented highertotal color difference values.

Therefore, the authors can conclude that the results of this studysuggest these photoactivated antimicrobial films could be applied aswraps or castings for fresh and processed products in order to avoidcontamination on the surface of foods and therefore contributing toincrease the shelf-life and safety of the food products. In addition, byusing low concentrations of chlorophyllin incorporated into gelatinfilm as a carrier, minimal impact on the visual sensory properties ofthe food is observed.

Acknowledgements

The authors would like to thank I. Galdeano for technical support.They are also grateful for the financial support of the GeneralitatValenciana (Project GV04B-194) and the Spanish Ministry of Educa-tion and Science (project AGL2003-07326-C02-01).

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