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Effect of modified atmosphere and active packaging on the shelf-life of fresh bluefin tuna fillets
Journal of Food Engineering 105 (2011) 429–435
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Effect of modified atmosphere and active packaging on the shelf-life of fresh bluefin tuna fillets Elena Torrieri a,⇑, Pier Antimo Carlino c, Silvana Cavella a, Vincenzo Fogliano a, Ilaria Attianese b,d, Giovanna Giuliana Buonocore b, Paolo Masi a,c a
Department of Food Science, University of Naples Federico II, Via Università 100, Parco Gussone, 80055 Portici (NA), Italy Institute of Composite and Biomedical Materials, National Research Council (CNR), P. le E. Fermi, 1, 80055 Portici (NA), Italy Centre of Food Innovation and Development in the Food Industry, University of Naples Federico II, Via Università 100, Parco Gussone, 80055 Portici (NA), Italy d Department of Materials and Production Engineering, University of Naples Federico II, P. le Tecchio 80, 80125, Naples, Italy b c
a r t i c l e
i n f o
Article history: Received 3 November 2010 Received in revised form 2 February 2011 Accepted 18 February 2011 Available online 23 February 2011 Keywords: Active film Antioxidant MAP Shelf-life Tuna fish
a b s t r a c t The aim of this work was to study the influence of the combined use of MAP and antioxidant-based active packaging on the shelf-life of fresh bluefin tuna fillets stored at 3 °C. Active packaging films were produced by embedding a-tocopherol into an unstabilized low density polyethylene (LDPE) matrix at three concentrations (0.1%, 0.5%, 1%). a-Tocopherol release kinetics, in vitro antioxidant activity, oxygen permeability and crystallinity degree were determined to characterize the film. Preliminary shelf-life tests were performed to select critical quality indices, the best gas composition and the best a-tocopherol concentrations in the active film. Then, the effectiveness of the chosen active packaging film in combination with MAP was assessed by monitoring critical quality indices of fresh bluefin tuna fillet during storage at 3 °C for 18 days. Obtained results showed that (i) 100% N2 atmosphere has a protective effect on haemoglobin and lipid oxidation processes monitored, (ii) active film is able to reduce fat oxidation, (iii) the combined effect of MAP and active packaging can be considered a valuable tool to increase the shelf-life of raw fish products. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fresh bluefin tuna (Thunnus thynnus) are heavily targeted by the Japanese raw fish market, where all bluefin species are highly prized for sushi and sashimi. In Italy, the processed fish market mainly consists of tuna in oil packed in glass jars or canned. However, local consumption of fresh bluefin tuna has greatly increased and tuna fillets packed as ready-to-use products could respond to the growing market of minimally processed food combining high quality and high convenience. Nevertheless, fresh bluefin tuna fillets are highly perishable products and appropriate packaging must be developed to extend product shelf-life (Davis, 1999). Lipid and haemoglobin oxidation are the main causes of their alteration during shelf-life. Lipid oxidation can be either enzymatic or nonenzymatic and causes development of off-flavours (Venugopal, 2006). On the other hand, haemoglobin oxidation lends the meat a purplish-red and brown colour due to deoxymyoglobin and metmyoglobin formation, respectively (Blakistone, 1999; Gill, 2003; Gill and Gill, 2005; Mancini and Hunt, 2005). Colour alteration ⇑ Corresponding author. Tel.: +39 081 2539456; fax: +39 081 7754942. E-mail address: [email protected] (E. Torrieri). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.02.038
makes products unattractive, affecting consumer preference at purchase (Carpenter et al., 2001). The most suitable packaging technology for these products is modified atmosphere packaging (MAP), an effective system to preserve quality and to maintain the hygienic and sensory characteristics of perishable fish (Corbo et al., 2005; Lopez-Galvez et al., 1995; Pastoriza et al., 1998; Reddy and Armstrog, 1992; Sivertsvik et al., 2002; Stammen et al., 1990; Torrieri et al., 2006). MAP combined with refrigeration showed positive effects on the quality of cod fillets, particularly when very fresh raw material is used (Bøknæs et al., 2000). Further, MAP has been found to increase the shelf-life of seer fish (Scomberomorus commerson) (Yesudhason et al., 2009, 2010), Mediterranean swordfish (Kykkidou et al., 2009) and tuna fish burgers (Carlino et al., 2009). However, no information is available in the literature on preservation of bluefin tuna fillets by MAP. Antioxidant (AO) packaging films have been developed to slow down or minimize oxidative deterioration of packaged products during storage. The use of both synthetic and natural antioxidants embedded into plastic films to improve the shelf-life of packaged products has been explored elsewhere. Lee et al. (2004b) applied a 3-mm thick nisin and/or a-tocopherol coating on paper to confer
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an antimicrobial and antioxidative property for use in the food packaging industry. Nerin et al. (2006) described the promising results of a new natural antioxidant active packaging system obtained by embedding rosemary extract into plastic film. The film was able to inhibit both haemoglobin and lipid oxidation in beef, improving the visual appearance of meat during its shelf-life. Moore et al. (2003) studied the effects of both synthetic and natural antioxidants (0.1%w/w) in low density polyethylene (LDPE) film on the preservation of fresh beef colour. They showed that after nine days of storage at 4 °C, the meat packed with the butylated hydroxyanisole-LDPE film had higher ‘‘a’’ (redness) values than any other film treatment, whereas a-tocopherol-containing film did not show any protective effect with respect to control film (without an antioxidant). Moreover, Graciano-Verduco et al. (2010) showed that the use of LDPE films containing a-tocopherol in concentrations between 19 and 30 mg/g are able to maintain the oxidative stability of corn oil during 16 weeks of storage at 30 °C. Torres-Arreola et al. (2007) report that LDPE films containing the synthetic antioxidant butylated hydroxytoluene may be used to prevent lipid oxidation and to prolong the shelf life of sierra fish. Performance of a new active packaging film containing natural antioxidants (from barley husks) with respect to lipid oxidation in frozen Atlantic salmon (Salmo salar L.) (Pereira de Abreu et al., 2010) and in Atlantic halibut (Hippoglossus hippoglossus) (Pereira de Abreu et al., 2011) were evaluated. The results obtained confirm the effectiveness of natural antioxidants derived from barley husks (7 mg dm2) in slowing down lipid hydrolysis and increasing the oxidative stability of salmon flesh and Atlantic Halibut. The combined use of MAP and antioxidants embedded into plastic films has received little attention. Camo et al. (2008) proved that rosemary-based and oregano-based active films resulted in enhanced oxidative stability of lamb steaks packaged in modified atmosphere and exposed to light illumination at 1 °C. In particular, active films with oregano were significantly more efficient than those with rosemary. However, to the best of our knowledge, use of tocopherol-based active film to package tuna fish products has not been reported to date. Thus, the present work aimed to obtain an active packaging film containing a-tocopherol and assess its combined effect with MAP on shelf-life extension of packaged fresh bluefin fillets. Active films were characterized with particular regard to a-tocopherol release kinetics from the films into a food simulant at 3 and 25 °C, their oxygen barrier properties and their effectiveness by means of in vitro tests. Shelf-life tests of fresh bluefin fillets packaged and stored at 3 °C were performed. In particular, their microbiological, chemical and physical characteristics were monitored up to 18 days.
2. Materials and methods 2.1. Materials Unstabilized low density polyethylene (LDPE) was supplied by Polimeri Europa (Italy). The natural antioxidant a-tocopherol and ethanol (96%v/v) as well as trichloroacetic acid, thiobarbituric acid peptone and malonaldehyde (MDA) were purchased from Sigma Aldrich (Milan, Italy). Bluefin tuna (Thunnus thynnus) were captured in the Mediterranean sea in April 2009 and were reared for five months in cages off the island of Procida (Naples, Italy). In November 2009, immediately after slaughter, the fish were gutted, washed and bled in salt water and ice, and finally the tuna fillets were frozen at 60 °C and stored at 25 °C. Three tuna fillets (around 20 kg) were packaged on ice in insulated polystyrene boxes, delivered to the university laboratory and kept in a freezer at 25 °C.
Polystyrene tray laminated with a multilayer barrier film (V = 500 cc; CoopBox, Bologna, Italy) and sealed with a film of PA/EVOH/PE (PO2 = 1.3 cc/m2 24 h atm; PH2O = 5.1 g/m2 24 h atm; thickness = 54 lm, CoopBox, Bologna, Italy) were used for shelf-life tests. 2.2. Film preparation The active films were prepared through a two-step process. The first step consisted of melt blending LDPE matrix with a-tocopherol by using an internal mixer (Thermo Scientific, Haake PolyLab QC, Karlsruhe, Germany) at 20 rpm at T = 140 °C for 6 min. The melt was pressed with a P300P hot press (Collin, Germany). The obtained sheets were then cut into pellets and fed into a co-rotating laboratory twin-screw extruder (Prism Eurolab 16, ThermoERMO Electron Corporation, Stone, UK) equipped with a sheet die of width 10 cm used to obtain the active antioxidant films. The temperature of the feeding, intermeshing and final zones of the extruder was maintained at 135–150–145 °C, respectively. Different amounts of a-tocopherol (0, 0.1, 0.5 and 1% w/w) were added to LDPE in order to obtain the control and active films coded as: LDPE, LDPE/0.1TOC, LDPE/0.5TOC, LDPE/1TOC. 2.3. Active film characterization 2.3.1. a-Tocopherol release tests Release kinetics of a-tocopherol from the three different films (LDPE/0.1TOC, LDPE/0.5TOC, LDPE/1TOC) into the chosen food simulant were determined. As food simulant, 96% ethanol was used instead of the normally proposed olive oil in order to avoid the quantification problems arising from its originally high a-tocopherol content (Sirò et al., 2006). Known amounts of active films (ffi1.5 g) were brought into contact with 80 ml of 96% ethanol at two different temperatures, T = 25 °C and T = 3 °C. The release kinetics were evaluated by monitoring the antioxidant concentration in the surrounding solution as a function of time until a constant value was reached. a-Tocopherol concentration in ethanol was determined using an HPLC (Agilent Model 1100, Milan, Italy) following the chromatographic method proposed by Sirò et al. (2006). The calibration curve was constructed for peak area against a-tocopherol concentration of standard solutions from 10 to 500 ppm. 2.3.2. In vitro antioxidant activity test Antioxidant activity of a-tocopherol released from LDPE films into ethanol was tested in a model system following the method proposed by Ruberto and Baratta (2000). The method is based on the spectrophotometric determination of the rate of conjugated diene formation from linoleic acid, in the absence and in the presence of a potential antioxidant. Known amounts of a-tocopherol released at 25 °C in ethanol from the three investigated films were added and peroxidation was monitored by recording the absorbance at 232 nm for 15 min. Effectiveness was measured based on the percentage antioxidant index (AI%) calculated using the formula: AI% = (1 T/C) 100 where C is the absorbance value at 232 nm of the oxidized linoleic acid after 15 min and T is the absorbance of the oxidized linoleic acid in the presence of the samples containing the released a-tocopherol under the same conditions. 2.3.3. Oxygen permeability Oxygen permeability of LDPE films was determined by means of Ox-Tran (Mocon, Model 2/20, Neuwied Germany). Samples with a surface area of 5 cm2 were tested at 23 °C, setting the relative humidity (RH) at the downstream and upstream side of the film at 50%. Each test was made in duplicate.
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2.3.4. Analysis of crystallinity degree A Differential Scanning Calorimeter DSC 2920 (TA Instrument) was used to measure the crystallinity of the films. Thermograms were recorded in three steps: (1) heating from 0 to 180 °C, (2) cooling from 180 to 0 °C and (3) a second heating from 0 to 180 °C. The heating and cooling rates were 10 °C/min. The crystallinity was calculated from the ratio of the melting enthalpy of the samples to the melting enthalpy of 100% crystalline polyethylene (293 J/g) (Wunderlich, 1999). 2.4. Shelf-life testing 2.4.1. Packaging and storage condition – Preliminary test In order to select critical quality indices and the best gas composition, tuna fillets were cut into slices by using a bandsaw (KT210-Ecom, Movinox, Acquaviva Picena (AP), Italy). Each slice was 1.5 cm thick and weighed 175 g. The slices were then laid on a polystyrene tray laminated with a multilayer barrier film and sealed with a film of PA/EVOH/PE. One slice was packed for each tray. Samples were modified atmosphere packed by using a packaging machine (Minipack Torre, TSM, 105, Cava dei Tirreni (SA), Italy). The ratio between the volume of gas and weight of food product (V/W ratio) was 2.5:1. Samples of bluefin tuna fillet were packaged in air and MAP (60% O2– 40% CO2; 100% N2) and stored for 9 days at 3 °C. High oxygen and high carbon dioxide amount were extensively reported to be protective for fresh food product at high content of haemoglobin (Gill and Gill, 2005). 100% N2 atmosphere was chosen because low oxygen concentration are protective for lipid oxidation (Lee et al., 2008). After 0, 2, 5, 7 and 9 days microbiological analysis, headspace composition in terms of O2% and CO2%, pH, colorimetric analysis (L⁄, a⁄, b⁄) and fat oxidation measurement (TBARS) were performed. In order to select the best a-tocopherol concentrations in the active film, one slice of samples was laid on a polystyrene tray laminated with a multilayer barrier film and the exposed surface was completely covered with the active film (15 cm 7 cm) LDPE/ 0.1TOC, LDPE/0.5TOC, LDPE/1TOC. Then, samples were packed in air by using a packaging machine using the PA/EVOH/PE to seal the tray. Samples were stored for 11 days at 3 °C and critical quality indices selected after the preliminary test previously described were monitored after 0, 4, 7 and 11 days. 2.4.2. Packaging and storage condition – Main test Once the best gas composition, the optimal a-tocopherol concentration and the critical quality indices had been selected, the effect of MAP and active packaging on product shelf life was studied. Samples were divided into four lots as detailed: lot I: samples packed in air with film not containing a-tocopherol (LDPE); lot II: samples packed in air with active film (LDPE/0.5TOC); lot III: samples packed in MAP (100% N2) with film not containing a-tocopherol (LDPE); lot IV: samples packed in MAP (100% N2) with active film (LDPE/0.5TOC). All samples were stored for 18 days at 3 °C and critical quality indices were analyzed after 0, 1, 4, 7, 11, 15, 18 days. 2.4.3. Microbiological analysis Standard enumeration methods were used to determine the microbial populations of the fish during chilled storage. Two samples of fish (25 g) were taken from each package, then diluted with 225 ml of peptone salt solution (0.85% NaCl–0.1% peptone) and homogenized with a Stomacher for 2 min. The sample was then serially diluted as needed for plating. The following media and incubation conditions were used: Plate Count Agar (PCA, Oxoid) for aerobic mesophilic bacteria (AMB) by pour plating, incubated at 30 °C for 72 h; Violet Red Bile Glucose Agar (VRBGA, Oxoid) for Enterobacteriaceae (E) counts, incubated at 37 °C for 24 h. Microbial counts were performed in duplicate and expressed as CFU/g, MRS agar (Oxoid) for Lactic Acid Bacteria (LAB) counts, incubated anaer-
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obically at 30 °C for 48 h; Streptomycin Thallous Acetate Agar (STAA, Oxoid) with STA selective supplement (Oxoid) for Brochothrix thermosphacta counts, incubated at 25 °C for 48 h. 2.4.4. Chemical analysis Gas analysis: O2 and CO2 concentrations (%v/v) in the package headspace were monitored by means of a portable PBI Dansensor A/S (Check Mate 9900 O2/CO2; Ringsted, Denmark) analyzer (accuracy ±0.1%) by sampling with a needle 3 ml of gas from the package headspace. One measurement was carried out on each sample. pH determination: pH was measured on samples obtained by mixing homogenized fish flesh with water (ratio 1:2) by using a pHmeter Cyber Scan pH/lon 510 (Eutech Instruments Pte Ltd. Ayer Rajah Crescent Singapore). The pHmeter was equipped with a Schott electrode which was previously calibrated with buffer solutions (pH 4 and pH 7) at 20 °C. Four measurements were carried out on each sample. TBARS analysis: Thiobarbituric acid reactive substances were determined by using the extraction procedure of Lemon (1975) and Kilic and Richards (2003). A 0.5 g sample was blended with 5 ml of extraction solution containing 25% trichloroacetic acid (TCA) and 10 ml of water. The samples were homogenized with an Ultra-Turrax for 30 s. The homogenate was filtered through Whatman 1 filter paper and the filtrate (3.5 ml) was mixed with 1.5 ml of thiobarbituric acid (TBA) solutions (0.6%) and vortexed. The mixture was heated at 70 °C for 15 min in a water bath, cooled, and then centrifuged at 2000 rpm for 5 min. Absorbance was determined at 532 nm against a blank containing 1 ml of TCA extraction solution and 1 ml of TBA solution. Calibration curves were prepared using malonaldehyde (MDA) standard working solutions. A stock MDA solution was obtained after hydrolysis of 690 mg of 1,1,3,3-tetraethoxypropane (TEP) in 10 ml of 0.1 N hydrochloric acid (HCl). The reaction was carried out in a 100-ml screw-capped bottle. The hydrolyzed TEP solution was then accurately diluted to 100 ml with ultrapure water. Appropriate working solutions were prepared from the stock MDA solution. The tubes used for the calibrations, including the blank and the corresponding samples to be analyzed, were always incubated at the same time. The reaction and the detection were as described above. The TBARS values are expressed as nmoles of malonaldehyde/g of freeze dried samples.
2.4.5. Physical analysis Colorimetric measurement: colour of the bluefin tuna fillets was measured with a tristimulus colorimeter (Minolta Chroma Meter, model CR-300) with a circular measurement area (D = 8 mm). The colorimeter was calibrated using a white standard plate (L = 100). The L⁄, a⁄ and b⁄ values were measured on the product surface. Five readings were carried out at each position.
2.4.6. Experimental design and statistical analysis for shelf life study A full factorial design was used to study the effect of time, atmosphere, active film on the quality indices of bluefin tuna fillets. There were seven levels of storage time (0, 1, 4, 7, 11, 15, 18), two levels of atmospheres (air, 100% N2) and two levels of active film (LDPE, LDPE/0.5TOC). Three replicates were performed for each sample for a total of 84 samples. Results are reported as mean value ± standard deviation. ANOVA analysis was performed on the data to evaluate the effect of time (A), atmosphere (B), active packaging (C) and the interaction effect (AxB; BxC; AxC; AxBxC) on the quality attributes. Duncan’s test was performed to find out the source of the significant differences within samples. Significance of differences was defined at p 6 0.05. All statistical analyses were performed using the SPSS computer package (SPSS Inc. 11.5, Chicago, 2002).
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Fig. 2. Oxygen permeability values of the investigated antioxidant films obtained at T = 23 °C and at RH% = 50%.
3.1.2. Oxidation test ‘in vitro’ The effectiveness of antioxidant activity of a-tocopherol released at the equilibrium in 96% ethanol from films LDPE/0.1TOC, LDPE/0.5TOC and LDPE/1TOC was evaluated on the oxidation of a linoleic acid. The AI% values obtained were 18%, 22% and 35%, respectively. This result shows the capacity of the released antioxidant to inhibit the oxidation of the unsaturated fatty acids. As expected, its effectiveness increases as the a-tocopherol concentration in the film increases. On the contrary, Lee et al. (2004a) proved that the amount of a-tocopherol contained in their co-extruded film had no effect on retarding lipid oxidation in the model product. On monitoring the linoleic acid content during storage time of a model system (water/linoleic acid), they found that at 45 °C, over a four-day storage period, there appeared to be no differences between the levels of linoleic acid in the model product packaged in the control and in the a-tocopherol-impregnated laminate pouch structures. This may be attributed to extensive fatty acid oxidation under the storage conditions. Fig. 1. a-Tocopherol release in ethanol 96% (v/v) at T = 4 °C (a) and at T = 25 °C (b) from LDPE/0.1TOC (s) LDPE/0.5TOC (j) and LDPE/1TOC (4).
3. Results and discussion 3.1. Active film characterization 3.1.1. a-Tocopherol release tests The amount of a-tocopherol released at 3 °C and 25 °C in 96% ethanol was monitored against time (Fig. 1a and b, respectively). As expected, the release kinetics at 25 °C are faster than those obtained at the lower temperature; at T = 3 °C, a constant value had still not been reached after 25 days. Moreover, the asymptotic value reached for each film depends on the initial quantity of atocopherol added to the polymer: at 25 °C the released concentrations for LDPE/0.1TOC, LDPE/0.5TOC and LDPE/1TOC films were 5, 20 and 70 ppm, respectively. As reported by Sirò et al. (2006), due to the high extraction effect of ethanol, the asymptotic value reached for each film corresponds to the total original a-tocopherol content of the plastic bags after manufacture. Thus, the total amounts of a-tocopherol contained in the three investigated films is 0.3, 1 and 3.8 mg/g and it can be concluded that approximatively 70% of the originally added a-tocopherol has been lost after film processing, as also experienced by other authors (Sirò et al., 2006; Wessling et al., 2000).
3.1.3. Oxygen permeability Since the addition of large amounts of additives may cause changes in film characteristics, the oxygen barrier properties of the LDPE/TOC material was investigated. Data reported in Fig. 2 show that no significant differences can be observed between the neat LDPE and the active films. This may be related to the similar degree of crystallinity observed for these films (data not shown). The data concerned yield a positive result since an increase in the oxygen transmission rate, which has been found elsewhere (Wessling et al., 2000), also in this application could influence the potential positive effect of the antioxidant in the active film on shelf-life extension. 3.2. Preliminary shelf-life testing 3.2.1. Selection of critical quality indices and modified atmosphere packaging 3.2.1.1. Microbiological analysis. Initial aerobic mesophilic counts in fresh bluefin fillets were 4.2 102 CFU/g, whereas they were absent in 10 g of sample products packaged in air and in MAP (100% N2 and 60% O2–40% CO2) at 3 °C after 2, 5, 7 and 9 days of storage. Moreover, Entorobacteriaceae, B. thermosphacta and lactic bacteria were absent in 0.1 g on all samples after nine days of storage, showing that due to the high quality of the initial product microbiological alteration did not represent a critical aspect for
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product shelf life. For this reason, microbiological indices were not considered as critical for quality. Moreover, results showed that for a storage temperature of 3 °C, high CO2 concentrations were not helpful for bacteriostatic effect. 3.2.1.2. Chemical analysis. During storage, gas composition into the package headspace was monitored. In particular, it was observed that O2 concentration in the pack headspace of samples packed in air and in MAP decreased from an initial value of 21% to 5% after nine days of storage for air-packed samples, from 60% to 45% for samples stored in MAP (60% O2–40% CO2), and from 1% to 0% after one day of storage for samples stored in MAP (100% N2). Generally, an O2 decrease can be used as an index of microbial contamination (Torrieri et al., 2006), but in this case, because no growth of microorganism was observed during storage, the decrease can only be justified by oxidation processes. For samples packed in air, CO2 increased to 1.6% after 2 days and remained constant up to nine days of storage, whereas for samples stored in MAP (60% O2–40% CO2) the CO2 remain almost constant (38 ± 2%), even if a evident pack depression was observed after five days of storage, indicating CO2 solubilization in the product. This shows that high CO2 concentrations were even detrimental to product quality. No CO2 accumulation was observed during storage for samples packed in MAP (100% N2). The pH was constant throughout storage with no differences among samples (p > 0.05) and assumed an average value of 5.60 ± 0.07. Therefore, the pH parameter is not useful as a physico-chemical index of quality decay or in predicting the shelf life of bluefin tuna fillet samples. The TBARS values of samples packed with 100% N2 were lower than TBARS values of control samples (Fig. 5) and samples packed with 60% O2–40% CO2 (data not showed). Therefore, TBARS was chosen as critical index for studying lipid oxidation and 100% N2 was chosen as the optimal atmosphere to pack the product. 3.2.1.3. Physical analysis. Values of CIE a⁄ and b⁄ for samples packed in air and MAP (60% O2–40% CO2; 100% N2) throughout storage are reported in Fig. 3a and b. Samples packed in air showed a significant decrease in a⁄ from an initial value of 8 ± 1 to as low as 1 ± 1 after nine days of storage. After two days of storage a⁄ assumed a value of 5 ± 2 that was already significantly different from the value assumed at time zero (p < 0.0001). A browning of colour tuna meat is the cause of the a⁄ decrement. This result is explained by the oxidation mechanism of the myoglobin. In fact, when the fish is cut up, oxygen comes into contact with myoglobin in the exposed tuna meat surface. The oxygen is absorbed and reacts with the myoglobin to form a bright red pigment (oxy-myoglobin) which brings about the attractive red colour of fresh tuna meat. However, due to a continued exposure to oxygen during storage, the red colour of the meat gradually changes into various shades of brown due to oxidation and conversion of the oxy-myoglobin (Fe2+–O2) to the brown ferric (Fe3+) met-myoglobin (Tajima and Shikama, 1987). Although a protective effect of high oxygen concentrations on oxyhaemoglobin, oxidation is normally reported for fresh food product at high content of haemoglobin (Gill and Gill, 2005) and, in particular, it was reported for refrigerating tuna steaks (LopezGalvez et al., 1995). Our results showed that samples packed in MAP 60% O2–40% CO2 had the same behaviour as air-packed samples, displaying no protective effect of the high oxygen concentration on myoglobin oxidation. On the other hand, samples packed in MAP 100% N2 showed a constant a⁄ value during storage (p > 0.05), thus indicating a protective effect of this atmosphere on colour change (Fig. 3a). Less significant was the effect of time and atmosphere on b⁄ (Fig. 3b). In particular, b⁄ increased from 8 ± 1 to
Fig. 3. Effect of modified atmosphere package on colorimetric coordinates a⁄ (a) and b⁄ (b) of bluefin tuna fillet stored at 3 °C. (j) air; (e) 60% O2–30% CO2; (N) 100% N2.
12 ± 1 after 9 days of storage for samples packed in air and with 60% O2–40% CO2, and to a value of 9 ± 1 after 9 days of storage for samples packed with 100% N2. Experimental results showed that a⁄ and b⁄ are critical quality indices to study product shelf life and confirmed that 100% N2 atmosphere was the optimal atmosphere in which to pack the product.
3.2.2. Selection of best active film On the base of previous results, colorimetric analysis (a⁄ and b⁄) and lipid oxidation (TBARS) were selected as critical quality indices and used to select the optimal concentration of a-tocopherol present in the film. The results obtained for air-packed samples (data not shown) proved that parameter a⁄ and b⁄ and the lipid oxidation were unaffected by tocopherol concentration. On the basis of these results and given that, on visual inspection, the LDPE/1TOC appeared somewhat more yellow in colour than the reference material and less visually attractive (Wessling et al., 2000), film at 0.5% w/w (LDPE/0.5TOC) was selected to study product shelf-life.
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Fig. 5. n mole of MDA/g dw of bluefin tuna fillet after 0, 9 and 18 days of storage at 3 °C. (A: 100% N2; B: 100% N2 + LDPE; C: 100% N2 + LDPE/0.5TOC; D: air + LDPE; E: air + LDPE/0.5TOC.)
Fig. 4. Effect of active film and modified atmosphere package on colorimetric coordinates a⁄ (a) and b⁄ (b) of bluefin tuna fillet stored at 3 °C. (j) Air + LDPE; (h) air + LDPE/0.5TOC; ()100% N2 + LDPE (e) 100% N2 + LDPE/0.5TOC.
3.3. Main test: effect of active film and MAP on critical quality indices Fig. 4a and b shows the variation of colorimetric parameters a⁄ and b⁄ of fresh bluefin tuna fillets packed with active film (LDPE, LDPE/0.5TOC) and modified atmosphere packed (air-100% N2) as measured against storage time. LDPE film was used as control for the active film (LDPE/0.5TOC). Samples were packed also in air to assess the utility of MAP in presence of active film. ANOVA results highlight a significant effect of the modified atmosphere package (p < 0.05) on colorimetric parameters a⁄ and b⁄ at any storage time, whereas the effect of active packaging and interaction between MAP and active packs were not statistically significant (p > 0.05). Samples packed with 100% N2 showed a higher value of a⁄ and a lower value of b⁄ at any storage time than samples packed in air. In particular, for samples packed with 100% N2, the colorimetric parameter a⁄ increased from time zero to time four days and then
remained constant with time. Indeed, the colour of the samples packed with nitrogen was bright red compared to the bloom red colour of the control samples at time zero. A slow diffusion kinetics of the a-tocopherol from the film to the product surface may be the reason for the antioxidant not affecting myoglobin oxidation. Our results are in accordance with Moore et al. (2003) who studied the effects of tocopherol (0.1%w/ w) in polyethylene film on fresh beef colour, and showed that after nine days of storage at 4 °C a-tocopherol film showed no protective effect with respect to control film (without AO). As reported by the authors, natural antioxidants may be less likely to migrate from the film disk to the surface of the beef, thereby limiting their effect. Nevertheless for all samples packed in air ANOVA results showed that the effect of time was not statistically significant (p > 0.05). These last results can be compared with data reported in Fig. 3 that show a significant effect of time on colorimetric parameters of samples packed in air without film. Whereas, irrespective to the amount of tocopherol contained into the film, its presence on the surface of the tuna fillet showed a protective effect on product colour change, probably due to a less oxygen amount at product surface. Data related to fat oxidation of samples packed in MAP and with active films are reported in Fig. 5. It is worth to notice that the presence of the active film showed a protective effect on lipid oxidation when combined with the 100% N2 MAP (p < 0.005). These results confirm the hypothesis that the action of the antioxidant is related to the different kinetics of oxidation and diffusion. Thus, when the oxidation kinetics is slowed down by the protective atmosphere, the action of the antioxidant becomes evident. On the other hand, in air the antioxidant film has no protective effect, most likely because the oxidation process is too fast compared to the diffusion kinetics of the a-tocopherol from the film to the sample surface.
4. Conclusion Our results showed that by using only modified packaging it was possible to extend product shelf-life from 2 days for control samples (air without film) up to 18 days at 3 °C. Moreover, the combined use of MAP and the active film resulted in a less oxidized product after 18 days of storage at 3 °C. These results, which should be considered preliminary until further investigation is carried out, show considerable promise and highlight the importance of optimizing active film in terms of initial antioxidant concentra-
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tions and/or obtaining faster release kinetics of the antioxidant into food products at optimal storage conditions. Acknowledgments Part of this research was supported by the MISE (ALIMENTA) project. Special thanks are due to the Microbiological division of the Department of Food Science of the University of Naples Federico II, to Dr. Giulia Graziani and Dr. Giancarlo Di Marzo for their contribution to the experimental work. References Blakistone, B.A., 1999. Meats and poultry. In: Blakistone, B.A. (Ed.), Principles and Applications of Modified Atmosphere Packaging of Foods. An Aspen Publication, Gaithersburg, Maryland, pp. 240–290. Bøknæs, N., Österberg, C., Nielsen, J., Dalgraad, P., 2000. Influence of freshness and frozen storage temperature on quality of thawed cod fillets stored in modified atmosphere packaging. Lebensmitt.-Wissenscha und-Technolologie 33, 244– 248. Camo, J., Beltran, J.A., Roncales, P., 2008. Extension of the display life of lamb with an antioxidant active packaging. Meat Science 80, 1086–1091. Carlino, P.A., Formisano, F., Torrieri, E., Fogliano, V., Martello, A., Cavella, S., Masi, P., 2009. Optimization of bluefin tuna fishburger MAP by mixture design and triangular surface analysis. In: Buonocore, G.G., Torrieri, E. (Eds.), Shelf Life International Meeting: SLIM2008, Special Issue of Italian Journal of Food Science. Chiriotti Editori, Pinerolo, Italy, pp. 58–61. Carpenter, C.E., Cornforth, D.P., Whittier, D., 2001. Consumer preferences for beef colour and packaging did not affect eating satisfaction. Meat Science 57, 359– 363. Corbo, M.R., Altieri, C., Bevilacqua, A., Campanello, D., D’Amato, D., Sinigaglia, M., 2005. Estimating packaging atmosphere-temperature effects on the shelf life of cod fillets. European Food Research Technology 220, 509–513. Davis, H.K., 1999. Fish and shellfish. In: Blakistone, B.A. (Ed.), Principles and Applications of Modified Atmosphere Packaging of Foods. An Aspen Publication, Gaithersburg, Maryland, pp. 194–239. Gill, A.O., Gill, C.O., 2005. Preservative packaging for fresh meats, poultry, and fin fish. In: Han, J.H. (Ed.), Innovation in Food Packaging. Elsevier Academic Press, pp. 205–226. Gill, C.O., 2003. Active packaging in practice. meat. In: Ahvenainem, H. (Ed.), Novel Food Packaging Technology. Woodhead Publishing Limited and CRC Press LLC, pp. 378–396. Graciano-Verduco, A.Z., Soto-Valdez, H., Peralta, E., Cruz-Zarate, P., Islas-Rubio, A.R., Sanchez-Valdes, S., Sanchez-Escalante, A., Gonzalez-Mendez, N., Gonzales Rios, H., 2010. Migration of a-tocopherol from LDPE films to corn oil and its effect on the oxidative stability. Food Research International 43, 1073–1078. Kilic, M.B., Richards, M.P., 2003. Lipid oxidation in poultry döner kebab: prooxidative anti-oxidative factors. Journal of Food Science 68, 686–689. Kykkidou, S., Giatrakou, V., Papavergou, A., Kontominas, M.G., Savvaidis, I.N., 2009. Effect of thyme essential oil and packaging treatments on fresh Mediterranean swordfish fillets during storage at 4 °C. Food Chemistry 115, 169–175. Lee, Y.S., Shin, H.S., Han, J.K., Lee, M., Giacin, J.R., 2004a. Effectiveness of antioxidantimpregnated film in retarding lipid oxidation. Journal of the Science of Food and Agriculture 84, 993–1000. Lee, C.H., An, D.S., Lee, S.C., Park, H.J., Lee, D.S., 2004b. A coating for use as an antimicrobial and antioxidative packaging material incorporating nisin and atocopherol. Journal of Food Engineering 62, 323–329. Lee, D.S., Yam, K.L., Piergiovanni, L., 2008. Vacuum/modified atmosphere packaging. In: Lee, D.S., Yam, K.L., Piergiovanni, L. (Eds.), Food Packaging Science and Technology. CRC Press Taylor & Francis Group, pp. 397–424. Lemon, D.W., 1975. An improved TBA Test for Rancidity. New Series Circular No. 51. Halifax Laboratory, Halifax, Nova Scotia, Canada. Lopez-Galvez, D., de la Hoz, L., Ordofiez, J.A., 1995. Effect of carbon dioxide and oxygen enriched atmospheres on microbiological and chemical changes in
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refrigerated tuna (Thunnus alalunga) steaks. Journal Agricultural Food Chemistry 43, 483–490. Mancini, R.A., Hunt, M.C., 2005. Current research in meat colour. Meat Science 71, 100–121. Moore, M.E., Han, L.Y., Acton, J.C., Ogale, A.A., Barmore, C.R., Dawson, P.L., 2003. Effect of antioxidants in polyethylene film on fresh beef colour. Journal of Food Science 68, 99–104. Nerin, C., Tovar, L., Djenane, D., Camo, J., Salafranca, J., Beltran, J.A., Roncales, P., 2006. Stabilization of beef meat by a new active packaging containing natural antioxidants. Journal of Agricultural and Food Chemistry 54, 7840–7846. Pastoriza, L., Sampedro, G., Herrera, J.J., Cabo, M.L., 1998. Influence of sodium chloride and modified atmosphere packaging on microbiological, chemical, and sensorial properties in ice storage of slices of hake (Merluccius merluccius). Food Chemistry 61, 23–28. Pereira de Abreu, D.A., Paseiro Losada, P., Maroto, J., Cruz, J.M., 2010. Evaluation of the effectiveness of a new active packaging film containing natural antioxidants (from barley husks) that retard lipid damage in frozen Atlantic salmon (Salmo salar L.). Food Research International 43, 1277–1282. Pereira de Abreu, D.A., Paseiro Losada, P., Maroto, J., Cruz, J.M., 2011. Lipid damage during frozen storage of Atlantic halibut (Hippoglossus hippoglossus) in active packaging film containing antioxidants. Food Chemistry 126, 315–320. Reddy, N.R., Armstrog, D.A., 1992. Shelf life extension and safety concerns about fresh fishery products packaged under modified atmosphere: a review. Journal of Food Safety 12, 87–118. Ruberto, G., Baratta, M.T., 2000. Antioxidant activity of selected essential oil components in two lipid model systems. Food Chemistry 69, 167–174. Sirò, I., Fenyvesi, E., Szente, L., De Meulenaer, B., Devlieghere, F., Orgovanyi, J., Senyi, J., Barta, J., 2006. Release of alpha-tocopherol from antioxidative low-density polyethylene film into fatty food simulant: influence of complexation in betacyclodextrin. Food Additives and Contaminants 23 (8), 845–853. Sivertsvik, M., Jeksrud, W.K., Rosnes, J.T., 2002. A review of modified atmosphere packaging of fish and fishery products: significance of microbial growth, activities and safety. International Journal of Food Science and Technology 37, 107–127. Stammen, K., Gerdes, D., Caporaso, F., 1990. Modified atmosphere packaging of seafood. Critical Reviews in Food Science and Nutrition 29, 330–331. Tajima, G., Shikama, K., 1987. Autoxidation of oxymyoglobin. An overall stoichiometry including subsequent side reaction. Journal of Biological Chemistry 262, 12603–12606. Torres-Arreola, W., Soto-Valdez, H., Peralta, E., Cardenas-Lopez, J.L., EzquerraBrauer, J.M., 2007. Effect of a low-density polyethylene film containing butylated hydroxytoluene on lipid oxidation and protein quality of Sierra fish (Scomberomorus sierra) muscle during frozen storage. Journal of Agricultural and Food Chemistry 55, 6140–6146. Torrieri, E., Cavella, S., Villani, F., Masi, P., 2006. Influence of modified atmosphere packaging on the chilled shelf life of gutted farmed bass (Dicentrarchus labrax). Journal of Food Engineering 77, 1078–1086. Venugopal, V., 2006. Postharvest quality changes and safety hazards. In: Vanugopal, V. (Ed.), Seafood Processing: Adding Value Through Quick Freezing, Retortable Packaging and Cook-chilling. Taylor & Francis, CRC, Boca Raton, FL, USA, pp. 23– 60. Wessling, C., Nielsen, T., Leufvên, A., 2000. The influence of -tocopherol concentration on the stability of linoleic acid and the properties of lowdensity polyethylene. Packaging Technology and Science 13, 19–28. Wunderlich, B., 1999. Macromolecular Physics. Crystal Structure, Morphology, Defect, Vol. 1. Academic Press, New York (Chapter 8). Yesudhason, P., Gopal, T.K.S., Ravishankar, C.N., Lalitha, K.V., Kumar, A., 2009. Effect of modified atmosphere packaging on chemical, textural, microbiological and sensory quality of seer fish (Scomberomorus commerson) steaks packaged in thermoformed trays at 0–2 °C. Journal of Food Processing and Preservation 33, 777–797. Yesudhason, P., Gopal, T.K.S., Ravishankar, C.N., Lalitha, K.V., Kumar, A., 2010. Effect of potassium sorbate and modified atmosphere packaging on the shelf life extension of seer fish (Scomberomorus commerson) steaks during iced storage. Journal of Food Biochemistry 34, 399–424.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Effect of modified atmosphere and active packaging on the shelf-life of fresh bluefin tuna fillets Elena Torrieri a,⇑, Pier Antimo Carlino c, Silvana Cavella a, Vincenzo Fogliano a, Ilaria Attianese b,d, Giovanna Giuliana Buonocore b, Paolo Masi a,c a
Department of Food Science, University of Naples Federico II, Via Università 100, Parco Gussone, 80055 Portici (NA), Italy Institute of Composite and Biomedical Materials, National Research Council (CNR), P. le E. Fermi, 1, 80055 Portici (NA), Italy Centre of Food Innovation and Development in the Food Industry, University of Naples Federico II, Via Università 100, Parco Gussone, 80055 Portici (NA), Italy d Department of Materials and Production Engineering, University of Naples Federico II, P. le Tecchio 80, 80125, Naples, Italy b c
a r t i c l e
i n f o
Article history: Received 3 November 2010 Received in revised form 2 February 2011 Accepted 18 February 2011 Available online 23 February 2011 Keywords: Active film Antioxidant MAP Shelf-life Tuna fish
a b s t r a c t The aim of this work was to study the influence of the combined use of MAP and antioxidant-based active packaging on the shelf-life of fresh bluefin tuna fillets stored at 3 °C. Active packaging films were produced by embedding a-tocopherol into an unstabilized low density polyethylene (LDPE) matrix at three concentrations (0.1%, 0.5%, 1%). a-Tocopherol release kinetics, in vitro antioxidant activity, oxygen permeability and crystallinity degree were determined to characterize the film. Preliminary shelf-life tests were performed to select critical quality indices, the best gas composition and the best a-tocopherol concentrations in the active film. Then, the effectiveness of the chosen active packaging film in combination with MAP was assessed by monitoring critical quality indices of fresh bluefin tuna fillet during storage at 3 °C for 18 days. Obtained results showed that (i) 100% N2 atmosphere has a protective effect on haemoglobin and lipid oxidation processes monitored, (ii) active film is able to reduce fat oxidation, (iii) the combined effect of MAP and active packaging can be considered a valuable tool to increase the shelf-life of raw fish products. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fresh bluefin tuna (Thunnus thynnus) are heavily targeted by the Japanese raw fish market, where all bluefin species are highly prized for sushi and sashimi. In Italy, the processed fish market mainly consists of tuna in oil packed in glass jars or canned. However, local consumption of fresh bluefin tuna has greatly increased and tuna fillets packed as ready-to-use products could respond to the growing market of minimally processed food combining high quality and high convenience. Nevertheless, fresh bluefin tuna fillets are highly perishable products and appropriate packaging must be developed to extend product shelf-life (Davis, 1999). Lipid and haemoglobin oxidation are the main causes of their alteration during shelf-life. Lipid oxidation can be either enzymatic or nonenzymatic and causes development of off-flavours (Venugopal, 2006). On the other hand, haemoglobin oxidation lends the meat a purplish-red and brown colour due to deoxymyoglobin and metmyoglobin formation, respectively (Blakistone, 1999; Gill, 2003; Gill and Gill, 2005; Mancini and Hunt, 2005). Colour alteration ⇑ Corresponding author. Tel.: +39 081 2539456; fax: +39 081 7754942. E-mail address: [email protected] (E. Torrieri). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.02.038
makes products unattractive, affecting consumer preference at purchase (Carpenter et al., 2001). The most suitable packaging technology for these products is modified atmosphere packaging (MAP), an effective system to preserve quality and to maintain the hygienic and sensory characteristics of perishable fish (Corbo et al., 2005; Lopez-Galvez et al., 1995; Pastoriza et al., 1998; Reddy and Armstrog, 1992; Sivertsvik et al., 2002; Stammen et al., 1990; Torrieri et al., 2006). MAP combined with refrigeration showed positive effects on the quality of cod fillets, particularly when very fresh raw material is used (Bøknæs et al., 2000). Further, MAP has been found to increase the shelf-life of seer fish (Scomberomorus commerson) (Yesudhason et al., 2009, 2010), Mediterranean swordfish (Kykkidou et al., 2009) and tuna fish burgers (Carlino et al., 2009). However, no information is available in the literature on preservation of bluefin tuna fillets by MAP. Antioxidant (AO) packaging films have been developed to slow down or minimize oxidative deterioration of packaged products during storage. The use of both synthetic and natural antioxidants embedded into plastic films to improve the shelf-life of packaged products has been explored elsewhere. Lee et al. (2004b) applied a 3-mm thick nisin and/or a-tocopherol coating on paper to confer
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an antimicrobial and antioxidative property for use in the food packaging industry. Nerin et al. (2006) described the promising results of a new natural antioxidant active packaging system obtained by embedding rosemary extract into plastic film. The film was able to inhibit both haemoglobin and lipid oxidation in beef, improving the visual appearance of meat during its shelf-life. Moore et al. (2003) studied the effects of both synthetic and natural antioxidants (0.1%w/w) in low density polyethylene (LDPE) film on the preservation of fresh beef colour. They showed that after nine days of storage at 4 °C, the meat packed with the butylated hydroxyanisole-LDPE film had higher ‘‘a’’ (redness) values than any other film treatment, whereas a-tocopherol-containing film did not show any protective effect with respect to control film (without an antioxidant). Moreover, Graciano-Verduco et al. (2010) showed that the use of LDPE films containing a-tocopherol in concentrations between 19 and 30 mg/g are able to maintain the oxidative stability of corn oil during 16 weeks of storage at 30 °C. Torres-Arreola et al. (2007) report that LDPE films containing the synthetic antioxidant butylated hydroxytoluene may be used to prevent lipid oxidation and to prolong the shelf life of sierra fish. Performance of a new active packaging film containing natural antioxidants (from barley husks) with respect to lipid oxidation in frozen Atlantic salmon (Salmo salar L.) (Pereira de Abreu et al., 2010) and in Atlantic halibut (Hippoglossus hippoglossus) (Pereira de Abreu et al., 2011) were evaluated. The results obtained confirm the effectiveness of natural antioxidants derived from barley husks (7 mg dm2) in slowing down lipid hydrolysis and increasing the oxidative stability of salmon flesh and Atlantic Halibut. The combined use of MAP and antioxidants embedded into plastic films has received little attention. Camo et al. (2008) proved that rosemary-based and oregano-based active films resulted in enhanced oxidative stability of lamb steaks packaged in modified atmosphere and exposed to light illumination at 1 °C. In particular, active films with oregano were significantly more efficient than those with rosemary. However, to the best of our knowledge, use of tocopherol-based active film to package tuna fish products has not been reported to date. Thus, the present work aimed to obtain an active packaging film containing a-tocopherol and assess its combined effect with MAP on shelf-life extension of packaged fresh bluefin fillets. Active films were characterized with particular regard to a-tocopherol release kinetics from the films into a food simulant at 3 and 25 °C, their oxygen barrier properties and their effectiveness by means of in vitro tests. Shelf-life tests of fresh bluefin fillets packaged and stored at 3 °C were performed. In particular, their microbiological, chemical and physical characteristics were monitored up to 18 days.
2. Materials and methods 2.1. Materials Unstabilized low density polyethylene (LDPE) was supplied by Polimeri Europa (Italy). The natural antioxidant a-tocopherol and ethanol (96%v/v) as well as trichloroacetic acid, thiobarbituric acid peptone and malonaldehyde (MDA) were purchased from Sigma Aldrich (Milan, Italy). Bluefin tuna (Thunnus thynnus) were captured in the Mediterranean sea in April 2009 and were reared for five months in cages off the island of Procida (Naples, Italy). In November 2009, immediately after slaughter, the fish were gutted, washed and bled in salt water and ice, and finally the tuna fillets were frozen at 60 °C and stored at 25 °C. Three tuna fillets (around 20 kg) were packaged on ice in insulated polystyrene boxes, delivered to the university laboratory and kept in a freezer at 25 °C.
Polystyrene tray laminated with a multilayer barrier film (V = 500 cc; CoopBox, Bologna, Italy) and sealed with a film of PA/EVOH/PE (PO2 = 1.3 cc/m2 24 h atm; PH2O = 5.1 g/m2 24 h atm; thickness = 54 lm, CoopBox, Bologna, Italy) were used for shelf-life tests. 2.2. Film preparation The active films were prepared through a two-step process. The first step consisted of melt blending LDPE matrix with a-tocopherol by using an internal mixer (Thermo Scientific, Haake PolyLab QC, Karlsruhe, Germany) at 20 rpm at T = 140 °C for 6 min. The melt was pressed with a P300P hot press (Collin, Germany). The obtained sheets were then cut into pellets and fed into a co-rotating laboratory twin-screw extruder (Prism Eurolab 16, ThermoERMO Electron Corporation, Stone, UK) equipped with a sheet die of width 10 cm used to obtain the active antioxidant films. The temperature of the feeding, intermeshing and final zones of the extruder was maintained at 135–150–145 °C, respectively. Different amounts of a-tocopherol (0, 0.1, 0.5 and 1% w/w) were added to LDPE in order to obtain the control and active films coded as: LDPE, LDPE/0.1TOC, LDPE/0.5TOC, LDPE/1TOC. 2.3. Active film characterization 2.3.1. a-Tocopherol release tests Release kinetics of a-tocopherol from the three different films (LDPE/0.1TOC, LDPE/0.5TOC, LDPE/1TOC) into the chosen food simulant were determined. As food simulant, 96% ethanol was used instead of the normally proposed olive oil in order to avoid the quantification problems arising from its originally high a-tocopherol content (Sirò et al., 2006). Known amounts of active films (ffi1.5 g) were brought into contact with 80 ml of 96% ethanol at two different temperatures, T = 25 °C and T = 3 °C. The release kinetics were evaluated by monitoring the antioxidant concentration in the surrounding solution as a function of time until a constant value was reached. a-Tocopherol concentration in ethanol was determined using an HPLC (Agilent Model 1100, Milan, Italy) following the chromatographic method proposed by Sirò et al. (2006). The calibration curve was constructed for peak area against a-tocopherol concentration of standard solutions from 10 to 500 ppm. 2.3.2. In vitro antioxidant activity test Antioxidant activity of a-tocopherol released from LDPE films into ethanol was tested in a model system following the method proposed by Ruberto and Baratta (2000). The method is based on the spectrophotometric determination of the rate of conjugated diene formation from linoleic acid, in the absence and in the presence of a potential antioxidant. Known amounts of a-tocopherol released at 25 °C in ethanol from the three investigated films were added and peroxidation was monitored by recording the absorbance at 232 nm for 15 min. Effectiveness was measured based on the percentage antioxidant index (AI%) calculated using the formula: AI% = (1 T/C) 100 where C is the absorbance value at 232 nm of the oxidized linoleic acid after 15 min and T is the absorbance of the oxidized linoleic acid in the presence of the samples containing the released a-tocopherol under the same conditions. 2.3.3. Oxygen permeability Oxygen permeability of LDPE films was determined by means of Ox-Tran (Mocon, Model 2/20, Neuwied Germany). Samples with a surface area of 5 cm2 were tested at 23 °C, setting the relative humidity (RH) at the downstream and upstream side of the film at 50%. Each test was made in duplicate.
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2.3.4. Analysis of crystallinity degree A Differential Scanning Calorimeter DSC 2920 (TA Instrument) was used to measure the crystallinity of the films. Thermograms were recorded in three steps: (1) heating from 0 to 180 °C, (2) cooling from 180 to 0 °C and (3) a second heating from 0 to 180 °C. The heating and cooling rates were 10 °C/min. The crystallinity was calculated from the ratio of the melting enthalpy of the samples to the melting enthalpy of 100% crystalline polyethylene (293 J/g) (Wunderlich, 1999). 2.4. Shelf-life testing 2.4.1. Packaging and storage condition – Preliminary test In order to select critical quality indices and the best gas composition, tuna fillets were cut into slices by using a bandsaw (KT210-Ecom, Movinox, Acquaviva Picena (AP), Italy). Each slice was 1.5 cm thick and weighed 175 g. The slices were then laid on a polystyrene tray laminated with a multilayer barrier film and sealed with a film of PA/EVOH/PE. One slice was packed for each tray. Samples were modified atmosphere packed by using a packaging machine (Minipack Torre, TSM, 105, Cava dei Tirreni (SA), Italy). The ratio between the volume of gas and weight of food product (V/W ratio) was 2.5:1. Samples of bluefin tuna fillet were packaged in air and MAP (60% O2– 40% CO2; 100% N2) and stored for 9 days at 3 °C. High oxygen and high carbon dioxide amount were extensively reported to be protective for fresh food product at high content of haemoglobin (Gill and Gill, 2005). 100% N2 atmosphere was chosen because low oxygen concentration are protective for lipid oxidation (Lee et al., 2008). After 0, 2, 5, 7 and 9 days microbiological analysis, headspace composition in terms of O2% and CO2%, pH, colorimetric analysis (L⁄, a⁄, b⁄) and fat oxidation measurement (TBARS) were performed. In order to select the best a-tocopherol concentrations in the active film, one slice of samples was laid on a polystyrene tray laminated with a multilayer barrier film and the exposed surface was completely covered with the active film (15 cm 7 cm) LDPE/ 0.1TOC, LDPE/0.5TOC, LDPE/1TOC. Then, samples were packed in air by using a packaging machine using the PA/EVOH/PE to seal the tray. Samples were stored for 11 days at 3 °C and critical quality indices selected after the preliminary test previously described were monitored after 0, 4, 7 and 11 days. 2.4.2. Packaging and storage condition – Main test Once the best gas composition, the optimal a-tocopherol concentration and the critical quality indices had been selected, the effect of MAP and active packaging on product shelf life was studied. Samples were divided into four lots as detailed: lot I: samples packed in air with film not containing a-tocopherol (LDPE); lot II: samples packed in air with active film (LDPE/0.5TOC); lot III: samples packed in MAP (100% N2) with film not containing a-tocopherol (LDPE); lot IV: samples packed in MAP (100% N2) with active film (LDPE/0.5TOC). All samples were stored for 18 days at 3 °C and critical quality indices were analyzed after 0, 1, 4, 7, 11, 15, 18 days. 2.4.3. Microbiological analysis Standard enumeration methods were used to determine the microbial populations of the fish during chilled storage. Two samples of fish (25 g) were taken from each package, then diluted with 225 ml of peptone salt solution (0.85% NaCl–0.1% peptone) and homogenized with a Stomacher for 2 min. The sample was then serially diluted as needed for plating. The following media and incubation conditions were used: Plate Count Agar (PCA, Oxoid) for aerobic mesophilic bacteria (AMB) by pour plating, incubated at 30 °C for 72 h; Violet Red Bile Glucose Agar (VRBGA, Oxoid) for Enterobacteriaceae (E) counts, incubated at 37 °C for 24 h. Microbial counts were performed in duplicate and expressed as CFU/g, MRS agar (Oxoid) for Lactic Acid Bacteria (LAB) counts, incubated anaer-
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obically at 30 °C for 48 h; Streptomycin Thallous Acetate Agar (STAA, Oxoid) with STA selective supplement (Oxoid) for Brochothrix thermosphacta counts, incubated at 25 °C for 48 h. 2.4.4. Chemical analysis Gas analysis: O2 and CO2 concentrations (%v/v) in the package headspace were monitored by means of a portable PBI Dansensor A/S (Check Mate 9900 O2/CO2; Ringsted, Denmark) analyzer (accuracy ±0.1%) by sampling with a needle 3 ml of gas from the package headspace. One measurement was carried out on each sample. pH determination: pH was measured on samples obtained by mixing homogenized fish flesh with water (ratio 1:2) by using a pHmeter Cyber Scan pH/lon 510 (Eutech Instruments Pte Ltd. Ayer Rajah Crescent Singapore). The pHmeter was equipped with a Schott electrode which was previously calibrated with buffer solutions (pH 4 and pH 7) at 20 °C. Four measurements were carried out on each sample. TBARS analysis: Thiobarbituric acid reactive substances were determined by using the extraction procedure of Lemon (1975) and Kilic and Richards (2003). A 0.5 g sample was blended with 5 ml of extraction solution containing 25% trichloroacetic acid (TCA) and 10 ml of water. The samples were homogenized with an Ultra-Turrax for 30 s. The homogenate was filtered through Whatman 1 filter paper and the filtrate (3.5 ml) was mixed with 1.5 ml of thiobarbituric acid (TBA) solutions (0.6%) and vortexed. The mixture was heated at 70 °C for 15 min in a water bath, cooled, and then centrifuged at 2000 rpm for 5 min. Absorbance was determined at 532 nm against a blank containing 1 ml of TCA extraction solution and 1 ml of TBA solution. Calibration curves were prepared using malonaldehyde (MDA) standard working solutions. A stock MDA solution was obtained after hydrolysis of 690 mg of 1,1,3,3-tetraethoxypropane (TEP) in 10 ml of 0.1 N hydrochloric acid (HCl). The reaction was carried out in a 100-ml screw-capped bottle. The hydrolyzed TEP solution was then accurately diluted to 100 ml with ultrapure water. Appropriate working solutions were prepared from the stock MDA solution. The tubes used for the calibrations, including the blank and the corresponding samples to be analyzed, were always incubated at the same time. The reaction and the detection were as described above. The TBARS values are expressed as nmoles of malonaldehyde/g of freeze dried samples.
2.4.5. Physical analysis Colorimetric measurement: colour of the bluefin tuna fillets was measured with a tristimulus colorimeter (Minolta Chroma Meter, model CR-300) with a circular measurement area (D = 8 mm). The colorimeter was calibrated using a white standard plate (L = 100). The L⁄, a⁄ and b⁄ values were measured on the product surface. Five readings were carried out at each position.
2.4.6. Experimental design and statistical analysis for shelf life study A full factorial design was used to study the effect of time, atmosphere, active film on the quality indices of bluefin tuna fillets. There were seven levels of storage time (0, 1, 4, 7, 11, 15, 18), two levels of atmospheres (air, 100% N2) and two levels of active film (LDPE, LDPE/0.5TOC). Three replicates were performed for each sample for a total of 84 samples. Results are reported as mean value ± standard deviation. ANOVA analysis was performed on the data to evaluate the effect of time (A), atmosphere (B), active packaging (C) and the interaction effect (AxB; BxC; AxC; AxBxC) on the quality attributes. Duncan’s test was performed to find out the source of the significant differences within samples. Significance of differences was defined at p 6 0.05. All statistical analyses were performed using the SPSS computer package (SPSS Inc. 11.5, Chicago, 2002).
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Fig. 2. Oxygen permeability values of the investigated antioxidant films obtained at T = 23 °C and at RH% = 50%.
3.1.2. Oxidation test ‘in vitro’ The effectiveness of antioxidant activity of a-tocopherol released at the equilibrium in 96% ethanol from films LDPE/0.1TOC, LDPE/0.5TOC and LDPE/1TOC was evaluated on the oxidation of a linoleic acid. The AI% values obtained were 18%, 22% and 35%, respectively. This result shows the capacity of the released antioxidant to inhibit the oxidation of the unsaturated fatty acids. As expected, its effectiveness increases as the a-tocopherol concentration in the film increases. On the contrary, Lee et al. (2004a) proved that the amount of a-tocopherol contained in their co-extruded film had no effect on retarding lipid oxidation in the model product. On monitoring the linoleic acid content during storage time of a model system (water/linoleic acid), they found that at 45 °C, over a four-day storage period, there appeared to be no differences between the levels of linoleic acid in the model product packaged in the control and in the a-tocopherol-impregnated laminate pouch structures. This may be attributed to extensive fatty acid oxidation under the storage conditions. Fig. 1. a-Tocopherol release in ethanol 96% (v/v) at T = 4 °C (a) and at T = 25 °C (b) from LDPE/0.1TOC (s) LDPE/0.5TOC (j) and LDPE/1TOC (4).
3. Results and discussion 3.1. Active film characterization 3.1.1. a-Tocopherol release tests The amount of a-tocopherol released at 3 °C and 25 °C in 96% ethanol was monitored against time (Fig. 1a and b, respectively). As expected, the release kinetics at 25 °C are faster than those obtained at the lower temperature; at T = 3 °C, a constant value had still not been reached after 25 days. Moreover, the asymptotic value reached for each film depends on the initial quantity of atocopherol added to the polymer: at 25 °C the released concentrations for LDPE/0.1TOC, LDPE/0.5TOC and LDPE/1TOC films were 5, 20 and 70 ppm, respectively. As reported by Sirò et al. (2006), due to the high extraction effect of ethanol, the asymptotic value reached for each film corresponds to the total original a-tocopherol content of the plastic bags after manufacture. Thus, the total amounts of a-tocopherol contained in the three investigated films is 0.3, 1 and 3.8 mg/g and it can be concluded that approximatively 70% of the originally added a-tocopherol has been lost after film processing, as also experienced by other authors (Sirò et al., 2006; Wessling et al., 2000).
3.1.3. Oxygen permeability Since the addition of large amounts of additives may cause changes in film characteristics, the oxygen barrier properties of the LDPE/TOC material was investigated. Data reported in Fig. 2 show that no significant differences can be observed between the neat LDPE and the active films. This may be related to the similar degree of crystallinity observed for these films (data not shown). The data concerned yield a positive result since an increase in the oxygen transmission rate, which has been found elsewhere (Wessling et al., 2000), also in this application could influence the potential positive effect of the antioxidant in the active film on shelf-life extension. 3.2. Preliminary shelf-life testing 3.2.1. Selection of critical quality indices and modified atmosphere packaging 3.2.1.1. Microbiological analysis. Initial aerobic mesophilic counts in fresh bluefin fillets were 4.2 102 CFU/g, whereas they were absent in 10 g of sample products packaged in air and in MAP (100% N2 and 60% O2–40% CO2) at 3 °C after 2, 5, 7 and 9 days of storage. Moreover, Entorobacteriaceae, B. thermosphacta and lactic bacteria were absent in 0.1 g on all samples after nine days of storage, showing that due to the high quality of the initial product microbiological alteration did not represent a critical aspect for
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product shelf life. For this reason, microbiological indices were not considered as critical for quality. Moreover, results showed that for a storage temperature of 3 °C, high CO2 concentrations were not helpful for bacteriostatic effect. 3.2.1.2. Chemical analysis. During storage, gas composition into the package headspace was monitored. In particular, it was observed that O2 concentration in the pack headspace of samples packed in air and in MAP decreased from an initial value of 21% to 5% after nine days of storage for air-packed samples, from 60% to 45% for samples stored in MAP (60% O2–40% CO2), and from 1% to 0% after one day of storage for samples stored in MAP (100% N2). Generally, an O2 decrease can be used as an index of microbial contamination (Torrieri et al., 2006), but in this case, because no growth of microorganism was observed during storage, the decrease can only be justified by oxidation processes. For samples packed in air, CO2 increased to 1.6% after 2 days and remained constant up to nine days of storage, whereas for samples stored in MAP (60% O2–40% CO2) the CO2 remain almost constant (38 ± 2%), even if a evident pack depression was observed after five days of storage, indicating CO2 solubilization in the product. This shows that high CO2 concentrations were even detrimental to product quality. No CO2 accumulation was observed during storage for samples packed in MAP (100% N2). The pH was constant throughout storage with no differences among samples (p > 0.05) and assumed an average value of 5.60 ± 0.07. Therefore, the pH parameter is not useful as a physico-chemical index of quality decay or in predicting the shelf life of bluefin tuna fillet samples. The TBARS values of samples packed with 100% N2 were lower than TBARS values of control samples (Fig. 5) and samples packed with 60% O2–40% CO2 (data not showed). Therefore, TBARS was chosen as critical index for studying lipid oxidation and 100% N2 was chosen as the optimal atmosphere to pack the product. 3.2.1.3. Physical analysis. Values of CIE a⁄ and b⁄ for samples packed in air and MAP (60% O2–40% CO2; 100% N2) throughout storage are reported in Fig. 3a and b. Samples packed in air showed a significant decrease in a⁄ from an initial value of 8 ± 1 to as low as 1 ± 1 after nine days of storage. After two days of storage a⁄ assumed a value of 5 ± 2 that was already significantly different from the value assumed at time zero (p < 0.0001). A browning of colour tuna meat is the cause of the a⁄ decrement. This result is explained by the oxidation mechanism of the myoglobin. In fact, when the fish is cut up, oxygen comes into contact with myoglobin in the exposed tuna meat surface. The oxygen is absorbed and reacts with the myoglobin to form a bright red pigment (oxy-myoglobin) which brings about the attractive red colour of fresh tuna meat. However, due to a continued exposure to oxygen during storage, the red colour of the meat gradually changes into various shades of brown due to oxidation and conversion of the oxy-myoglobin (Fe2+–O2) to the brown ferric (Fe3+) met-myoglobin (Tajima and Shikama, 1987). Although a protective effect of high oxygen concentrations on oxyhaemoglobin, oxidation is normally reported for fresh food product at high content of haemoglobin (Gill and Gill, 2005) and, in particular, it was reported for refrigerating tuna steaks (LopezGalvez et al., 1995). Our results showed that samples packed in MAP 60% O2–40% CO2 had the same behaviour as air-packed samples, displaying no protective effect of the high oxygen concentration on myoglobin oxidation. On the other hand, samples packed in MAP 100% N2 showed a constant a⁄ value during storage (p > 0.05), thus indicating a protective effect of this atmosphere on colour change (Fig. 3a). Less significant was the effect of time and atmosphere on b⁄ (Fig. 3b). In particular, b⁄ increased from 8 ± 1 to
Fig. 3. Effect of modified atmosphere package on colorimetric coordinates a⁄ (a) and b⁄ (b) of bluefin tuna fillet stored at 3 °C. (j) air; (e) 60% O2–30% CO2; (N) 100% N2.
12 ± 1 after 9 days of storage for samples packed in air and with 60% O2–40% CO2, and to a value of 9 ± 1 after 9 days of storage for samples packed with 100% N2. Experimental results showed that a⁄ and b⁄ are critical quality indices to study product shelf life and confirmed that 100% N2 atmosphere was the optimal atmosphere in which to pack the product.
3.2.2. Selection of best active film On the base of previous results, colorimetric analysis (a⁄ and b⁄) and lipid oxidation (TBARS) were selected as critical quality indices and used to select the optimal concentration of a-tocopherol present in the film. The results obtained for air-packed samples (data not shown) proved that parameter a⁄ and b⁄ and the lipid oxidation were unaffected by tocopherol concentration. On the basis of these results and given that, on visual inspection, the LDPE/1TOC appeared somewhat more yellow in colour than the reference material and less visually attractive (Wessling et al., 2000), film at 0.5% w/w (LDPE/0.5TOC) was selected to study product shelf-life.
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Fig. 5. n mole of MDA/g dw of bluefin tuna fillet after 0, 9 and 18 days of storage at 3 °C. (A: 100% N2; B: 100% N2 + LDPE; C: 100% N2 + LDPE/0.5TOC; D: air + LDPE; E: air + LDPE/0.5TOC.)
Fig. 4. Effect of active film and modified atmosphere package on colorimetric coordinates a⁄ (a) and b⁄ (b) of bluefin tuna fillet stored at 3 °C. (j) Air + LDPE; (h) air + LDPE/0.5TOC; ()100% N2 + LDPE (e) 100% N2 + LDPE/0.5TOC.
3.3. Main test: effect of active film and MAP on critical quality indices Fig. 4a and b shows the variation of colorimetric parameters a⁄ and b⁄ of fresh bluefin tuna fillets packed with active film (LDPE, LDPE/0.5TOC) and modified atmosphere packed (air-100% N2) as measured against storage time. LDPE film was used as control for the active film (LDPE/0.5TOC). Samples were packed also in air to assess the utility of MAP in presence of active film. ANOVA results highlight a significant effect of the modified atmosphere package (p < 0.05) on colorimetric parameters a⁄ and b⁄ at any storage time, whereas the effect of active packaging and interaction between MAP and active packs were not statistically significant (p > 0.05). Samples packed with 100% N2 showed a higher value of a⁄ and a lower value of b⁄ at any storage time than samples packed in air. In particular, for samples packed with 100% N2, the colorimetric parameter a⁄ increased from time zero to time four days and then
remained constant with time. Indeed, the colour of the samples packed with nitrogen was bright red compared to the bloom red colour of the control samples at time zero. A slow diffusion kinetics of the a-tocopherol from the film to the product surface may be the reason for the antioxidant not affecting myoglobin oxidation. Our results are in accordance with Moore et al. (2003) who studied the effects of tocopherol (0.1%w/ w) in polyethylene film on fresh beef colour, and showed that after nine days of storage at 4 °C a-tocopherol film showed no protective effect with respect to control film (without AO). As reported by the authors, natural antioxidants may be less likely to migrate from the film disk to the surface of the beef, thereby limiting their effect. Nevertheless for all samples packed in air ANOVA results showed that the effect of time was not statistically significant (p > 0.05). These last results can be compared with data reported in Fig. 3 that show a significant effect of time on colorimetric parameters of samples packed in air without film. Whereas, irrespective to the amount of tocopherol contained into the film, its presence on the surface of the tuna fillet showed a protective effect on product colour change, probably due to a less oxygen amount at product surface. Data related to fat oxidation of samples packed in MAP and with active films are reported in Fig. 5. It is worth to notice that the presence of the active film showed a protective effect on lipid oxidation when combined with the 100% N2 MAP (p < 0.005). These results confirm the hypothesis that the action of the antioxidant is related to the different kinetics of oxidation and diffusion. Thus, when the oxidation kinetics is slowed down by the protective atmosphere, the action of the antioxidant becomes evident. On the other hand, in air the antioxidant film has no protective effect, most likely because the oxidation process is too fast compared to the diffusion kinetics of the a-tocopherol from the film to the sample surface.
4. Conclusion Our results showed that by using only modified packaging it was possible to extend product shelf-life from 2 days for control samples (air without film) up to 18 days at 3 °C. Moreover, the combined use of MAP and the active film resulted in a less oxidized product after 18 days of storage at 3 °C. These results, which should be considered preliminary until further investigation is carried out, show considerable promise and highlight the importance of optimizing active film in terms of initial antioxidant concentra-
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