Chapter 4 Show Me the Money

Innovative Food Science and Emerging Technologies 29 (2015) 280–287

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Effect of pomegranate juice dipping and chitosan coating enriched with Zataria multiflora Boiss essential oil on the shelf-life of chicken meat during refrigerated storage Behnaz Bazargani-Gilani, Javad Aliakbarlu ⁎, Hossein Tajik Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

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Article history: Received 6 December 2014 Received in revised form 11 April 2015 Accepted 13 April 2015 Available online 28 April 2015 Keywords: Pomegranate juice Chitosan Zataria multiflora essential oil Chicken meat Oxidation

a b s t r a c t The objective of present study was to investigate the effect of pomegranate juice (PJ) and chitosan (CH) coating enriched with Zataria multiflora essential oil (ZEO) on the shelf-life of chicken breast meat during refrigerated storage. Treatments examined were the following: Control, PJ, PJ–CH, PJ–CH–Z 1%, and PJ–CH–Z 2%. The samples were stored at 4 °C for 20 days and analyzed at 5-day intervals. All of treatments significantly decreased total viable counts, Pseudomonas spp., lactic acid bacteria, Enterobacteriaceae, Psychrotrophic bacteria and yeasts– molds as compared control during the storage period. Peroxide value, Thiobarbituric acid reactive substance values and protein oxidation significantly were lower in all of treatments than control. PJ gave a pleasant effect on sensory attributes and chitosan coating enriched with ZEO significantly improved sensory scores. In conclusion, PJ can be suggested as a replacement to synthetic preservatives as well as synthetic flavorings in chicken breast meat. Industrial relevance: Chicken meat is susceptible to rapid spoilage due to high level protein and moisture. Then, food industries are recently finding methods to extend its shelf-life. The various chemical preservatives are generally undesired by consumers because of their adverse effects. Therefore, natural additives such as pomegranate juice, not only give appropriate color and flavor to foods but also they can extend the shelf-life of foods. The objective of this study was to introduce a new and palatable product resulting from dipping chicken breast meat in pomegranate fruit juice and its preservation with chitosan coating enriched with Z. multiflora Boiss essential oil under refrigerated storage. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The fresh meat is very sensitive to spoilage by microbial growth and oxidative reactions. High level protein and moisture cause microbial spoilage of meat while the aerobic condition induces oxidation of lipid and protein. Decreasing microbial growth and delaying lipid and protein oxidation during storage can increase the shelf-life of meat (Vaithiyanathan, Naveena, Muthukumar, Girish, & Kondaiah, 2011). Nowadays, synthetic preservatives are being applied to prevent the microbial growth and as well as retarding the oxidation reactions in meat. The consumers are unsatisfied from various chemical preservatives because of their side effects such as carcinogenicity and teratogenicity. The excessive demand for natural preservatives results in their extended utility (Giatrakou & Savvaidis, 2012). Acidification using organic acids and natural acidic fruit juices such as pomegranate juice is an extensively used method in food processing to extend the shelf-life (Sengun & Karapinar, 2004; Vijayakumar & ⁎ Corresponding author. Tel.: +98 4432972620; fax: +98 4432771926. E-mail address: [email protected] (J. Aliakbarlu).

http://dx.doi.org/10.1016/j.ifset.2015.04.007 1466-8564/© 2015 Elsevier Ltd. All rights reserved.

Wolf-Hall, 2002). Pomegranate (Punica granatum L.) from the Punicaceae family is an important commercial fruit crop that is excessively cultivated in Asia, North Africa, the Mediterranean and the Middle East (Sarkhosh, Zamani, Fatahi, & Ebadi, 2006). Pomegranate juice products are one of the most popular flavorings used to give flavor to several foods such as chicken, fish, salads and appetizers, in Iran and Turkey (Karabiyikli & Kisla, 2012). Pleasant flavor of pomegranate juice results from the combination of various taste, aroma and mouthfeel sensations. The distinguished taste is due to mainly the presence of sugars (glucose and fructose) and organic acids (primarily citric and malic acids). The aroma contains volatile compounds, including alcohols, aldehydes, ketones, and terpenes (Vázquez‐Araújo, Koppel, Chambers Iv, Adhikari, & Carbonell‐Barrachina, 2011). Recently, the great antioxidant potency from different components of pomegranate fruit such as juice, peel and seeds have been discovered (Gil, TomásBarberán, Hess-Pierce, Holcroft, & Kader, 2000). The antioxidant activity of pomegranate juice is higher than other fruit juices and beverages (Seeram et al., 2008). This antioxidant activity is correlated to the high level of phenolic compounds, including anthocyanins (3-glucosides and 3,5-diglucosides of delphinidin, cyanidin, and pelargonidin),

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ellagic acid, punicalin, punicalagin, pedunculagin and various flavanols (Alighourchi, Barzegar, & Abbasi, 2008; Miguel, Fontes, Antunes, Neves, & Martins, 2004). Pomegranate anthocyanins are sensitive compounds to oxidation that will be degraded by enormous destructive reactions during storage and processing (Wrolstad, Durst, & Lee, 2005). Chitosan, a high molecular weight cationic polysaccharide, produced by the deacetylation of chitin, is widely used in phenolic preservation of some fruits, such as pomegranate, litchi (De Reuck, Sivakumar, & Korsten, 2009) and raspberries (Zhang & Quantick, 1998) because of its excellent film forming and anti-fungal, bio-safe and biochemical properties (Lin & Chao, 2001). Recently, these properties of chitosan have been improved by incorporation of essential oils into chitosan films or coatings. Incorporation of essential oils into chitosan films or coatings can not only increase the film's antimicrobial and antioxidant properties but also decrease water vapor permeability and retard lipid oxidation of the product on which the film is used (Giatrakou, Ntzimani, & Savvaidis, 2010; Ojagh, Rezaei, Razavi, & Hosseini, 2010). Zataria multiflora Boiss (Persian name: Avishane shirazi) belongs to the family of Lamiaceae, and is a medicinal plant that grows extensively in tropical regions of Iran, Pakistan and Afghanistan. The essential oil of this plant (ZEO) consists great quantities of phenolic oxygenated monoterpens that cause antioxidant, antibacterial and antifungal activities (Aliakbarlu, Sadaghiani, & Mohammadi, 2013; Saei-Dehkordi, Tajik, Moradi, & Khalighi-Sigaroodi, 2010). The use of essential oils for food preservation is often restricted because of their application costs and other defects, such as their vigorous aroma and potential toxicity. An interesting alternative to reduce the amounts of essential oils while maintaining their effectiveness could be to incorporate these compounds into the formulation of edible coatings (Petrou, Tsiraki, Giatrakou, & Savvaidis, 2012). Therefore, the objective of this work was to introduce the new product resulting from dipping chicken breast meat in pomegranate fruit juice and its preservation with chitosan coating enriched with Z. multiflora Boiss essential oil under refrigerated storage (4 °C). 2. Materials and methods 2.1. Chicken samples Skinless and boneless fresh chicken breast meat (ca. 400 g) was purchased from a local poultry processing plant 1 h after slaughter and immediately transferred to the laboratory of Department of Food Hygiene and Quality Control, Urmia University, in insulated polystyrene boxes on ice flakes. The chicken meat samples were subsequently stored at 4 °C until use. 2.2. Preparation of pomegranate fruit juice (PJ) Fresh pomegranate fruits (P. granatum L. cv. Rabbab-e Neyriz) were obtained from a local super market. The fruits were washed and cut into four pieces. The seeds/arils were manually separated and ground in a mixer for 30 s and then passed through muslin cloth. After filtering by a Millipore filter with a 0.22 μm nylon membrane under vacuum at 25 °C, the freshly prepared juice was used for treatments. 2.3. Extraction and analysis of ZEO The plant, Z. multiflora Boiss, was purchased from the local groceries in Shiraz and identified at the Institute of Medicinal Plants, Karaj, Iran. The dried aerial parts were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The extracted oil was dried over anhydrous sodium sulfate, followed by filtering and stored in airtight glass vials covered with aluminum foil at 4 °C. Gas Chromatography–Mass Spectroscopy (GC–MS) analysis of ZEO was performed according to conditions reported in the previous work of authors (Bazargani-Gilani, Tajik, & Aliakbarlu, 2014).

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2.4. Preparation of coating solutions and treatment of chicken meat Chitosan (medium molecular weight, Sigma-Aldrich Chemical Co.) solution was prepared with 1.5% (w/v) chitosan in 1% (v/v) acetic acid. To achieve complete dispersion of chitosan, the solution was stirred at room temperature for overnight. The solution in beakers was placed on a hotplate/magnetic stirrer and glycerol was added to chitosan at 0.75 mL/g concentration as a plasticizer and stirred for 10 min. The resultant chitosan coating solution was filtrated through a Whatman No. 3 filter paper to remove any undissolved particles. Then the ZEO, mixed with Tween 80 (Aldrich Chemical Co., Steinheim, Germany), was added to the chitosan solution (Ojagh et al., 2010; Petrou et al., 2012). The final coating solution consisted of 1.5% chitosan, 1% acetic acid, 0.75% glycerol, 0.2% Tween 80 singly or incorporated with 1% and 2% ZEO. Meat samples were randomly assigned into five groups including the control (sterile distilled water) and four groups treated with the following solution: PJ, PJ–CH, PJ–CH–ZEO 1% and PJ–CH–ZEO 2%. Each sample was immersed for 2 min in dipping solution (sterile distilled water and PJ) and then drained well and followed by a second immersion in the coating solution for 2 min (Ojagh et al., 2010; Vaithiyanathan et al., 2011). Then the meat samples were removed and allowed to drain for 5 h at 10 °C on a pre-sterilized metal net under a biological containment hood in order to form the edible coatings and then the treated samples were aerobically packaged in a low density polyethylene pouches and stored at 4 °C for subsequent quality assessments. Chemical, microbiological and sensorial analyses were performed at 5-day intervals to determine the overall quality of samples for 20 days. 2.5. Microbiological analysis Chicken meat (10 g) was mixed with 90 mL of 0.1% sterile peptone water (Merck, Darmstadt, Germany) in a sterile stomacher bag and stomached for 1 min. For microbial enumeration, 0.1 mL of serial dilutions of chicken homogenates was spread on the surface of agar plates. Total viable counts (TVC) 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, Fluka, Germany) and incubated at 20 °C for 2 days. Lactic acid bacteria (LAB) were counted on de Man Rogosa Sharpe agar (MRS, QUELAB, Canada) incubated at 35 °C for 2 days. Enterobacteriaceae were enumerated by the pour-overlay method using Violet Red Bile Glucose (VRBG) agar (Merck, Darmstadt, Germany). The plates were incubated at 37 °C for 24 h. Psychrotrophic bacteria were determined on Plate Count Agar and the plates were incubated at 7 °C for 10 days. Finally, yeasts–molds were enumerated on Rose Bengal Chloramphenicol (RBC) selective agar (Merck, Darmstadt, Germany) plates using surface spreading technique and plates were incubated at 25 °C for 3–5 days in the dark. Three replicates of at least three appropriate dilutions depending on the sampling day were enumerated. Microbiological data were transformed into logarithms of the number of colony forming units (cfu/g). 2.6. Physicochemical analysis 2.6.1. Determination of pH value The pH value was recorded using a pH meter (PH-Meter E520, Metrohm Herisau, Switzerland). Meat sample (5 g) was homogenized with 25 mL of distilled water for 30 s and the homogenate was used for pH determination. 2.6.2. Determination of peroxide value Peroxide value (PV) was measured as suggested by the International Dairy Federation (IDF) (Shantha & Decker, 1993). The sample (0.30 g) was mixed with 9.8 mL chloroform–methanol in a glass tube and vortexed for 2–4 s. Ammonium thiocyanate solution (10 mM)

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(0.05 mL) was added and the sample was vortexed for 2–4 s. Then, 0.05 mL iron (II) solution was added and the sample was vortexed for 2–4 s. After that, the mixture was incubated for 5 min at room temperature and the absorbance was measured at 500 nm using an UV–visible spectrophotometer (LKB Novaspec II; Pharmacia, Sweden). PV was expressed as milliequivalents of O2 per kilogram of fat.

deviation (SD). Microbiological counts were converted to log CFU/g. Statistical analysis of data was performed using the SPSS software package (IBM SPSS statistics 21). Tukey's test and Independent-Sample T test with a significance set at P b 0.05 was used to compare differences among mean values. 3. Results and discussion

2.6.3. Determination of thiobarbituric acid reactive substance (TBARS) The TBARS value was determined colorimetrically by the modified method of Pikul et al. (1989). A portion (10 g) of sample was weighed and homogenized with 1 mL BHT(1 mg/mL) and 35 mL trichloroacetic acid (5%). The homogenate was transferred to a flask and then 100 mL distilled water was added and distilled. After collecting 50 mL distillate, the solution was filtered through a filter paper (Whatman No. 1). Five milliliter of filtrate was mixed with 5 mL of TBA solution (0.02 M) and placed in a water bath at 100 °C for 60 min. After cooling, the absorbance was measured at 532 nm against a water blank. TBARS value was expressed as milligram of malonaldehyde equivalents per kilogram of sample. TEP (1,1,3,3-tetraethoxypropane) was used for preparation of standard curve. 2.6.4. Determination of total carbonyls Protein oxidation, as measured by the total carbonyl content, was evaluated by derivatisation with 2,4-dinitrophenylhydrazine (DNPH) (Oliver, Ahn, Moerman, Goldstein, & Stadtman, 1987). Chicken samples (1 g) were homogenized 1:10 (w/v) in 20 mM sodium phosphate buffer containing 6 M NaCl (pH 6.5) using an ultraturrax homogenizer for 30 s. Two equal aliquots of 0.2 mL were taken from the homogenates and dispensed in 2 mL Eppendorf tubes. Proteins were precipitated by cold 10% TCA (1 mL) and subsequent centrifugation for 5 min at 4200 g. One pellet was treated with 1 mL 2 M HCl (protein concentration measurement) and the other with an equal volume of 0.2% (w/v) DNPH in 2 M HCl (carbonyl concentration measurement). Both samples were incubated for 1 h at room temperature. Then, samples were precipitated by 10% TCA (1 mL) and washed three times with 1 mL ethanol:ethyl acetate (1:1, v/v) to remove residue DNPH. The pellets were then dissolved in 1.5 mL of 20 mM sodium phosphate buffer containing 6 M guanidine HCl (pH 6.5), mixed and centrifuged for 2 min at 4200 g to remove insoluble particles. Protein concentration was calculated from the absorption at 280 nm using Bovine Serum Albumin (BSA) (SigmaAldrich Chemical Co.) as standard. The amount of carbonyls was expressed as nanomole of carbonyl per milligram of protein using an absorption coefficient of 21.0 nM−1 cm−1 at 370 nm for protein hydrazones. 2.7. Sensory analysis Each sample (boneless and skinless muscle, ca. 200 g) was cooked in a microwave (Kenwood) oven at high power (700 W) for 10 min. A panel of ten judges experienced (laboratory-trained) in meat 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 properties of cooked chicken. Fresh chicken breast meat was used as reference. The panelists were asked to evaluate taste, odor, color, texture and overall acceptability of the cooked samples. Sensory characteristics were determined using 9 point hedonics scale (0–9). The scale points were: excellent, 9; very good, 8; good, 7; acceptable, 6; poor (first off-odor, off-taste development) b6; 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 (Petrou et al., 2012). 2.8. Statistical analysis Two replications of the study were conducted and all analyses were run in triplicate. Results are reported as mean values ± standard

3.1. GC–MS analysis of ZEO The results of GC–MS analysis of ZEO showed that the major constituents of ZEO were carvacrol (59.17%) and linalool (23.67%) (BazarganiGilani et al., 2014). Other researchers reported that thymol was the most abundant component in ZEO (Saei-Dehkordi et al., 2010). 3.2. Microbiological analysis Changes in TVC, Pseudomonas spp., LAB, Enterobacteriaceae, Psychrotrophic bacteria and yeasts–molds of samples are shown in Fig. 1a–f. The initial (day 0) TVC of chicken meat was ca. 4.85 log cfu/g (Fig. 1a), which was in agreement with the results (4.3 log cfu/g) for fresh chicken meat (Economou, Pournis, Ntzimani, & Savvaidis, 2009). Fig. 1a shows a significant effect of the treatments (PJ, PJ–CH, PJ–CH–Z 1%, PJ–CH–Z 2%) and storage time on TVC (P b 0.05). Chicken samples reached or exceeded the value of 7.0 log cfu/g for TVC, which was considered as the upper acceptability limit for fresh meat (Senter, Arnold, & Chew, 2000) on days 5 (control) and 15 (PJ) while chicken samples of other treatments never reached this limit value after a storage period of 20 days. Thus, compared to the control samples, a microbiological shelf-life extension of 10 or 15 days was achieved for PJ, PJ–CH, PJ–CH–Z 1% and PJ–CH–Z 2% samples. The 10 and 15 days shelf-life extension for these treatments could be due to the antimicrobial action of PJ components especially condensed tannins and protein precipitable phenolics. A previous study showed that the total phenolics and total anthocyanins of PJ were 154.90 (mg per 100 g) and 28.90 mg cyaniding-3-glucoside per 100 g of juice, respectively (Bazargani-Gilani et al., 2014). The phenolics inhibited the microbial growth in samples treated with PJ, by protein binding or enzyme inhibition (Kumar & Vaithiyanathan, 1990). Similar reports have also been presented in fresh ground beef by using grape seed extract and pine bark extract (Ahn, Grün, & Mustapha, 2007). In present study, PJ–CH–Z 2% treatment was the most effective in TVC as well as in other investigated microbial groups 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. Antimicrobial activity of ZEO was attributed to phenolic compounds such as thymol and carvacrol. As previously stated, antimicrobial activity of PJ, is attributed to its major phenolic components such as tannins and phenolics. 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, Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001). The mechanism of action of EOs, including ZEO, 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). It is now well established that Pseudomonas spp. may form a significant part of the spoilage microflora of chicken meat stored under refrigeration (Jay, Loessner, & Golden, 2005). Pseudomonas spp., are known to compete other bacterial groups (Gram-positive or Gram-negative) for nutrients by forming siderophores, that may inhibit growth of both spoilage microorganisms and pathogens (Wei, Wolf, & Hammes, 2006). Proteolysis is an important phenomenon involved in the meat spoilage. The microflora of chicken meat particularly pseudomonads are responsible for proteolysis and the subsequent slime

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Fig. 1. Changes in TVC (a), Pseudomonas spp., (b), LAB (c), Enterobacteriacea (d), Psychrotrophic bacteria (e) and yeasts–molds (f) of chicken breast meat during storage at 4 °C. Treatments: Control (C), pomegranate juice (PJ), pomegranate juice with chitosan (PJ–CH), pomegranate juice with chitosan and ZEO 1% (PJ–CH–Z 1%) and pomegranate juice with chitosan and ZEO 2% (PJ–CH–Z 2%). Different letters indicate a statistically significant difference (P b 0.05) for each time.

production. This event starts when the bacterial counts reach 107– 108 CFU/g and the contents of glucose and gluconate are exhausted (Nychas, Dillon, & Board, 1988; Nychas & Tassou, 1997). Initial Pseudomonas spp. count was 4.04 log CFU/g (Fig. 1b), increasing during storage to reach final population of ca. 8.85 log CFU/g (control samples), whereas respective counts for PJ, PJ–CH, PJ–CH–Z 1% and PJ–CH–Z 2% were about 0.8, 1.76, 2.65 and 2.91 log CFU/g lower than in the control samples. Pseudomonas spp. population in all treatments was significantly (P b 0.05) lower than control. Samples containing ZEO were the most effective treatments for the inhibition of Pseudomonas spp., probably due to the combined antimicrobial actions of PJ, chitosan and ZEO. This finding is in agreement with the results reported for a poultry product treated with chitosan and thyme oil (Giatrakou et al., 2010). In other study, a combined application of nisin and EDTA inhibited Pseudomonas spp. growth in fresh chicken meat (Economou et al., 2009). LAB (Fig. 1c) as facultative anaerobic species was found to be member of the microbial flora of the chicken meat (Giatrakou et al., 2010). Initial population of LAB was 4.87 and increased to 8.62 log CFU/g on final day of storage. However, significantly lower LAB counts (P b 0.05) were recorded for PJ, PJ–CH and PJ–CH–Z samples stored during the entire storage period under refrigeration. Of all the antimicrobial treatments in this study, PJ–CH and PJ–CH–Z 1 and 2% treatments were found to be the most effective in inhibiting growth of

LAB in meat samples, almost resulting in a 1.5 log cycle decrease during the storage period, probably attributed to the combined antimicrobial action of PJ, chitosan and ZEO. In one study, the combined use of rosemary essential oil and chitosan on fresh pork sausages resulted in a 2.0 log CFU/g reduction of the final population of LAB (Georgantelis, Ambrosiadis, Katikou, Blekas, & Georgakis, 2007). In our study, Enterobacteriaceae, a facultative anaerobic bacterial group, formed a substantial part of the chicken meat microbial flora and reached final counts of 7.68 logs on day-20 (Fig. 1d). PJ, PJ–CH and PJ–CH–Z treatments produced significantly lower (P b 0.05) Enterobacteriaceae counts (approximately 1–2 log cycles) as compared to the control samples (day-20). Similarly, other researchers have reported that PJ inactivated Enterobacteriacea especially Escherichia coli (Dahham, Ali, Tabassum, & Khan, 2010; Salam, Tarik, Danfeng, & Mehrdad, 2011). Chitosan in combination with sulphite selectively inactivated Enterobacteriaceae to an undetectable level in fresh pork sausages (Roller et al., 2002). The Gram-negative Psychrotrophic bacteria (PTC) are the major group of microorganisms responsible for spoilage of aerobically stored fresh meat at chilled temperatures (Ibrahim Sallam, 2007). In our study, the initial PTC (day 0) of samples ranged from 3.19 log CFU/g, in PJ–CH–Z 2% samples, to 4.10 log CFU/g in controls (Fig. 1e). Additionally, the growth pattern of PTC showed that control being the highest at day

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lipase that cause an increase in volatile bases (e.g., ammonia and trimethylamine) during prolonged storage (Chaijan, Benjakul, Visessanguan, & Faustman, 2005). Our results are in agreement with those reported for reduced-fat sausages (Lin & Chao, 2001), ready-tocook chicken product (containing chitosan and essential oil) (Giatrakou et al., 2010), and cooked chicken patties (containing pomegranate juice) (Naveena, Sen, Vaithiyanathan, Patil, & Kondaiah, 2010). However, increasing pH values have been reported for chicken breast meat containing pomegranate juice phenolic solution during refrigerated storage (Vaithiyanathan et al., 2011). 3.4. Peroxide value

Fig. 2. Changes in pH of chicken breast meat during storage under refrigeration. Treatments: Control (C), pomegranate juice (PJ), pomegranate juice with chitosan (PJ–CH), pomegranate juice with chitosan and ZEO 1% (PJ–CH–Z 1%) and pomegranate juice with chitosan and ZEO 2% (PJ–CH–Z 2%). Different letters indicate a statistically significant difference (P b 0.05) for each time.

20 (10.37 log CFU/g), followed by PJ treatment (8.97 log CFU/g), PJ–CH (8.43 log CFU/g), PJ–CH–Z 1% (7.45 log CFU/g) and the lowest count (7 log CFU/g) was detected in PJ–CH–Z 2% samples. Similarly, it has been reported that pomegranate juice phenolics inactivated Psychrotrophic bacteria in chicken breast meat under refrigerated storage (Vaithiyanathan et al., 2011). With regard to yeasts/molds species known to be involved in the spoilage of chicken meat, all of the antimicrobial treatments examined in the present study caused significantly lower (P b 0.05) counts for yeasts/molds in PJ, PJ–CH, PJ–CH–Z 1% and PJ–CH–Z 2% chicken samples as compared to the control samples during the storage period (Fig. 1f). Antifungal activity of PJ against different kinds of molds and yeasts has been reported (Dahham et al., 2010). 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., 2007) led to a reduction of almost 3 and 2 log cycles, respectively. Results of our study (microbiological data) showed that PJ can be used as a potent antimicrobial agent in food preservation. Also, the results indicated that chitosan as edible coating can improve the antimicrobial effects of PJ in food. Moreover, chitosan dipping when combined with ZEO resulted in a more effective natural antimicrobial treatment. 3.3. Determination of pH value Changes in pH values of samples during storage under refrigeration are shown in Fig. 2. The initial pH of fresh chicken meat (pH 5.9) was in agreement with the results of a previous work (Economou et al., 2009). The pH values of control samples increased during the storage period, whereas other samples showed decreasing throughout storage probably due to the presence of acidified antimicrobial treatments such as PJ, CH and ZEO. The increasing of pH in the control samples may be due to action of endogenous or microbial enzymes such as protease and

Table 1 shows the changes in peroxide value (PV) in chicken breast meat during refrigerated storage for 20 days. In control samples, the PV increased from 0.065 to 0.33 meq peroxides/kg lipid after 5 days of storage and decreased thereafter to 0.13 after 20 days of storage. The PV of PJ, PJ–CH, PJ–CH–Z 1% and PJ–CH–Z 2%, increased from 0.065 to 0.265, 0.05 to 0.21, 0.065 to 0.165 and 0.045 to 0.15 meq peroxides/kg lipid after 10 days of storage and decreased thereafter to 0.13, 0.13, 0.11, 0.099 and 0.065 after 20 days of storage, respectively. The increase was probably due to the faster rate of formation of peroxides during days 5 and 10 of storage than degradation of peroxides into secondary oxidation products. The degradation of peroxides is observed in breast meat after 5 and 10 days of storage. Similar patterns were found by other researchers in salted raw minced chicken breast meat (Teets & Were, 2008) and in raw chicken breast meat (Soyer, Özalp, Dalmış, & Bilgin, 2010) during frozen storage. Not only PJ retarded initiating of lipid oxidation for 5 days in all of treatments, but also decreased amount of PV in all of treatments significantly (P b 0.05) as compared to control. This can be correlated to high amount of phenolic compounds in PJ. Our findings were in agreement with the results of other studies (Topuz, Yerlikaya, Ucak, Gumus, & Büyükbenli, 2014; Yasoubi, Barzegar, Sahari, & Azizi, 2010). Furthermore, the results showed that chitosan and ZEO improved antioxidant activity of PJ and PJ–CH–ZEO 2% samples significantly (P b 0.05) exhibited the most decline in PV. This can be attributed to protecting effect of coating against oxidation of phenolic compounds of PJ (Varasteh, Arzani, Barzegar, & Zamani, 2012). 3.5. TBARS values The results of TBARS analyses of chicken samples during refrigerated storage are presented in Table 2. Different treated samples (PJ, PJ–CH, PJ–CH–Z 1% and PJ–CH–Z 2%) significantly (P b 0.05) reduced malondialdehyde (MDA) values, as compared to the control (C) ones. In general, lipid oxidation of control and treated chicken samples was low and below 0.5 mg MDA/kg showing no oxidative rancidity during the storage period. In control and all of the treatments an increasing trend in TBARS values was observed until day 5 and 15, respectively that followed by a decrease until day 20 of storage. This pattern was similar to that reported in chicken breast meat (Chouliara, Karatapanis, Savvaidis, & Kontominas, 2007) which might be attributed to initial formation of MDA and its possible decomposition during the later stages of storage. However, PJ in all of treatments decreased lipid

Table 1 Changes in peroxide value (meq O2/kg fat) of chicken breast meat during refrigerated storage. Treatments: Control (C), pomegranate juice (PJ), pomegranate juice with chitosan (PJ–CH), pomegranate juice with chitosan and ZEO 1% (PJ–CH–Z 1%) and pomegranate juice with chitosan and ZEO 2% (PJ–CH–Z 2%). Different letters in each column indicate a statistically significant difference (P b 0.05). Storage time (day) Treatment C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

0

5 a

0.065 ± 0.02 0.065 ± 0.007a 0.05 ± 0.01a 0.065 ± 0.007a 0.045 ± 0.007a

10 a

0.33 ± 0.04 0.11 ± 0.01b 0.08 ± 0.01c 0.075 ± 0.007c 0.055 ± 0.007d

15 b

0.215 ± 0.01 0.265 ± 0.02a 0.21 ± 0.01b 0.165 ± 0.02c 0.15 ± 0.01c

20 a

0.18 ± 0.02 0.16 ± 0.01a 0.11 ± 0.02b 0.1 ± 0.007b 0.08 ± 0.01c

0.13 ± 0.02a 0.13 ± 0.01a 0.11 ± 0.01b 0.099 ± 0.02b 0.065 ± 0.02c

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Table 2 Changes in TBARS value (mg MDA/kg) of chicken breast meat during refrigerated storage. Treatments: Control (C), pomegranate juice (PJ), pomegranate juice with chitosan (PJ–CH), pomegranate juice with chitosan and ZEO 1% (PJ–CH–Z 1%) and pomegranate juice with chitosan and ZEO 2% (PJ–CH–Z 2%). Different letters in each column indicate a statistically significant difference (P b 0.05). Storage time (day) Treatment

0

5

10

15

20

C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

0.042 ± 0.006a 0.018 ± 0.01a 0.028 ± 0.01a 0.018 ± 0.01a 0.014 ± 0.006a

0.22 ± 0.006a 0.017 ± 0.002b 0.016 ± 0.01b 0.014 ± 0.002b 0.011 ± 0.0007b

0.16 ± 0.007a 0.07 ± 0.001b 0.06 ± 0.0007b 0.03 ± 0.002c 0.017 ± 0.001d

0.137 ± 0.007b 0.161 ± 0.01a 0.146 ± 0.006b 0.137 ± 0.007b 0.127 ± 0.006c

0.128 ± 0.006a 0.112 ± 0.01ab 0.106 ± 0.009b 0.09 ± 0.001bc 0.08 ± 0.001c

oxidation significantly (P b 0.05) as compared to the control. The large amount of phenolics contained in PJ may cause its strong antioxidant ability. Our results were in agreement with previous studies (Naveena, Sen, Vaithiyanathan, Babji, & Kondaiah, 2008; Vaithiyanathan et al., 2011). Chitosan enriched with ZEO (1 and 2%) significantly (P b 0.05) increased antioxidant activity of PJ against lipid oxidation. This increase was probably due to protecting effect of chitosan on numerous degradative reactions of total phenolic content of PJ during storage (Varasteh et al., 2012). 3.6. Protein oxidation The protein carbonyl content, as a measure of the extent of protein oxidation, is shown in Fig. 3. Carbonyl compounds are formed as a result of the oxidative deterioration of side chains of lysine, proline, arginine and histidine residues (Stadtman & Levine, 2003). However, less attention has been given to protein oxidation as compared lipid oxidation. Results of protein oxidation indicated that the treated (PJ, PJ–CH, PJ– CH–Z 1% and PJ–CH–Z 2%) chicken samples produced lower (P b 0.05) carbonyl content, as compared to the control (C) samples throughout refrigerated storage. Similar to lipid oxidation, phenolic compounds of PJ can also exhibit antioxidant activity by reducing protein oxidation (Naveena et al., 2008). Phenolics with free hydroxyl group may have spared SH group of proteins from further oxidation in treated sample and resulted in higher amount of SH concentration than in untreated samples. The results showed that chitosan and ZEO improved antioxidant activity of PJ so that treatment PJ–CH–ZEO 2% significantly (P b 0.05) exhibited the most decline in carbonyl content at the end of storage period. This can be correlated to the protecting effect of coating on phenolic compounds of PJ.

Persian Fesenjan and Khoreshte Anar are the most popular food containing pomegranate that are made with chicken meat in the north of Iran. The results of color score showed that dipping chicken breasts in pomegranate juice kept them moist, infused them with sweet and sour fruit flavor and imparted an exciting pink tinge everyone liked to see in a chicken breast. Also the findings of texture analysis indicated that the natural acids in pomegranate juice helped to tenderize chicken meat and added a brittleness and palatability to them. Missed scores in taste attribute are due to off-flavor of samples during storage. However, the taste, odor and overall acceptability scores started declining after days 5, 10, 15 and 20 in C, PJ, PJ–CH, PJ–CH–Z (1%, 2%) treatments, respectively. These results were in agreement with microbial and chemical findings of samples which may be due to high microbial load, production of lipid and protein oxidation products such as ammonia that may lead to production of off-odor and offflavor which may be a cause for poor score for these samples. 4. Conclusion It can be concluded that the pomegranate juice has the ability to delay microbial and chemical changes, extend the shelf-life and exhibit desirable sensory attributes including taste, color, odor, texture and overall acceptability in chicken breast meat. Therefore, considering the consumer preference for natural additives, pomegranate juice can be used as a natural antioxidant, antimicrobial, flavoring, texturing and coloring additive in chicken breast as well as other kinds of meat products. The results also revealed that chitosan and ZEO have potential to maintain and prolong these characteristics of pomegranate juice during storage. The coatings allow longer storage at refrigerated temperatures (4 °C) because they can reduce phenolic degradation of PJ and maintain nutritional quality. Finally, PJ–CH–Z 2% had the best

3.7. Sensory analysis Sensory evaluation showed that treatments containing PJ were significantly (P b 0.05) more pleasant than control in all sensory attributes during storage period (Table 3). The results also indicated that single PJ treatment earned the highest score among other treatments in all sensory attributes up to 10 days of storage. This can be correlated to the unique flavor of pomegranate juice. Pomegranate juice was described as providing sweet and sour taste, musty/earthy and fruity odors, with an astringent mouthfeel. Overall, the flavor of pomegranate fruit derives from a combination of various taste, aroma and mouthfeel sensations, which are perceived simultaneously by the brain and stimulate appropriate satisfaction and pleasure (Schwab, Davidovich‐ Rikanati, & Lewinsohn, 2008). The overall aroma of pomegranate fruit derives from a mixture of various volatiles that comprise mainly alcohols, aldehydes, terpenes and hexanal derivates (hexanal, hexanol and (Z)-3-hexenol) and terpenes (β-pinene, limonene, α-terpineol and β-caryophyllene) and they can be considered as key aroma volatiles in pomegranate fruits which together provide green, woody, earthy, fruity, floral, sweet and musty notes (Vázquez‐Araújo et al., 2011). Therefore, pomegranate juice products are one of the most popular flavorings used to give flavor to several foods such as chicken, fish, salads and appetizers in Iran and Turkey (Karabiyikli & Kisla, 2012).

Fig. 3. Changes in carbonyl content of chicken breast meat during refrigerated storage. Treatments: Control (C), pomegranate juice (PJ), pomegranate juice with chitosan (PJ–CH), pomegranate juice with chitosan and ZEO 1% (PJ–CH–Z 1%) and pomegranate juice with chitosan and ZEO 2% (PJ–CH–Z 2%). Each point is the mean of three samples taken from each treatment. Different letters indicate a statistically significant difference (P b 0.05) for each time.

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Table 3 Changes in sensory attributes of chicken breast meat during refrigerated storage. Sensory attributes

Treatment

Storage time (day) 0

5

10

15

20

Taste

C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

7 ± 0.66b 8.7 ± 0.67Aa 7.6 ± 0.69Ab 7.5 ± 0.84Ab 7.5 ± 0.84Ab

– 8.8 ± 0.42Aa 7.9 ± 0.56Ab 7.9 ± 0.82Ab 7.9 ± 1.07Ab

– – 7.7 ± 0.94Aa 6.8 ± 1.03Aab 6 ± 1.15Bb

– – – 6.8 ± 0.78Aa 6.3 ± 0.82Ba

– – – – –

Color

C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

7.1 ± 1.37Ab 8.4 ± 0.69Aa 7.9 ± 0.73Aab 7.5 ± 0.70Aab 7.5 ± 0.84Aab

5.3 ± 0.82Bc 8.3 ± 0.67Aa 7.8 ± 0.78Aab 7.2 ± 0.63Ab 7.2 ± 0.63ABb

4.8 ± 1.81BCa 5.3 ± 2.21Ba 6.1 ± 1.52Ba 6.2 ± 1.13Ba 6.4 ± 1.26BCa

3.7 ± 1.059Cb 5.8 ± 0.78Ba 5.4 ± 0.69BCa 5.7 ± 0.48Ba 6.2 ± 0.63BCa

1.6 ± 0.69Dc 4.3 ± 0.94Bb 4.8 ± 0.63Cab 5.4 ± 0.69Ba 5.5 ± 0.70Ca

Odor

C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

7 ± 0.94Ab 8.2 ± 1.03Aa 7.6 ± 1.07Aab 7.8 ± 0.78Aab 8.1 ± 0.73Aab

1.1 ± 0.31Bb 8 ± 0.81Aa 7.6 ± 0.96Aa 8 ± 0.81Aa 7.7 ± 0.94Aa

1 ± 0Bd 2.5 ± 0.84Bc 5.3 ± 1.56Bb 7.1 ± 1.1Aa 7.4 ± 1.34ABa

1 ± 0Bc 2.1 ± 1.1Bbc 3.3 ± 1.41Cb 5.2 ± 1.03Ba 6.2 ± 0.78BCa

1 ± 0Bc 1.7 ± 0.67Bbc 2.2 ± 1.03Cb 5.2 ± 0.91Ba 6 ± 0.81Ca

Texture

C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

7.2 ± 1.03Ac 9 ± 0Aa 8.4 ± 0.51Aab 8.1 ± 0.56Ab 8.2 ± 0.63Aab

6.4 ± 0.96Ab 8.8 ± 0.63Aa 8.2 ± 0.63Aa 8.1 ± 0.31Aa 8.1 ± 0.31Aa

5 ± 0.81Bb 6.4 ± 0.84Ba 6.3 ± 0.67Ba 6.4 ± 0.69Ba 6.5 ± 0.97Ba

2.5 ± 0.52Cb 5.2 ± 0.42Ca 5.7 ± 0.82Ba 5.9 ± 0.73BCa 5.7 ± 0.67BCa

1.8 ± 0.78Cb 2.4 ± 1.07Db 3. ± 1.41Cb 5.5 ± 0.52Ca 5.6 ± 0.69Ca

Overall

C PJ PJ–CH PJ–CH–Z 1% PJ–CH–Z 2%

7 ± 0.94Ab 8.7 ± 0.67Aa 8.2 ± 0.63Aa 8 ± 0.66Aa 7.8 ± 0.78Aab

5.2 ± 1.03Bc 8.7 ± 0.67Aa 8.2 ± 0.42Aab 7.9 ± 0.31Ab 7.8 ± 0.42Ab

1.8 ± 0.78Cc 5.5 ± 0.84Bb 6.2 ± 1.13Bab 6.4 ± 0.84Bab 7 ± 0.81ABa

1.7 ± 0.94Cc 5.1 ± 0.87Bb 5.6 ± 0.96Bab 6.1 ± 0.73Bab 6.7 ± 0.94Ba

1.4 ± 0.69Cc 2.1 ± 1.28Cbc 3 ± 1.15Cb 4.9 ± 1.1Ca 5.6 ± 1.17Ca

Treatments: Control (C), pomegranate juice (PJ), pomegranate juice with chitosan (PJ–CH), pomegranate juice with chitosan and ZEO 1% (PJ–CH–Z 1%) and pomegranate juice with chitosan and ZEO 2% (PJ–CH–Z 2%). Means within the same row (A, B, C) and the same column (a, b, c) with different letters are significantly different (P b 0.05).

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