Chitosan dipping or oregano oil treatments, singly or combined on modified atmosphere packaged chicken breast meat

International Journal of Food Microbiology 156 (2012) 264–271

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Chitosan dipping or oregano oil treatments, singly or combined on modified atmosphere packaged chicken breast meat S. Petrou, M. Tsiraki, V. Giatrakou, I.N. Savvaidis ⁎ Laboratory of Food Chemistry and Food Microbiology, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece

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Article history: Received 1 December 2011 Received in revised form 6 March 2012 Accepted 1 April 2012 Available online 6 April 2012 Keywords: Chitosan Oregano oil Poultry Chicken Antimicrobials

a b s t r a c t The present study examined the effect of natural antimicrobials: chitosan, oregano and their combination, on the shelf-life of modified atmosphere packaged chicken breast meat stored at 4 °C. Treatments examined in the present study were the following: M (control samples stored under modified atmosphere packaging), M–O (samples treated with oregano oil 0.25% v/w, stored under MAP), M–CH (samples treated with chitosan 1.5% w/v, stored under MAP) and M–CH–O (treated with chitosan 1.5% w/v and oregano oil 0.25% v/w, stored under MAP). Treatment, M–CH–O, significantly affected mesophilic Total Plate Counts (TPC), lactic acid bacteria (LAB), Brochothrix thermosphacta, Enterobacteriaceae, Pseudomonas spp., and yeasts-moulds during the storage period. Lipid oxidation (as determined by MDA values) of control and treated chicken samples was in general low and below 0.5 mg MDA/kg, showing no oxidative rancidity during the storage period. Addition of chitosan to the chicken samples produced higher (P b 0.05) lightness (L*) values as compared to the control samples. The results of this study indicate that the shelf-life of chicken fillets can be extended using, either oregano oil singly, and/or chitosan, by approximately 6 (M–O) and >15 (M–CH and M–CH–O) days. Interestingly, chitosan (M– CH) or chitosan–oregano (M–CH–O) treated chicken samples were sensorially acceptable during the entire refrigerated storage period of 21 days. It is noteworthy that the presence of chitosan in M–CH and M–CH–O samples did not negatively influence the taste of chicken samples, with M–CH samples receiving a higher score (compared to M–CH–O), probably as a result of a distinct and “spicy” lemon taste of chitosan, that was well received by the panelists. Based primarily on sensory data (taste attribute) M–CH and M–O treatments extended the shelf-life of chicken fillets by 6 days, while M–CH–O treatment resulted in a product with a shelf-life of 14 days, maintaining acceptable sensory characteristics. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Chicken breast meat (fillet) is favored by consumers worldwide, prone to rapid spoilage, therefore, food industries are nowadays seeking technologies to increase its shelf-life. Spoilage of fresh poultry products is an economic burden to the producer, consequently, developing methods to prolong the shelf-life and overall safety/quality represents a major task of the poultry processing industry. Furthermore, as consumer's demand for more “healthier” meals (free of conventional chemical preservatives) has increased in the last decade, novel packaging (e.g. active) and processing technologies, in some cases, combined with “natural” antimicrobials such as essential oils (EOs) chitosan, nisin etc., have been suggested (Giatrakou and Savvaidis, 2012). Chitosan, a deacetylated form of chitin, is a polysaccharide found in the shells of crab and shrimps and the cell walls of fungi. Chitosan has been proved to be non-toxic, biodegradable and biocompatible.

⁎ Corresponding author. Tel.: + 30 26510 08343; fax: + 30 26510 08795. E-mail address: [email protected] (I.N. Savvaidis). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.04.002

Chitosan shows a broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria and fungi (Prashanth and Tharanathan, 2007). There is ample evidence that edible chitosan coatings on fruits and vegetables can be used to control fungal or bacterial growth during post-harvest storage and distribution (El-Ghaouth et al., 1991; Devlieghere et al., 2004; Chien et al., 2007). Other potential applications of chitosan as biopreservative have also been investigated in various meat products (Darmadji and Izumimoto, 1994; Roller et al., 2002; Yingyuad et al., 2006; Georgantelis et al., 2007a; 2007b; Kanatt et al., 2008; Soultos et al., 2008; Giatrakou et al., 2010). EOs possess antibacterial, antioxidant, antiviral and antifungal properties (Burt, 2004; Holley and Patel, 2005). Among the EOs from various aromatic plants, oregano essential oil (EO) has increasingly gained the interest of researchers as a potential “natural” antimicrobial to be used in food processing. Before using EOs as “natural preservatives” (Nychas, 1995) and due to their strong sensory characteristics, careful evaluation is needed, so that when added to the food product, EOs appeal sensorially acceptable to the consumer. Several studies have demonstrated the beneficial effects of chitosan, applied either singly, or in combination to food systems over the last decade. In one of these studies, Roller et al. (2002) developed a novel

S. Petrou et al. / International Journal of Food Microbiology 156 (2012) 264–271

preservation system involving the combined use of chitosan glutamate, carnocin and sulphite for the preservation of pork sausages. Trials showed that the batch, containing chitosan combined with sulphite, retarded the growth of spoilage organisms and deteriorated less rapidly, judged by a sensory panel to be more acceptable than all the other batches. Pranoto et al. (2005) reported that incorporation of garlic oil (100 μl/g), potassium sorbate (100 mg/g) and nisin (51.000 IU/g of chitosan) in chitosan films was found to have antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. In another study, Zivanovic et al. (2005) incorporated EOs (oregano, anise, basil and coriander) in chitosan films, in order to enhance the antimicrobial properties of the film against two pathogens (L. monocytogenes and Escherichia coli O157:H7), inoculated on bologna slices. The antibacterial effects of the EOs were similar when applied alone or incorporated in the films. Pure chitosan films reduced L. monocytogenes by 2 logs, whereas the films with 1% and 2% oregano EO decreased the numbers of L. monocytogenes by 3.6–4 logs and E. coli by 3 logs. Georgantelis et al. (2007a) studied the effect of rosemary extract, αtocopherol and chitosan (10 g/kg) on microbial parameters and lipid oxidation of fresh pork sausages. Shelf-life of samples containing chitoaan was almost doubled compared to the remaining samples, and the best antimicrobial and antioxidant effects were obtained from the combination of chitosan with rosemary extract. Finally, Kanatt et al. (2008) studied the preservative effect of mint and chitosan (0.1%) mixture on pork cocktail salami. Results showed that addition of chitosan to mint extract did not interfere with the antioxidant activity of mint, and that the shelf-life of pork cocktail salami, as determined by bacterial count and oxidative rancidity, was enhanced in chitosan-mint treated samples stored at 0–3 °C. To the best of our knowledge, the application of chitosan as a dipping agent singly, or in combination with oregano EO, has not been studied to date, in fresh poultry meat. Thus, the objective of the present work was to determine the effect of chitosan and oregano oil, applied individually, and/or in combination, on microbiological, physicochemical and sensory parameters of modified atmosphere packaged fresh chicken breast meat. 2. Material and methods 2.1. Chicken samples Fresh chicken breast meat (skinless and boneless fillet, ca. 200 g or 16 cm × 8 cm each, Pindos S.A., Ioannina, Greece) was provided by a local poultry processing company, within 1 h after slaughter in insulated polystyrene boxes on ice flakes. Chicken samples were subsequently kept under refrigeration in a cooling incubator (4 ± 0.5 °C) before the addition of the antimicrobials (see below). 2.2. Preparation of chitosan and oregano oil solutions Chitosan of low molecular weight (MW; 340) in powder form from crab shells was purchased from Aldrich Company (Athens, Greece). Moisture content was less than 10% and chitosan had a deacetylation degree of 75–85% (Manufacturer's data). A stock solution of chitosan was prepared by dissolving 1.5 g in 100 ml of 1% (w/v) glacial acetic acid and stirred overnight at room temperature (final chitosan concentration = 1.5% w/v). Pure oregano oil (Kokkinakis S.A., Athens, Greece) was used (see below). The oil consisted of (major components): Carvacrol: 57.7%; p-cymene: 28.7%; γ- terpinene: 6.4% and thymol 2.8% (Manufacturer's data). 2.3. Application of the antimicrobials to the chicken samples The antimicrobials were added to the chicken fillets, either singly, or sequentially using the following procedure: A chicken fillet (ca. 200 g) was transferred aseptically into an open sterile packaging pouch,

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containing 100 ml of chitosan solution (1.5% w/v). Each fillet was individually dipped and remained into the chitosan solution for 1.5 min. Immediately after dipping, the excess solution was drained off and each sample was packaged into a clean sterile pouch. Oregano oil (sterile, as previously determined, in our laboratory by measuring its total plate counts) was added onto the chicken samples, undiluted at a concentration of 0.25% v/w using a micropipette. Finally, for the combined antimicrobial treatment, chitosan solution was applied first to the samples, followed by oregano oil, both added at concentrations previously applied. Treatments included: M, (control samples, under modified atmosphere packaging, in the absence of antimicrobial agents), M–O, (under MAP treated with oregano oil 0.25% v/w), M–CH, (under MAP treated with chitosan 1.5% w/v), and finally, M–CH–O, (under MAP treated with chitosan and oregano oil, at concentrations, previously applied, respectively). It must be noted that the selection of the final optimum concentration of each antimicrobial, applied to the chicken fillets, was established following preliminary microbiological analysis (determination of mesophilic total plate counts) and sensory evaluation of the chicken samples treated with the aforementioned antimicrobials (results not shown). 2.4. Packaging of samples Fresh boneless and skinless chicken breast meat was packed in pouches (low density polyethylene/polyamide/low density polyethylene; VER PACK, Thessaloniki, Greece). The pouches had an inside volume of 800 ml, resulting in an approximate 3:1 volumetric ratio between the package and meat. Samples were packaged and sealed in a modified atmosphere packaging machine (BOSS model N48, Bad Homburg, Germany) using a gas combination of 30% CO2 and 70% NO2 produced by a PBI-Dansensor model mix 9000 gas mixer (Ringsted, Denmark) with a high-O2 barrier lid stock film (75 μm in thickness, having an oxygen permeability of 52.2 cm3/m2/day/atm, at 75% relative humidity (RH), 23 °C, a carbon dioxide permeability of 191 cm3/m2/day/ atm at 0% RH, 23 °C and a water vapor permeability of 2.4 g/m 2/day at 100% RH, 23 °C). The breast meat was then subdivided into 4 lots. Lots were assigned and treated as follows: Control = M; stored under MAP, M–O; stored under MAP, treated with oregano oil 0.25% v/w, M– CH; stored under MAP, treated with chitosan 1.5% w/v, and M–CH–O; stored under MAP, treated with chitosan 1.5% w/v and oregano oil 0.25% v/w. Meat samples (control and treated) were kept under refrigeration (4± 0.5 °C) for a period of 12 (M), 18 (M–O) and 21 days (M– CH, M–CH–O). 2.5. Microbiological analysis Chicken meat (25 g) was mixed with 225 ml of 0.1% sterile peptone water (Merck, Darmstadt, Germany) in a sterile filtered stomacher bag (Seward Ltd., London, UK). The mixture was stomached for 1 min. For microbial enumeration, 0.1 ml samples of serial dilutions (1:10, diluent, 0.1% peptone water) of chicken homogenates were spread on the surface of agar plates. Mesophilic total plate counts (TPC) were determined using Plate Count Agar (PCA, Merck, Darmstadt, Germany), after incubation for 2 days at 30 °C. Pseudomonas spp. were enumerated on cetrimide fusidin cephaloridine agar (CFC, Oxoid code CM 559, supplemented with SR 103, Oxoid, Basingstoke, UK) and incubated at 20 °C for 2 days. Brochothrix thermosphacta was determined on streptomycin sulphate-thallous acetate-cycloheximide (actidione) agar (Oxoid code CM0881, supplemented with selective supplement SR 0151) after incubation at 20 °C for 3 days. As a reference, strain, B. thermosphacta 847 (ATCC11589) was used for confirmation of this group; Gram, catalase tests were run and tested positive, whereas oxidase test was tested negative. Lactic acid bacteria (LAB) were enumerated on de Man Rogosa Sharpe agar (MRS, pH 6.2, Oxoid code CM361, Basingstoke, UK) incubated at 25 °C for 5 days. Enterobacteriaceae were enumerated by the pour-overlay method using Violet Red Bile Glucose (VRBG) agar (CM485, Oxoid). Plates

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were incubated at 37 °C for 24 h. Purple colonies surrounded by the purple zone, were enumerated and recorded as Enterobacteriaceae. Finally, yeasts-moulds were enumerated on Rose Bengal Chloramphenicol (RBC) selective agar (Merck, Germany) plates using surface spreading technique and plates were incubated at 25 °C 3–5 days in the dark. Three replicates of at least three appropriate dilutions depending on the sampling day were enumerated. Plates from dilutions having from 30 to 300 colonies were counted and microbiological data were transformed into logarithms of the number of colony forming units (cfu/g). All plates were examined visually for typical colony types and morphology characteristics associated with each growth medium. 2.6. Physicochemical analysis The pH value was recorded using a pH meter (Hanna Instruments, HI 9219, Woonsocket, RI, USA). Some 25 g of the chicken breast meat was homogenized thoroughly with 225 ml of distilled water and the homogenate was used for pH determination. Determination of lipid oxidation was carried out according to the procedure of Schmedes and Hølmer (1989). The 2-thiobarbituric acid (TBA) values were expressed as mg malondialdehyde (MDA) per kg of chicken sample. Superficial color alterations were monitored with a colorimeter (HunterLab, model DP-9000, Reston, VA, USA) as described by Du et al. (2002). Color parameters a* (redness) b* (yellowness) and L* were measured directly on the surface of the chicken samples (skinless product) after opening the packages. 2.7. Sensory analysis Each chicken sample (boneless and skinless fillet, ca. 200 g) was cooked in a microwave oven at high power (700 W) for 10 min. A panel of seven judges experienced (laboratory-trained) in poultry evaluation was used for sensory evaluation. All panelists who evaluated the sensory attributes of cooked chicken had previously participated in training sessions to become familiar with the sensory characteristics of cooked chicken. Fresh chicken breast meat was used as reference. Panelists were asked to evaluate taste, odor and appearance intensities of the cooked samples. Acceptability as a composite of odor, taste and appearance was estimated using a scale ranging from 0 to 9. The scale points were: excellent, 9; very good, 8; good, 7; acceptable, 6; poor (first off-odor, off-taste development) b 6; a score of 6 was taken as the lower limit of acceptability. The product was defined as unacceptable after development of first off-odor or off-taste. 2.8. Statistical analysis Experiments were replicated twice (n = 2) on different occasions with different chicken meat samples. Analyses were run in triplicate for each replicate. Results are reported as mean values ± standard deviation (S.D.). Data were subjected to analysis of variance (ANOVA). The least significant difference (LSD) procedure was used to test for differences between means (P b 0.05). Microbiological counts were converted to log cfu/g and were subjected to analysis of variance (ANOVA) using the software Stat graphics (Statistical Graphics Corp., Rockville, MD, USA). 3. Results and discussion 3.1. Microbiological changes of chicken meat stored under MAP in the absence and presence of antimicrobials Changes in TPC, LAB, B. thermosphacta, Enterobacteriaceae, Pseudomonas spp., and yeasts/moulds of M, M–O, M–CH and M–CH–O chicken samples are shown in Fig. 1a–f. The initial (day 0) mesophilic TPC (Fig. 1) of chicken meat (control M) was ca. 4.85 log cfu/g, in

agreement with results (4.3 log cfu/g) for fresh (skinless fillet) chicken meat (Economou et al., 2009). ANOVA showed a significant effect of the antimicrobial treatments (M–O, M–CH, M–CH–O) and storage time on TPC (P b 0.05) (Fig. 1a). Chicken samples reached or exceeded the value of 7.0 log cfu/g for TPC, which was considered as the upper acceptability limit for fresh meat (Senter et al., 2000) on days 11 (M), 16–17 (M–O) while M– CH and M–CH–O chicken samples never reached this limit value after a storage period of 21 days. Thus, compared to the control samples (M), a microbiological shelf-life extension of 5–6 or 10 days was achieved for M–O, M–CH and M–CH–O samples. The 5–6 and 10 days shelf-life extension for M–O and M–CH samples could be due to the antimicrobial action of oregano oil's components (especially carvacrol) and of chitosan, acting on spoilage microorganisms (Helander et al., 2001; Holley and Patel, 2005 and Prashanth and Tharanathan, 2007). Contributing to the oregano oil's antimicrobial activity (apart from carvacrol) other phenolic components such as p-cymene, γterpinene and thymol, are known to exhibit antibacterial properties (Burt, 2004). The mechanism of action of EOs, including oregano, is generally considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport and coagulation of cell contents (Burt, 2004). Recently, in a related study, a 5-day microbiological shelf-life extension was obtained for a poultry product (ready to cook chicken– pepper kebab) treated with either thyme oil (0.2% v/w) or chitosan (1.5% w/v) (Giatrakou et al., 2010). In other studies, Yingyuad et al. (2006) found that the combined use of vacuum packaging (VP) and chitosan solutions (2% and 2.5% w/v) kept the TPC count of grilled Thai-style pork meat below 4 log cfu/g for more than 28 days, achieving a shelf-life extension of more than 14 days, as compared to the control samples. Darmadji and Izumimoto (1994) reported a reduction of microbial counts by an average of 2.0 log cfu/g for beef patties coated with 1% (w/v) chitosan and stored at 4 °C under aerobic conditions, while Lin and Chao (2001) reported that Chinese style sausage containing chitosan 0.1% w/w had lower TPC than control samples during refrigerated storage (ca. 1 log cfu/g lower). In another study, the combined use of rosemary extract and chitosan on the preservation of fresh pork sausages led to a reduction of the TPC count (ca. by 1–2 log cfu/g), extending their shelf-life at 4 °C (Georgantelis et al., 2007a). Furthermore, Zivanovic et al. (2005) reported that the addition of oregano EO into the chitosan film not only improved the film's antimicrobial properties, but also effectively reduced pathogens on bologna slices. It must be noted that limited work so far has been reported on the application of chitosan, singly, or in combination with plant EOs on poultry meat. In the present study, and of the treatments examined, M–CH–O was the most effective in inhibiting the growth of mesophilic TPC (Fig. 1a) throughout the storage period. This result could be attributed to the inhibitory effect of the combined antimicrobials, suppressing the growth of Gram-positive, as well as Gram-negative spoilage organisms. Oregano oil's antimicrobial activity, as previously stated, is attributed to its major phenolic components such as carvacrol, p-cymene, γ-terpinene and thymol (Burt, 2004). Chitosan is believed to act on the cells of spoilage microorganisms and pathogens, by changing the permeability of the cytoplasmatic membrane, leading to the leakage of intracellular electrolytes and proteinaceous constituents, finally to the death of the cell (Helander et al., 2001; Prashanth and Tharanathan, 2007). LAB counts (Fig. 1b) for M samples showed an increasing trend during the entire storage period, whereas for M–O samples counts increased between days 12 and 15. Treatments M–CH and M–CH–O had similar LAB patterns producing significantly lower counts (P b 0.05) as compared to both control and M-O chicken samples (day 12). LAB populations were the highest among the microbial groups examined, due to their microaerophilic nature favoring their growth under MAP conditions. Chitosan either singly, or combined with oregano oil

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Fig. 1. Changes in mesophilic TPC (a), LAB (b), Brochothrix thermosphacta (c), Enterobacteriaceae (d), Pseudomonas spp. (e) and yeasts/moulds (f) of chicken breast meat during refrigerated storage under MAP (M; ■), under MAP with oregano oil (0.25% v/w (M–O treatment; ▲), under MAP with chitosan (1.5% w/v) (M-CH treatment; ♦) and under MAP with oregano oil (0.25% v/w) and chitosan (1.5% w/v) (M–CH–O treatment; ●). Each point is the mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show S.D.

suppressed LAB growth in chicken samples throughout the storage period, demonstrating a strong antimicrobial effect. Georgantelis et al. (2007a) reported that throughout refrigeration storage of Greekstyle sausages containing chitosan and rosemary extract, the LAB counts were approximately 2.0 log cfu/g lower than in the control samples. Moreover, LAB counts during the refrigeration storage of minced pork mixture with 0.6% w/v chitosan added were 2–2.5 log cfu/g lower, as compared to the control (Sagoo et al., 2002). Recently, Giatrakou et al. (2010) reported that a combined treatment consisting of chitosan (1.5% w/v) and thyme oil (0.2% v/w) resulted in significantly lower (P b 0.05) LAB populations (ca. by 4 log cfu/g) in a poultry product (ready-to-eat chicken kebab) stored under aerobic and MAP conditions.

B. thermosphacta, a facultative anaerobic bacterial group, involved in the spoilage of meat stored under MAP conditions (Jay et al., 2005) reached a relatively high population (approximately 6.3 log cfu/g) in M chicken samples on day-12 (Fig. 1c). All treated chicken samples had significantly lower (P b 0.05) counts (ca. 3.5–4.0 log cfu/g) than the control (M) samples. Interestingly, M–CH and M–CH–O treatments were effective in inhibiting the growth of this Gram-positive, meat spoilage bacterial group. It is noteworthy that under treatment M–CH–O a low final population of 2.0 log cfu/g (detection limit) was recorded on day-21 of storage, in agreement with results recently reported by Rodríguez-Calleja et al. (2012) who found that a combination of high hydrostatic pressure, a commercial liquid antimicrobial edible coating and MAP significantly affected B.

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thermosphacta (under the detection limit). In other studies, Soultos et al. (2008) reported that chitosan, added at concentrations of 0.5 and 1% (w/v) in fresh pork sausages reduced B. thermosphacta counts by 0.5 to 1.5 log cfu/g, respectively, on the first day of chill storage, while final populations were less than in the control samples. Giatrakou et al. (2010) recently reported that thyme oil or chitosan, either applied singly, or in combination were effective in inhibiting B. thermosphacta growth in a poultry product, reducing final populations by approximately 2–4.5 log cycles, as compared to the control samples. Our results support the hypothesis that the application of chitosan (dipping) and oregano EO on B. thermosphacta, could be beneficial in terms of controlling its spoilage potential in meat products, including poultry. In our study, Enterobacteriaceae, a psychrotrophic facultative anaerobic bacterial group, formed a substantial part of the chicken meat microbial flora and reached final counts of ca 6 logs on day-12 (Fig. 1d). As previously noted, M–O, M–CH and M–CH treatments produced significantly lower (P b 0.05) Enterobacteriaceae counts (approximately 3–4 log cycles) as compared to the control samples (day-12). Similarly, Roller et al. (2002) reported that chitosan in combination with sulphite selectively inactivated these species to an undetectable level (as determined by the plate-count method) in fresh pork sausages stored at 4 °C. Moreover, Ouattara et al. (2000) reported that indigenous Enterobacteriaceae in meat products (bologna, beef pastrami and cooked ham) were inhibited by the use of antimicrobial films, containing chitosan and acetic acid. Recently, thyme oil or chitosan, applied singly, or in combination, produced low Enterobacteriaceae counts in a ready-to-eat poultry product stored under MAP for 14 days (Giatrakou et al., 2010). It is now well established that Pseudomomas spp. and yeasts/moulds may form a significant part of the spoilage microflora of chicken meat stored under refrigeration (Jay et al., 2005). Pseudomonas spp., are known to antagonize other bacterial (Gram-positive or Gramnegative) groups for nutrients by forming siderophores, that may inhibit growth of both spoilage microorganisms or pathogens (Wei et al., 2006). However, the latter hypothesis, however, is only speculative and further research is needed to support our findings. As previously noted for LAB and B. thermosphacta, Pseudomonas spp. populations were significantly (P b 0.05) lower for M–O, M–CH and M– CH–O chicken samples as compared to the control (M) samples on day12 of storage (Fig. 1e), with M–CH–O treatment being the most effective for the inhibition of pseudomonads in chicken fillet samples, probably attributed to the combined antimicrobial action of chitosan and oregano EO, in agreement with results reported for a poultry product treated with chitosan and thyme oil (Giatrakou et al., 2010). In other studies, a combined application of nisin and EDTA (Economou et al., 2009) and yogurt dip with wax-coating (Göğüş et al., 2004) inhibited Pseudomonas spp. growth in fresh chicken meat. Finally, with regard to yeasts/moulds, species known to be involved in the spoilage of poultry meat (Hwang and Beuchat, 1995), all of the antimicrobial treatments examined in the present study produced significantly lower (P b 0.05) counts in M–O, M–CH and M–CH–O chicken samples as compared to the control samples up to day-12 of storage (Fig. 1f). Chitosan applied, either singly (M–CH), or in combination with oregano oil (M–CH–O) suppressed the growth of these species, and interestingly, their counts were below 3.0 logs at the end of storage period (21 days). In other studies involving preservation of fresh sausages stored under refrigeration, combinations of chitosan with sulphite (Roller et al., 2002) and chitosan with rosemary extract (Georgantelis et al., 2007a) led to a reduction of almost 3 and 2 log cycles, respectively, as compared to untreated samples. It is well established that the microbial flora, that develops primarily in raw poultry meat is on its surface, may depend on the initial number and bacterial types present, storage temperature, pH and packaging environment (ICMSF, 2005). Results of our study (microbiological data) support the hypothesis that chitosan, applied via a dipping procedure, leads to a homogenous dispersion of this antimicrobial agent in the

chicken samples and, by extrapolation, resulting in a more efficient action against the microbial flora (surface contamination) that causes spoilage of chicken meat. Moreover, chitosan dipping when combined with oregano EO results in a more effective natural antimicrobial treatment. 3.2. Physicochemical changes of chicken meat stored under MAP in the absence and presence of antimicrobials The initial pH of fresh chicken meat was ca. 6.2 (results not shown), in agreement with results of Economou et al. (2009), A small drop of pH values (P > 0.05) was recorded for untreated (M) samples, whereas respective values for treated (M–O, M–CH and M– CH–O) samples were in the range of 6.1–6.3, showing no statistically significant differences between them (P > 0.05) during storage. Changes in lipid oxidation of treated (M–O, M–CH and M–CH–O) chicken samples produced lower (P b 0.05) MDA values, as compared to the control (M) samples, shown in Fig. 2. Lipid oxidation (as determined by MDA values) of control (M) and treated (M–O, M–CH and M–CH–O) chicken samples was in general low and below 0.5 mg MDA/kg, showing no oxidative rancidity during the storage period. M–CH–O treatment resulted in the lowest (P b 0.05) MDA values on day-12 of storage (Fig. 2) leading to final values of approximately 0.1 mg MDA/kg (day-21). Chitosan may retard oxidative rancidity in muscle foods, by acting as a chelator on transition metal ions, such as ferrous ions, which can initiate lipid peroxidation and start chain reactions that lead to deterioration of flavor and taste in foods (Yen et al., 2008). To our knowledge, few studies have examined the combined effect of chitosan and EOs on lipid oxidation of meats, including poultry. In one of these studies, chitosan with rosemary extract was shown to prevent lipid oxidation in sausages (Georgantelis et al., 2007a), beef burgers (Georgantelis et al., 2007b), pork salamis with chitosan and mint extract (Kanatt et al., 2008), and recently a combination of chitosan and thyme oil in a poultry product (Giatrakou et al., 2010). Changes in color parameters L*, a*, and b* are shown in Fig. 3a–c. Control, untreated (M) samples resulted in the lowest L* values, while treated samples with added oregano (M–O) or chitosan (M– CH, M–CH–O) had significantly higher values (P b 0.05) on day-12 of 0.5

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Storage Fig. 2. Changes in TBA (MDA/kg) of chicken breast meat during refrigerated storage under MAP (M; ■), under MAP with oregano oil (0.25% v/w (M–O treatment; ▲), under MAP with chitosan (1.5% w/v) (M–CH treatment; ♦) and under MAP with oregano oil (0.25% v/w) and chitosan (1.5% w/v) (M–CH–O treatment; ●). Each point is the mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show S.D.

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storage period (Fig. 3a). Addition of chitosan to the chicken samples produced higher L* values (P b 0.05) as compared to M samples, resulting in an increase of lightness. Similarly to our results, Jo et al. (2001) reported that sausages containing chitosan had higher L* values than the control samples during the storage period. Giatrakou et al. (2010) found that addition of chitosan to a readyto-eat chicken product resulted in higher L* values, as compared to the untreated samples, whereas Darmadji and Izumimoto (1994) found that minced (beef) meat samples containing chitosan had lower L* values than the controls. Changes in a* and b* values for M, M–O, M–CH and M–CH–O chicken samples, varied with no significant differences (P > 0.05) among the treatments examined (Fig. 3b–c). Darmadji and Izumimoto (1994) also observed similar results in the a* values during storage between control and chitosan-added meat samples. Jo et al. (2001) and Youn et al. (1999) reported that chitosan affected the a* values of sausages, resulting in a redder surface color. Additionally, Georgantelis et al. (2007b) noted that the combination of chitosan and rosemary extract acted synergistically and improved the redness of beef burgers, during frozen storage, while individual use of chitosan or rosemary extract improved color stability as compared to the controls. Color acceptability of chicken breast fillets, treated with chitosan (M–CH, M–CH–O) by consumers could be related to the increase in a* value (redness) and the lower myoglobin content of chicken breast compared to red meat. These results are in agreement with those of Rodríguez-Calleja et al. (2012) who found that a combination of high hydrostatic pressure, a commercial liquid antimicrobial edible coating and MAP did not affect color acceptability of chicken breast fillets. Youn et al. (1999) reported that addition of chitosan (0.5%) increased the b* value, suggesting that the natural color of chitosan affected on the surface color of the sausage. Giatrakou et al. (2010) reported that b* (yellowness) values were varied with no specific pattern produced by any of the treatments (combination of thyme oil and chitosan) in a poultry product. In general meat color is perceived by consumers as indicative of freshness and meat type (e.g. chicken, beef etc.) may affect color characteristics. The rate of discoloration in fresh meat is related to the rate of pigment oxidation, oxygen consumption and to the effectiveness of the metmyoglobin reducing system (Ledward, 1991). Differences, therefore, in L*, a*, and b* values between our data (control and treated samples) and those results reported in the literature, could be attributed to the meat type used (chicken, beef, sausages etc.). Overall, both chitosan and oregano oil may act as preventive antioxidants in food products, including poultry meat. Oregano oil has a high total phenol (carvacrol) content, thus it could act as an efficient antioxidant, preventing oxidation of red heme-pigments to brown metmyoglobin (Kroll and Rawel, 2001). Moreover, chitosan has antioxidant properties and may maintain redness in muscle foods, due to its ability to act as a chelator on transition metal ions which catalyse oxidative reactions (i.e. oxidation of myoglobin) (Yen et al., 2008).

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3.3. Sensory changes of the chicken samples stored under MAP in the absence and presence of antimicrobials The results of the sensory evaluation (odor and taste acceptance) of cooked chicken samples, untreated (M) and treated (M–O, M–CH and M–CH–O) are presented (Fig. 4a–b). Odor and taste scores of chicken, irrespective of treatment, showed a similar pattern of decreasing acceptance (Fig. 4a–b). Generally, the taste attribute was a more sensitive parameter than odor, therefore, in our study the taste attribute was used for the determination of the shelf-life of cooked M, M–O, M–CH and M–CH–O chicken samples. On the initial day (0) of storage, cooked chicken breast meat had a pleasant taste and odor. Treatment of results with ANOVA gave a sensorial shelf-life (based on taste acceptance scores) of 11 days for M (control, untreated) chicken samples, while M–O samples exceeded the upper acceptability sensory limit (6.0) between days 16–17 of

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Storage Fig. 3. Changes in color parameters: L* lightness (a), a* redness (b) and b* yellowness (c) of chicken breast meat during refrigerated storage under MAP (M; ■), under MAP with oregano oil (0.25% v/w (M–O treatment; ▲), under MAP with chitosan (1.5% w/v) (M–CH treatment; ♦) and under MAP with oregano oil (0.25% v/w) and chitosan (1.5% w/v) (M–CH–O treatment; ●). Each point is the mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show S.D.

refrigerated storage. Moreover, samples containing chitosan and/or oregano oil (M–CH and M–CH–O) were sensorially acceptable for the entire period of 21 days (Fig. 4a–b). The results of this study indicate that chicken fillets' shelf-life can be extended using, either oregano oil singly, and/or chitosan, by approximately 6 (M–O) and >15 (M–CH and M–CH–O) days. Interestingly, chitosan (M–CH) or chitosan–oregano (M–CH–O) treated chicken

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Present results, in combination with current, limited knowledge on the potential use of natural antimicrobials, i.e. plant EOs and chitosan as “natural” preservatives in foods, leads to the general conclusion that their use (singly, or in combination) could expand its application for shelf-life extension of poultry meat. Furthermore it must be, however, stressed that extra care is needed by the consumer, with regard to the quality and safety of chicken meat, especially in the case of new poultry products e.g. chicken meat with added herbs or spices (paprika, basil) that are served at home or restaurants.

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Results of the present study demonstrate that the combined use of chitosan and oregano essential oil under MAP conditions, could a) inhibit growth of microbial spoilage flora, b) retard lipid oxidation, c) maintain lightness and d) improve the sensory quality of fresh chicken meat.

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Acknowledgements

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We thank European Union for financial support of the project “DOUBLE FRESH” (Proposal. /Contract no.: PL 023182). We acknowledge Drs. M. Zwietering, Z. Sosa Mejia and R. Beumer for providing useful suggestions in relation to this project.

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References

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Storage Fig. 4. Changes in acceptance sores of taste (a) and odor (b) of chicken breast meat during refrigerated storage under MAP (M; ■), under MAP with oregano oil (0.25% v/ w (M–O treatment; ▲), under MAP with chitosan (1.5% w/v) (M–CH treatment; ♦) and under MAP with oregano oil (0.25% v/w) and chitosan (1.5% w/v) (M–CH–O treatment; ●). Each point is the mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show S.D.

samples were sensorially acceptable during the entire refrigerated storage period of 21 days (Fig. 4a). It is noteworthy that the presence of chitosan in M–CH and M–CH–O samples did not negatively influence the taste of chicken samples, with M–CH samples receiving a higher score (compared to M–CH–O), probably as a result of a distinct and “spicy” lemon taste of chitosan, that was well received by the panelists. Chitosan dipping of chicken fillets produced a very pleasant taste in the chicken samples, promoting the natural freshness and aroma of the product. Moreover, oregano oil with chitosan (M–CH–O samples) resulted in an equally desirable (organoleptically acceptable) product. Similarly to the taste attribute, our results show that addition of chitosan or oregano oil to the chicken meat did not seem to affect the odor of cooked samples throughout the entire storage period. According to Soultos et al. (2008) and Roller et al. (2002) the addition of chitosan to sausages gave more acceptable odor and flavor as compared to the untreated samples. In addition, according to Yingyuad et al. (2006) aerobically- and VP-stored Thai-style grilled pork, reached unacceptable sensory scores after 7 and 14 days, respectively, while VP samples coated with chitosan maintained acceptable sensory quality throughout the storage period (28 days, 2 °C). In agreement to our results, it was recently, reported that a combination of chitosan and thyme oil extended a poultry product's shelf-life by 14 days (Giatrakou et al., 2010).

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