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Microbiological evaluation of an edible antimicrobial coating on minimally processed carrots
Food Control 17 (2006) 336–341 www.elsevier.com/locate/foodcont
Microbiological evaluation of an edible antimicrobial coating on minimally processed carrots A.M. Durango a
a,b
, N.F.F Soares
a,*
, N.J. Andrade
a
Food Science Department, Universidade Federal de Vic¸osa, 36570-000 Vic¸osa-MG, Brazil b Food Engineering Department, Universidad de Co´rdoba, Monterı´a, Colombia
Received 3 February 2004; received in revised form 23 October 2004; accepted 25 October 2004
Abstract This work aimed to develop an edible antimicrobial coating based on a starch–chitosan matrix to evaluate its effect on minimally processed carrot by means of microbiological analyses. Coatings based on 4% yam starch (w/w) + 2% glycerol (w/w) and coatings based on 4% yam starch (w/w) + 2% glycerol (w/w) + chitosan in 0.5% and 1.5% concentrations were prepared. Samples of minimally processed carrot slices were immersed into these coatings. All the samples were placed in expanded polystyrene trays, wrapped in polyvinylchloride film and stored at 10 C/15 days. During storage, all the samples had counting <100 CFU/g for Staphylococcus aureus and <3 MPN/g for Escherichia coli. Starch + 0.5% chitosan coating controlled the growth of mesophilic aerobes, yeasts and molds and psychrotrophs during the first five days of storage, ultimately presenting reductions of only 0.64, 0.11 and 0.16 log cycles, respectively, compared to the control. Starch + 1.5% chitosan coated samples showed reductions in mesophilic aerobes, mold and yeast and psychrotrophic counting of 1.34, 2.50 and 1.30 log cycles, respectively, compared to the control. The presence of 1.5% chitosan in the coatings inhibited the growth of total coliforms and lactic acid bacteria throughout the storage period. The use of edible antimicrobial yam starch and chitosan coating is a viable alternative for controlling microbiological growth in minimally processed carrot. 2005 Elsevier Ltd. All rights reserved. Keywords: Edible antimicrobial coating; Chitosan; Starch
1. Introduction The greatest losses in food are due to microbiological alterations. Many chemical and physical processes have been developed to preserve food quality. Among such processes, adequate packaging is a fundamental factor in the conservation and marketing phases. Thus, packaging plays a prominent role in maintaining food quality (Debeaufort, Quezada-Gallo, & Voilley, 1998). Antimicrobial films and coatings have innovated the concept of active packaging and have been developed
*
Corresponding author. Address: Food Science Department, Universidade Federal de Vic¸osa, 36570-000 Vic¸osa-MG, Brazil. E-mail address: [email protected] (N.F.F Soares). 0956-7135/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.10.024
to reduce, inhibit or delay the growth of microorganisms on the surface of foods in contact with the packaged product (Appendini & Hotchkiss, 2002). In most fresh or processed foods, microbial contamination occurs at a higher intensity on the food surface, thus requiring an effective microbial growth control (Padgett, Han, & Dawson, 1998). Traditionally, antimicrobial agents are added directly to the foods, but their activity may be inhibited by many substances in the food itself, diminishing their efficiency. In such cases, the use of antimicrobial films or coatings can be more efficient than adding antimicrobial agents directly to the food since these may selectively and gradually migrate from the package onto the surface of the food, thereby high concentrations being maintained when most necessary (Ouattara, Simard, Piette, Be´gin, & Holley, 2000).
A.M. Durango et al. / Food Control 17 (2006) 336–341
Edible antimicrobial films and coatings have shown to be an efficient alternative in controlling food contamination. The growth of both deteriorating and pathogenic microorganisms may be prevented by incorporating antimicrobial agents into edible films or coatings (Debeaufort et al., 1998). The antimicrobial agents most commonly utilized in edible coatings are: sorbic acid, propionic acid, potassium sorbate, benzoic acid, sodium benzoate and citric acid (Quintavalla & Vicini, 2002). Bacteriocins, such as nisin and pediocin (Sebti & Coma, 2002); enzymes, such as peroxidase and lysozyme (Padgett et al., 1998); and polysaccharides displaying natural antimicrobial properties, such as chitosan, (Debeaufort et al., 1998) are also being used as antimicrobial agents. Chitosan, is a polysaccharide obtained by deacetylation chitin, originated from crustacean exoskeleton and fungal cell walls. Chitosan has widely been used in antimicrobial films and coatings due to its property of inhibiting the growth of many pathogenic bacteria and fungi (Romanazzi, Nigro, Ippolito, Di Venere, & Salerno, 2002). In some fungi, chitosan can produce alterations of membrane functions, by interaction with the strongly electronegative microbial surface leading to changes in permeability, metabolic disturbances, and eventually death (Fang, Li, & Shih, 1994). According to Muzzarelli et al. (1990) chitosan antimicrobial activity against bacteria, could be due to the polycationic nature of its molecule, which allows interaction and formation of polyelectrolyte complexes with acid polymers produced at the bacteria cell surface (lipopolysaccharides, teichoic and teichuronic acids or capsular polysaccharides). Chitosan based films and coatings tested on Listeria monocytogenes were found to inhibit the growth of this organism (Coma et al., 2002). El Ghaouth, Ponnampalam, Castaigne, and Arul (1992) showed that coatings based on 1% and 2% chitosan reduced the incidence of tomato deterioration, mainly that caused by Botrytis cinerea. Studies have shown that chitosan based coatings have the potential to increase the shelf life of fresh fruits and vegetables, inhibiting the growth of microorganisms, reduced ethylene production, increased internal carbonic gas and decreased oxygen levels (Lazaridou & Biliaderis, 2002). Among the polysaccharides used in the production of edible packaging, starch is the natural biopolymer most commonly used. Starch can be an interesting alternative for edible films and coatings because this polymer is cheap, abundant, biodegradable, edible and easy of use (Mali, Grossmann, Garcı´a, Martino, & Zaritzky, 2002). Studies carried out by Lawton (1996), show that starch based films and coatings exhibit different properties, attributed to the amylose content in the starch. Yam (Dioscorea sp) could be a good source of starch for the production of edible films and coatings, since
337
its starch contains about 30% of amylose, and amylose is responsible for the film forming capacity of starches (Mali et al., 2002). Carrot (Daucus carota L.) is one of the most popularly consumed vegetables, but marketing is limited by its fast deterioration during storage, due to physiological changes that reduce its shelf life (Peiyin & Barth, 1998). The product loses its firmness and develops odors characteristic of anaerobic catabolism, due to the high respiration rate and microbiological deterioration (Barry-Ryan, Pacussi, & OÕBeirne, 2000). Minimally processed carrot quickly lose their bright orange color during storage, developing a whitish appearance or white blush on its surface (Bolin & Huxsoll, 1991), thereby reducing consumersÕ acceptability. Both microbial proliferation and white blush on the surface of the product can be controlled by application of biopolymer based edible coatings (Cisneros-Zevallos, Saltveit, & Krochta, 1997). The objective of this work was to develop a yam starch and chitosan based edible antimicrobial coating to evaluate its effect on the microbiota normally present in minimally-processed carrot.
2. Materials and methods The experiment was conducted at the Packaging Laboratory of the Universidade Federal de Vic¸osa-MG, Brazil. The experimental design was arranged in a randomized complete design using a split plot scheme, with the variable treatments being coatings 1, 2, 3 and 4 in the plot and the variable times being 0, 5, 10 and 15 days in the split plot, with two repetitions in triplicate. 2.1. Edible coating The coatings were prepared using yam starch (Dioscorea sp) and chitosan. Yam starch was isolated according to Cruz and El Dash methodology (1984). Chitosan (85% deacetylated) was acquired from Padetec (Fortaleza Ceara´–Brazil). The coatings were prepared by heat-gelatinization using suspensions of 4% yam starch (w/w) and 2% glycerol (w/w) (Mali et al., 2002); chitosan was then added in concentrations of 0.5% and 1.5% (w/w) previously dissolved in 0.4% glacial acetic acid (w/w). An agitator (Ultra Turrax T 18 basic) was used at 10.000 rpm for 5 s to homogenize the suspensions. Starch coatings without chitosan were also prepared (Table 1). 2.2. Sample preparation and coating application Carrot (Daucus carota L.) from an unknown cultivar was purchased at the local market, washed, manually peeled and cut into slices (5 mm thick) using a vegetable
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Table 1 Composition of edible antimicrobial coatings used in minimally processed carrots Treatment
Starch (%)
Glycerol (%)
Chitosan (%)
Coating Coating Coating Coating
4.0 4.0 4.0
– 2.0 2.0 2.0
– – 0.5 1.5
1 (control) 2 3 4
3. Results During storage, all the samples showed absence of S. aureus and E. coli with counting of <100 CFU/g and <3 MPN/g, respectively. Coating 4 inhibited the growth of lactic acid bacteria and total coliforms, throughout the storage time presenting counting <10 CFU/g and <3 MPN/g, respectively. Compared to coating 1 (control), coating 4 showed a reduction of 4, 18 and 2.56 log cycles in the growth of lactic acid bacteria and total coliforms, respectively. (Fig. 1). During storage time, the samples with coatings 1, 2 and 3 showed similar acid lactic bacteria and total coliform counts. According to ANOVA analysis of the mesophilic aerobes, yeasts and molds and psychrotrophs count in minimally processed carrots, submitted to four coatings during 15 storage days at 10 C, the coatings and coating-time interaction showed significant differences at 5% probability (Table 2). The starch–1.5% chitosan coated sample (coating 4) showed the lowest counting with 3.88 log CFU/g and a reduction of 1.34 log cycles compared to the control, at the end of the experiment (Fig. 2). The starch coated sample (coating 2) had the highest mesophilic aerobes count during the first 10 days of storage. Although presenting a reduced mesophilic aerobes count when evaluated at 5 storage days, coating 3 failed to control the development of this microbial group, afterwards. At the end of the storage time, coating 3 showed a reduction of only 0.64 log cycles, compared to the control. The antimicrobial activity of the coatings on the yeasts and molds was most efficient in coating 4, containing 1.5% chitosan (Fig. 3). At this concentration, chitosan reduced 2.5 log cycles the counting of this microbiological group in the carrot, after 15 storage days. Coating 3, at a concentration of 0.5% chitosan, controlled the development of yeasts and molds in minimally processed carrot, during the first 5 days of storage. After this time, these samples had counting similar to the control.
processor (Robout Coup). The carrot slices were placed in sanitized nylon bags (200 mg/L active chlorine) and rinsed (3 mg/L active chlorine), at 5 C, for 10 min and centrifuged at 800g for 10 min. Samples containing 80 g of sliced carrots were submerged in different coatings for 3 min and air flow dried at 20 C for 3 h. The control (noncoated carrots) was submerged in sterile distilled water under similar conditions. All the samples were placed in expanded polystyrene trays, wrapped in polyvinylchloride (PVC) film and stored at 10 C for 15 days. 2.3. Microbiological analyses To evaluate the microbiological efficiency of the antimicrobial coatings on the carrot microbiota, microbiological analyses of mesophilic aerobes, total and fecal coliforms, Escherichia coli, Staphylococcus aureus, yeasts and molds, lactic acid bacteria and psychrotrophs were carried out in the coated and noncoated carrot samples at 0, 5, 10 and 15 storage days, according to the methodology described by the Compendium of Methods for the Microbiological Examination of Foods (Vanderzant & Splittstoesser, 1992). At time zero, the coated carrots were analyzed after the treatment. 2.4. Statistical analyses The computer program Statistical Analysis System (SAS) was used in all the statistical analyses. Variance analysis (ANOVA) tests were applied to the split plots and regression analysis was performed at a level of 5% significance.
Coating 1 Coating 3
6
Coating 2 Coating 4
4
Coating 1
Coating 2
Coating 3
Coating 4
Log MPN/g
Log CFU/g
5 4 3 2
3 2 1
1 0
0 0
(A)
5
10 Time (days)
15
0
20
(B)
5
10
15
20
Time (days)
Fig. 1. Effect of coatings on lactic acid bacteria (A) and total coliforms (B) in minimally processed carrot stored at 10 C/15 days.
A.M. Durango et al. / Food Control 17 (2006) 336–341
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Table 2 Summary of variance analysis of mesophilic aerobes, yeasts and molds and psychrotrophic bacteria in minimally processed carrots, submitted to four coatings during 15 days of storage at 10 C FV
GL
QM Mesophilic aerobes
QM Yeasts and molds
QM Psychrotrophs
Coating Residue (a) Time Coating · time Residue (b)
3 4 3 9 12
1.6987** 0.0003 11.1527** 0.3210** 0.0047
6.0143** 0.0009 9.1639** 1.5102** 0.0018
2.6090** 0.0067 13.6104** 0.1697** 0.0013
**Significant at 5% probability (P < 0.05).
6
7
5
6 5 Log CFU/g
Log CFU/g
4
3
2
1
Coating 1
= 2,1258 + 0,2787X – 0,0055 X2 R2 = 0, 93
Coating 2
= 2,5695 + 0,4804X – 0,0224 X2 R2 = 0, 94
Coating 3
= 1,5950 + 0,3700X – 0,0110 X2 R2 = 0, 96
Coating 4
= 1,7213 + 0,3548X – 0,0142 X2 R2 = 0, 99 5 Time (days)
10
3 2 1
0 0
4
15
Coating 1
= 2,3140 + 0,3928X – 0,0118 X2 R2= 0,99
Coating 2
= 3,1265 + 0,4248X – 0,0170 X2 R2= 0,93
Coating 3
= 1,9985 + 0,4582X – 0,0154 X2 R2= 0,99
Coating 4
= 1,9887 + 0,3262X – 0,0114 X2 R2= 0,99
0 0
5
10
15
Time (days)
Fig. 2. Effect of coating on the growth of mesophyll aerobes in minimally processed carrot stored during 15 days at 10 C.
6
5
Coating 1
= 1,7840 + 0,1613X – 0,0023 X2 R2= 0,84
Coating 2
= 2,0525 + 0,1615X – 0,0047 X2 R2= 0,95
Coating 3
= 0,6462 + 0,2802X – 0,0005 X2 R2= 0,81
Coating 4
= 1,0500 + 0,0900X – 0,0100 X2 R2= 0,93
4. Discussion
Log CFU/g
4
3
2
1
0 0
5
10
Fig. 4. Psychrotrophic counting in minimally processed carrot submitted to four coatings during 15 days at 10 C.
15
Time (days)
Fig. 3. Effect of coatings on the growth of filamentous fungi and yeast in minimally processed carrot stored at 10 C for 15 days.
The effect of the coatings on minimally processed carrotÕs psychrotrophic microbiota was greater for coating 4 (Fig. 4), which presented the lowest psychrotrophic counting, 4.3 log CFU/g, at the end of the storage period. Starch coated samples (coating 2) presented the highest psychrotrophic counting throughout the storage period, starting at 3.0 log CFU/g, and reaching 5.8 log CFU/g after 15 days of storage. Coating 3 did not inhibit the growth of this microbiological group, compared to coating 1 (control), with a reduction of 0.16 log cycle at the end of 15 storage days.
Coating 4, consisting of 1.5% chitosan added—starch was the most efficient in controlling the microorganisms evaluated, normally present in minimally processed carrot, stored at 10 C for 15 days. At this concentration, the coating totally inhibited the growth of lactic acid bacteria and total coliforms, during storage time. Lactic acid bacteria have been associated to carrot deterioration (Zagory, 1999). According to Carlin, Nguyen-the, Cudennes, and Reich (1989), deterioration of minimally processed carrot after 14 days storage at 10 C was associated to Leuconostoc mesenteroides populations due to the process of lactic fermentation. Marchetti, Casadei, and Guerzoni (1992), found lactic acid bacteria counting of 3.16 · 105 CFU/g and 108 CFU/g in carrot salad after 7 day storage at 5 C. Coating 4 also had a satisfactory performance in controlling mesophilic aerobes, with a reduction of 1.34 log cycles at the end of the storage period. Such reduction is considerable, when compared to other methods applied to reduce the microbial load in foods. For instance, the sanitation method used in sliced carrot showed a reduction in the number of mesophilic aerobic bacteria of, approximately, one log cycle, using 200 mg/L of chlorine during the process (Silva, 2003). A lower efficiency of this process was found by Amanatidou, Slump, Gorris, and Smid (2000), in sliced carrot with a reduction of only 0.4 log cycle in the microbial
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load of mesophilic aerobes. The number of mesophilic aerobes found by Chervin and Boisseau (1992), in minimally processed carrots was 6.7 log CFU/g. Buick and Damoglou (1987) showed that 70% of the initial microbiota in minimally processed sliced carrot consists of Erwinia ssp, which attacks the carrot, causing the socalled bacterial wet rot (Mu¨eller, 1981). In this work, the coating containing 0.5% chitosan did not present a good performance in controlling deteriorating bacteria, despite the initial low number of mesophilic aerobes, lactic acid bacteria and psychrotrophs found in minimally processed carrot. In 1993, carrot was associated to an infection outbreak caused by enterotoxigenic E. coli in the US. In Lebanon, the prevalence of S. aureus in raw carrot has been 14.3% (Beuchat, 2002). Zheng and Zhu (2003) showed that 1% chitosan at a degree of deacetylation of 88.76% and molecular weight of 48.5 kDa, has an antimicrobial effect on E. coli and S. aureus at an inhibition rate of 100% on these microorganisms growth. Concentration of 1% chitosan presented reductions between 1 and 2 log cycles in the total counting of bacteria in small meat pies; concentrations of 0.2% and 0.5% did not have an inhibiting effect on the deteriorating microflora, but the number of bacteria before starting the experiment was high >107 CFU/g, and chitosan is likely to be more effective in low microbial populations (Darmadji & Izumimoto, 1994). Regard to the action of the coatings on yeasts and molds, coating 4 presented the highest fungicidal action. Studies have shown that the effect of chitosan on some fungi is mainly due to alterations in the functions of the cellular membrane (Fang et al., 1994). The yeasts most commonly isolated from vegetables belong to the genera Cryptococcus, Rhodotorula and Candida; among the moulds are Fusarium, Mucor, Rhizopus and Penicillium (Francis, Thomas, & OÕBeirne, 1999). The main disease caused by fungus in carrots is Sclerotinia rot. Cheah, Page, and Shepherd (1997) showed that chitosan applied as a 2% or 4% solution inhibits Sclerotinia storage rot on carrots, and that the effect was due, at least in part, to inhibition of fungal growth. In this work, coating 4 containing 1.5% chitosan was the most efficient on the psychrothrophic flora in minimally processed carrot. According to Garg, Churey, and Splittstoesser (1990) the shelf life of refrigerated vegetables is especially affected by the psychrotrophic population, with Pseudomonas sp. being the main psychrotrophic agent present in these foods. Several authors have found psychrotrophic counting in minimally processed carrot sold at supermarkets ranging between 106 and 109 UFC/mL (Rosa, 2002). Listeria monocytogenes is one of the pathogens most frequently associated with ready-to-use vegetables (Francis et al., 1999). Studies conducted by Coma et al. (2002) show that chitosan coatings have a bactericidal effect on Lis-
teria monocytogenes and that such activity was probably due to the positive charges of chitosan which interfere with the negatively charged residues of macromolecules at the Listeria cell surface, presumably by competing with calcium for electronegative sites on the membrane. Chitosan has been reported to inhibit various spoilage bacteria, through its capacities to both bind water and inactivate various enzymes, and through its ability to absorb nutrients normally used by bacteria (Ouattara et al., 2000). Coating 2, which contained only starch, favored the development of mesophilic and psychrotrophic bacteria, indicating that these microorganisms may have utilized this carbohydrate as a source of energy. 5. Conclusions The results of this experiment showed that the use of an antimicrobial coating consisting of chitosan-added yam starch is a viable alternative in controlling the microbiota present in minimally processed carrot, since the growth of lactic acid bacteria, total coliforms, psychrotrophs, yeasts and molds and mesophilic aerobes, was substantially inhibited by the application of 1.5% chitosan coating. Based on the concept of protection barrier technology, the use of such coating may contribute to improve safety in minimally processed carrot thereby prolonging its shelf life. Coating may be applied on minimally processed fruits and vegetables, combined to other types of controls, such as quality raw material, hygienic processing conditions and storage temperatures. The combination of these treatments as barrier offers a greater potential for shelf-life extension of minimally processed vegetables. Acknowledgements The authors thank CNPq and FAPEMIG for the financial support. References Amanatidou, A., Slump, R. A., Gorris, L. G. M., & Smid, D. (2000). High oxygen and high carbon dioxide modified atmospheres for shelf-life extension of minimally processed carrots. Journal of Food Science, 65(1), 61–66. Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food packaging. Innovative Food Science and Emerging Technologies, 3, 113–126. Barry-Ryan, C., Pacussi, J. M., & OÕBeirne, D. (2000). Quality of shredded carrots as affected by packaging film and storage temperature. Journal of Food Science, 65(4), 726–730. Beuchat, L. R. (2002). Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection, 4, 413–423.
A.M. Durango et al. / Food Control 17 (2006) 336–341 Bolin, H. R., & Huxsoll, C. C. (1991). Control of minimally processed carrot (Daucus carota) surface discoloration caused by abrasion peeling. Journal of Food Science, 56, 416–418. Buick, R. K., & Damoglou, A. (1987). The effect of vacuum packaging on the microbial spoilage and shelf-life of ready-to-use sliced carrots. Journal Science Food Agriculture, 38, 167–175. Carlin, F., Nguyen-the, C., Cudennes, P., & Reich, M. (1989). Microbiological spoilage of fresh, ready-to-use grated carrots. Science Aliment, 9, 371–386. Cheah, L. H., Page, B. B. C., & Shepherd, R. (1997). Chitosan coating for inhibition of sclerotinia rot in carrots. Journal Crop Horticulture Science, 25, 89–92. Chervin, C., & Boisseau, P. (1992). Quality maintenance of ready-toeat shredded carrots by gamma irradiation. Postharvest Biology and Technology, 2(2), 7–17. Cisneros-Zevallos, L., Saltveit, M. E., & Krochta, J. M. (1997). Hygroscopic coatings control surface white discoloration of peeled (minimally processed) carrots during storage. Journal of Food Science, 62(2), 363–366. Coma, V., MartiaL-Giros, A., Garreau, S., Copinet, A., Salin, F., & Deschamps, A. (2002). Edible antimicrobial films based on chitosan matrix. Journal of Food Science, 67(3), 1162–1168. Cruz, R., & El Dash, A. A. (1984). Isolamento e caracterizac¸a˜o de amido de chuchu. Revista Ceres, 31(175), 173–188. Darmadji, P., & Izumimoto, M. (1994). Effect of chitosan on meat preservation. Meat Science, 38, 243–254. Debeaufort, F., Quezada-Gallo, J. A., & Voilley, A. (1998). Edible films and coatings: tomorrow packaging: a review. Critical Reviews in Food Science, 38(4), 299–313. El Ghaouth, A., Ponnampalam, R., Castaigne, F., & Arul, J. (1992). Chitosan coating to extend the storage life of tomatoes. HortScience, 27(9), 1016–1018. Fang, S. W., Li, C. F., & Shih, D. Y. C. (1994). Antifungal activity of chitosan and its preservative effect on low-sugar candied kumwuat. Journal of Food Protection, 56, 136–140. Francis, G. A., Thomas, C., & OÕBeirne, D. (1999). The microbiological safety of minimally processed vegetables. International Journal of Food Science and Technology, 34, 1–23. Garg, N., Churey, J. J., & Splittstoesser, D. F. (1990). Effect of processing conditions on the microflora of fresh-cut vegetables. Journal Food Protection, 53(8), 701–703. Lawton, J. W. (1996). Effect of starch type on the properties of starch containing films. Carbohydrate Polymers, 29, 203–208. Lazaridou, A., & Biliaderis, C. G. (2002). Thermophysical properties of chitosan–starch and chitosan-pullulan films near the glass transition. Carbohydrate Polymers, 48, 179–190.
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Microbiological evaluation of an edible antimicrobial coating on minimally processed carrots A.M. Durango a
a,b
, N.F.F Soares
a,*
, N.J. Andrade
a
Food Science Department, Universidade Federal de Vic¸osa, 36570-000 Vic¸osa-MG, Brazil b Food Engineering Department, Universidad de Co´rdoba, Monterı´a, Colombia
Received 3 February 2004; received in revised form 23 October 2004; accepted 25 October 2004
Abstract This work aimed to develop an edible antimicrobial coating based on a starch–chitosan matrix to evaluate its effect on minimally processed carrot by means of microbiological analyses. Coatings based on 4% yam starch (w/w) + 2% glycerol (w/w) and coatings based on 4% yam starch (w/w) + 2% glycerol (w/w) + chitosan in 0.5% and 1.5% concentrations were prepared. Samples of minimally processed carrot slices were immersed into these coatings. All the samples were placed in expanded polystyrene trays, wrapped in polyvinylchloride film and stored at 10 C/15 days. During storage, all the samples had counting <100 CFU/g for Staphylococcus aureus and <3 MPN/g for Escherichia coli. Starch + 0.5% chitosan coating controlled the growth of mesophilic aerobes, yeasts and molds and psychrotrophs during the first five days of storage, ultimately presenting reductions of only 0.64, 0.11 and 0.16 log cycles, respectively, compared to the control. Starch + 1.5% chitosan coated samples showed reductions in mesophilic aerobes, mold and yeast and psychrotrophic counting of 1.34, 2.50 and 1.30 log cycles, respectively, compared to the control. The presence of 1.5% chitosan in the coatings inhibited the growth of total coliforms and lactic acid bacteria throughout the storage period. The use of edible antimicrobial yam starch and chitosan coating is a viable alternative for controlling microbiological growth in minimally processed carrot. 2005 Elsevier Ltd. All rights reserved. Keywords: Edible antimicrobial coating; Chitosan; Starch
1. Introduction The greatest losses in food are due to microbiological alterations. Many chemical and physical processes have been developed to preserve food quality. Among such processes, adequate packaging is a fundamental factor in the conservation and marketing phases. Thus, packaging plays a prominent role in maintaining food quality (Debeaufort, Quezada-Gallo, & Voilley, 1998). Antimicrobial films and coatings have innovated the concept of active packaging and have been developed
*
Corresponding author. Address: Food Science Department, Universidade Federal de Vic¸osa, 36570-000 Vic¸osa-MG, Brazil. E-mail address: [email protected] (N.F.F Soares). 0956-7135/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.10.024
to reduce, inhibit or delay the growth of microorganisms on the surface of foods in contact with the packaged product (Appendini & Hotchkiss, 2002). In most fresh or processed foods, microbial contamination occurs at a higher intensity on the food surface, thus requiring an effective microbial growth control (Padgett, Han, & Dawson, 1998). Traditionally, antimicrobial agents are added directly to the foods, but their activity may be inhibited by many substances in the food itself, diminishing their efficiency. In such cases, the use of antimicrobial films or coatings can be more efficient than adding antimicrobial agents directly to the food since these may selectively and gradually migrate from the package onto the surface of the food, thereby high concentrations being maintained when most necessary (Ouattara, Simard, Piette, Be´gin, & Holley, 2000).
A.M. Durango et al. / Food Control 17 (2006) 336–341
Edible antimicrobial films and coatings have shown to be an efficient alternative in controlling food contamination. The growth of both deteriorating and pathogenic microorganisms may be prevented by incorporating antimicrobial agents into edible films or coatings (Debeaufort et al., 1998). The antimicrobial agents most commonly utilized in edible coatings are: sorbic acid, propionic acid, potassium sorbate, benzoic acid, sodium benzoate and citric acid (Quintavalla & Vicini, 2002). Bacteriocins, such as nisin and pediocin (Sebti & Coma, 2002); enzymes, such as peroxidase and lysozyme (Padgett et al., 1998); and polysaccharides displaying natural antimicrobial properties, such as chitosan, (Debeaufort et al., 1998) are also being used as antimicrobial agents. Chitosan, is a polysaccharide obtained by deacetylation chitin, originated from crustacean exoskeleton and fungal cell walls. Chitosan has widely been used in antimicrobial films and coatings due to its property of inhibiting the growth of many pathogenic bacteria and fungi (Romanazzi, Nigro, Ippolito, Di Venere, & Salerno, 2002). In some fungi, chitosan can produce alterations of membrane functions, by interaction with the strongly electronegative microbial surface leading to changes in permeability, metabolic disturbances, and eventually death (Fang, Li, & Shih, 1994). According to Muzzarelli et al. (1990) chitosan antimicrobial activity against bacteria, could be due to the polycationic nature of its molecule, which allows interaction and formation of polyelectrolyte complexes with acid polymers produced at the bacteria cell surface (lipopolysaccharides, teichoic and teichuronic acids or capsular polysaccharides). Chitosan based films and coatings tested on Listeria monocytogenes were found to inhibit the growth of this organism (Coma et al., 2002). El Ghaouth, Ponnampalam, Castaigne, and Arul (1992) showed that coatings based on 1% and 2% chitosan reduced the incidence of tomato deterioration, mainly that caused by Botrytis cinerea. Studies have shown that chitosan based coatings have the potential to increase the shelf life of fresh fruits and vegetables, inhibiting the growth of microorganisms, reduced ethylene production, increased internal carbonic gas and decreased oxygen levels (Lazaridou & Biliaderis, 2002). Among the polysaccharides used in the production of edible packaging, starch is the natural biopolymer most commonly used. Starch can be an interesting alternative for edible films and coatings because this polymer is cheap, abundant, biodegradable, edible and easy of use (Mali, Grossmann, Garcı´a, Martino, & Zaritzky, 2002). Studies carried out by Lawton (1996), show that starch based films and coatings exhibit different properties, attributed to the amylose content in the starch. Yam (Dioscorea sp) could be a good source of starch for the production of edible films and coatings, since
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its starch contains about 30% of amylose, and amylose is responsible for the film forming capacity of starches (Mali et al., 2002). Carrot (Daucus carota L.) is one of the most popularly consumed vegetables, but marketing is limited by its fast deterioration during storage, due to physiological changes that reduce its shelf life (Peiyin & Barth, 1998). The product loses its firmness and develops odors characteristic of anaerobic catabolism, due to the high respiration rate and microbiological deterioration (Barry-Ryan, Pacussi, & OÕBeirne, 2000). Minimally processed carrot quickly lose their bright orange color during storage, developing a whitish appearance or white blush on its surface (Bolin & Huxsoll, 1991), thereby reducing consumersÕ acceptability. Both microbial proliferation and white blush on the surface of the product can be controlled by application of biopolymer based edible coatings (Cisneros-Zevallos, Saltveit, & Krochta, 1997). The objective of this work was to develop a yam starch and chitosan based edible antimicrobial coating to evaluate its effect on the microbiota normally present in minimally-processed carrot.
2. Materials and methods The experiment was conducted at the Packaging Laboratory of the Universidade Federal de Vic¸osa-MG, Brazil. The experimental design was arranged in a randomized complete design using a split plot scheme, with the variable treatments being coatings 1, 2, 3 and 4 in the plot and the variable times being 0, 5, 10 and 15 days in the split plot, with two repetitions in triplicate. 2.1. Edible coating The coatings were prepared using yam starch (Dioscorea sp) and chitosan. Yam starch was isolated according to Cruz and El Dash methodology (1984). Chitosan (85% deacetylated) was acquired from Padetec (Fortaleza Ceara´–Brazil). The coatings were prepared by heat-gelatinization using suspensions of 4% yam starch (w/w) and 2% glycerol (w/w) (Mali et al., 2002); chitosan was then added in concentrations of 0.5% and 1.5% (w/w) previously dissolved in 0.4% glacial acetic acid (w/w). An agitator (Ultra Turrax T 18 basic) was used at 10.000 rpm for 5 s to homogenize the suspensions. Starch coatings without chitosan were also prepared (Table 1). 2.2. Sample preparation and coating application Carrot (Daucus carota L.) from an unknown cultivar was purchased at the local market, washed, manually peeled and cut into slices (5 mm thick) using a vegetable
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Table 1 Composition of edible antimicrobial coatings used in minimally processed carrots Treatment
Starch (%)
Glycerol (%)
Chitosan (%)
Coating Coating Coating Coating
4.0 4.0 4.0
– 2.0 2.0 2.0
– – 0.5 1.5
1 (control) 2 3 4
3. Results During storage, all the samples showed absence of S. aureus and E. coli with counting of <100 CFU/g and <3 MPN/g, respectively. Coating 4 inhibited the growth of lactic acid bacteria and total coliforms, throughout the storage time presenting counting <10 CFU/g and <3 MPN/g, respectively. Compared to coating 1 (control), coating 4 showed a reduction of 4, 18 and 2.56 log cycles in the growth of lactic acid bacteria and total coliforms, respectively. (Fig. 1). During storage time, the samples with coatings 1, 2 and 3 showed similar acid lactic bacteria and total coliform counts. According to ANOVA analysis of the mesophilic aerobes, yeasts and molds and psychrotrophs count in minimally processed carrots, submitted to four coatings during 15 storage days at 10 C, the coatings and coating-time interaction showed significant differences at 5% probability (Table 2). The starch–1.5% chitosan coated sample (coating 4) showed the lowest counting with 3.88 log CFU/g and a reduction of 1.34 log cycles compared to the control, at the end of the experiment (Fig. 2). The starch coated sample (coating 2) had the highest mesophilic aerobes count during the first 10 days of storage. Although presenting a reduced mesophilic aerobes count when evaluated at 5 storage days, coating 3 failed to control the development of this microbial group, afterwards. At the end of the storage time, coating 3 showed a reduction of only 0.64 log cycles, compared to the control. The antimicrobial activity of the coatings on the yeasts and molds was most efficient in coating 4, containing 1.5% chitosan (Fig. 3). At this concentration, chitosan reduced 2.5 log cycles the counting of this microbiological group in the carrot, after 15 storage days. Coating 3, at a concentration of 0.5% chitosan, controlled the development of yeasts and molds in minimally processed carrot, during the first 5 days of storage. After this time, these samples had counting similar to the control.
processor (Robout Coup). The carrot slices were placed in sanitized nylon bags (200 mg/L active chlorine) and rinsed (3 mg/L active chlorine), at 5 C, for 10 min and centrifuged at 800g for 10 min. Samples containing 80 g of sliced carrots were submerged in different coatings for 3 min and air flow dried at 20 C for 3 h. The control (noncoated carrots) was submerged in sterile distilled water under similar conditions. All the samples were placed in expanded polystyrene trays, wrapped in polyvinylchloride (PVC) film and stored at 10 C for 15 days. 2.3. Microbiological analyses To evaluate the microbiological efficiency of the antimicrobial coatings on the carrot microbiota, microbiological analyses of mesophilic aerobes, total and fecal coliforms, Escherichia coli, Staphylococcus aureus, yeasts and molds, lactic acid bacteria and psychrotrophs were carried out in the coated and noncoated carrot samples at 0, 5, 10 and 15 storage days, according to the methodology described by the Compendium of Methods for the Microbiological Examination of Foods (Vanderzant & Splittstoesser, 1992). At time zero, the coated carrots were analyzed after the treatment. 2.4. Statistical analyses The computer program Statistical Analysis System (SAS) was used in all the statistical analyses. Variance analysis (ANOVA) tests were applied to the split plots and regression analysis was performed at a level of 5% significance.
Coating 1 Coating 3
6
Coating 2 Coating 4
4
Coating 1
Coating 2
Coating 3
Coating 4
Log MPN/g
Log CFU/g
5 4 3 2
3 2 1
1 0
0 0
(A)
5
10 Time (days)
15
0
20
(B)
5
10
15
20
Time (days)
Fig. 1. Effect of coatings on lactic acid bacteria (A) and total coliforms (B) in minimally processed carrot stored at 10 C/15 days.
A.M. Durango et al. / Food Control 17 (2006) 336–341
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Table 2 Summary of variance analysis of mesophilic aerobes, yeasts and molds and psychrotrophic bacteria in minimally processed carrots, submitted to four coatings during 15 days of storage at 10 C FV
GL
QM Mesophilic aerobes
QM Yeasts and molds
QM Psychrotrophs
Coating Residue (a) Time Coating · time Residue (b)
3 4 3 9 12
1.6987** 0.0003 11.1527** 0.3210** 0.0047
6.0143** 0.0009 9.1639** 1.5102** 0.0018
2.6090** 0.0067 13.6104** 0.1697** 0.0013
**Significant at 5% probability (P < 0.05).
6
7
5
6 5 Log CFU/g
Log CFU/g
4
3
2
1
Coating 1
= 2,1258 + 0,2787X – 0,0055 X2 R2 = 0, 93
Coating 2
= 2,5695 + 0,4804X – 0,0224 X2 R2 = 0, 94
Coating 3
= 1,5950 + 0,3700X – 0,0110 X2 R2 = 0, 96
Coating 4
= 1,7213 + 0,3548X – 0,0142 X2 R2 = 0, 99 5 Time (days)
10
3 2 1
0 0
4
15
Coating 1
= 2,3140 + 0,3928X – 0,0118 X2 R2= 0,99
Coating 2
= 3,1265 + 0,4248X – 0,0170 X2 R2= 0,93
Coating 3
= 1,9985 + 0,4582X – 0,0154 X2 R2= 0,99
Coating 4
= 1,9887 + 0,3262X – 0,0114 X2 R2= 0,99
0 0
5
10
15
Time (days)
Fig. 2. Effect of coating on the growth of mesophyll aerobes in minimally processed carrot stored during 15 days at 10 C.
6
5
Coating 1
= 1,7840 + 0,1613X – 0,0023 X2 R2= 0,84
Coating 2
= 2,0525 + 0,1615X – 0,0047 X2 R2= 0,95
Coating 3
= 0,6462 + 0,2802X – 0,0005 X2 R2= 0,81
Coating 4
= 1,0500 + 0,0900X – 0,0100 X2 R2= 0,93
4. Discussion
Log CFU/g
4
3
2
1
0 0
5
10
Fig. 4. Psychrotrophic counting in minimally processed carrot submitted to four coatings during 15 days at 10 C.
15
Time (days)
Fig. 3. Effect of coatings on the growth of filamentous fungi and yeast in minimally processed carrot stored at 10 C for 15 days.
The effect of the coatings on minimally processed carrotÕs psychrotrophic microbiota was greater for coating 4 (Fig. 4), which presented the lowest psychrotrophic counting, 4.3 log CFU/g, at the end of the storage period. Starch coated samples (coating 2) presented the highest psychrotrophic counting throughout the storage period, starting at 3.0 log CFU/g, and reaching 5.8 log CFU/g after 15 days of storage. Coating 3 did not inhibit the growth of this microbiological group, compared to coating 1 (control), with a reduction of 0.16 log cycle at the end of 15 storage days.
Coating 4, consisting of 1.5% chitosan added—starch was the most efficient in controlling the microorganisms evaluated, normally present in minimally processed carrot, stored at 10 C for 15 days. At this concentration, the coating totally inhibited the growth of lactic acid bacteria and total coliforms, during storage time. Lactic acid bacteria have been associated to carrot deterioration (Zagory, 1999). According to Carlin, Nguyen-the, Cudennes, and Reich (1989), deterioration of minimally processed carrot after 14 days storage at 10 C was associated to Leuconostoc mesenteroides populations due to the process of lactic fermentation. Marchetti, Casadei, and Guerzoni (1992), found lactic acid bacteria counting of 3.16 · 105 CFU/g and 108 CFU/g in carrot salad after 7 day storage at 5 C. Coating 4 also had a satisfactory performance in controlling mesophilic aerobes, with a reduction of 1.34 log cycles at the end of the storage period. Such reduction is considerable, when compared to other methods applied to reduce the microbial load in foods. For instance, the sanitation method used in sliced carrot showed a reduction in the number of mesophilic aerobic bacteria of, approximately, one log cycle, using 200 mg/L of chlorine during the process (Silva, 2003). A lower efficiency of this process was found by Amanatidou, Slump, Gorris, and Smid (2000), in sliced carrot with a reduction of only 0.4 log cycle in the microbial
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load of mesophilic aerobes. The number of mesophilic aerobes found by Chervin and Boisseau (1992), in minimally processed carrots was 6.7 log CFU/g. Buick and Damoglou (1987) showed that 70% of the initial microbiota in minimally processed sliced carrot consists of Erwinia ssp, which attacks the carrot, causing the socalled bacterial wet rot (Mu¨eller, 1981). In this work, the coating containing 0.5% chitosan did not present a good performance in controlling deteriorating bacteria, despite the initial low number of mesophilic aerobes, lactic acid bacteria and psychrotrophs found in minimally processed carrot. In 1993, carrot was associated to an infection outbreak caused by enterotoxigenic E. coli in the US. In Lebanon, the prevalence of S. aureus in raw carrot has been 14.3% (Beuchat, 2002). Zheng and Zhu (2003) showed that 1% chitosan at a degree of deacetylation of 88.76% and molecular weight of 48.5 kDa, has an antimicrobial effect on E. coli and S. aureus at an inhibition rate of 100% on these microorganisms growth. Concentration of 1% chitosan presented reductions between 1 and 2 log cycles in the total counting of bacteria in small meat pies; concentrations of 0.2% and 0.5% did not have an inhibiting effect on the deteriorating microflora, but the number of bacteria before starting the experiment was high >107 CFU/g, and chitosan is likely to be more effective in low microbial populations (Darmadji & Izumimoto, 1994). Regard to the action of the coatings on yeasts and molds, coating 4 presented the highest fungicidal action. Studies have shown that the effect of chitosan on some fungi is mainly due to alterations in the functions of the cellular membrane (Fang et al., 1994). The yeasts most commonly isolated from vegetables belong to the genera Cryptococcus, Rhodotorula and Candida; among the moulds are Fusarium, Mucor, Rhizopus and Penicillium (Francis, Thomas, & OÕBeirne, 1999). The main disease caused by fungus in carrots is Sclerotinia rot. Cheah, Page, and Shepherd (1997) showed that chitosan applied as a 2% or 4% solution inhibits Sclerotinia storage rot on carrots, and that the effect was due, at least in part, to inhibition of fungal growth. In this work, coating 4 containing 1.5% chitosan was the most efficient on the psychrothrophic flora in minimally processed carrot. According to Garg, Churey, and Splittstoesser (1990) the shelf life of refrigerated vegetables is especially affected by the psychrotrophic population, with Pseudomonas sp. being the main psychrotrophic agent present in these foods. Several authors have found psychrotrophic counting in minimally processed carrot sold at supermarkets ranging between 106 and 109 UFC/mL (Rosa, 2002). Listeria monocytogenes is one of the pathogens most frequently associated with ready-to-use vegetables (Francis et al., 1999). Studies conducted by Coma et al. (2002) show that chitosan coatings have a bactericidal effect on Lis-
teria monocytogenes and that such activity was probably due to the positive charges of chitosan which interfere with the negatively charged residues of macromolecules at the Listeria cell surface, presumably by competing with calcium for electronegative sites on the membrane. Chitosan has been reported to inhibit various spoilage bacteria, through its capacities to both bind water and inactivate various enzymes, and through its ability to absorb nutrients normally used by bacteria (Ouattara et al., 2000). Coating 2, which contained only starch, favored the development of mesophilic and psychrotrophic bacteria, indicating that these microorganisms may have utilized this carbohydrate as a source of energy. 5. Conclusions The results of this experiment showed that the use of an antimicrobial coating consisting of chitosan-added yam starch is a viable alternative in controlling the microbiota present in minimally processed carrot, since the growth of lactic acid bacteria, total coliforms, psychrotrophs, yeasts and molds and mesophilic aerobes, was substantially inhibited by the application of 1.5% chitosan coating. Based on the concept of protection barrier technology, the use of such coating may contribute to improve safety in minimally processed carrot thereby prolonging its shelf life. Coating may be applied on minimally processed fruits and vegetables, combined to other types of controls, such as quality raw material, hygienic processing conditions and storage temperatures. The combination of these treatments as barrier offers a greater potential for shelf-life extension of minimally processed vegetables. Acknowledgements The authors thank CNPq and FAPEMIG for the financial support. References Amanatidou, A., Slump, R. A., Gorris, L. G. M., & Smid, D. (2000). High oxygen and high carbon dioxide modified atmospheres for shelf-life extension of minimally processed carrots. Journal of Food Science, 65(1), 61–66. Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food packaging. Innovative Food Science and Emerging Technologies, 3, 113–126. Barry-Ryan, C., Pacussi, J. M., & OÕBeirne, D. (2000). Quality of shredded carrots as affected by packaging film and storage temperature. Journal of Food Science, 65(4), 726–730. Beuchat, L. R. (2002). Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection, 4, 413–423.
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