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Effect of combination of ozonation and vacuum packaging on shelf life extension of fresh chicken legs during storage under refrigeration
Accepted Manuscript Effect of combination of ozonation and vacuum packaging on shelf life extension of fresh chicken legs during storage under refrigeration Ioanna N. Gertzou, Ioannis K. Karabagias, Panagiotis E. Drosos, Kyriakos A. Riganakos PII:
S0260-8774(17)30272-8
DOI:
10.1016/j.jfoodeng.2017.06.026
Reference:
JFOE 8931
To appear in:
Journal of Food Engineering
Received Date: 29 October 2016 Revised Date:
20 June 2017
Accepted Date: 22 June 2017
Please cite this article as: Gertzou, I.N., Karabagias, I.K., Drosos, P.E., Riganakos, K.A., Effect of combination of ozonation and vacuum packaging on shelf life extension of fresh chicken legs during storage under refrigeration, Journal of Food Engineering (2017), doi: 10.1016/j.jfoodeng.2017.06.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of combination of ozonation and vacuum packaging on shelf life extension
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of fresh chicken legs during storage under refrigeration
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by
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Ioanna N. Gertzou, Ioannis K. Karabagias, Panagiotis E. Drosos, and Kyriakos A. Riganakos*
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Laboratory of Food Chemistry, Department of Chemistry, University of Ioannina, 45110-Ioannina,
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*Corresponding author: Tel.: +302651008341; Fax: +302651008795 E-mail address: [email protected] (K.A. Riganakos)
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ACCEPTED MANUSCRIPT Abstract
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The objective of the present study was to investigate the combined effect of different ozone dose (2,
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5, and 10 mg/L) and of vacuum packaging on shelf life extension of fresh chicken legs, packaged
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with polyamide/polyethylene bags and stored at 4±1 °C, for 16 days. Parameters considered were:
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microbiological (TVC, Pseudomonas spp., LAB, Yeasts and moulds, and Enterobacteriaceae),
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physicochemical (pH, colour parameters) and sensory (odour, appearance, texture, and taste)
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attributes. The results obtained in this study showed that physicochemical parameters varied
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significantly (P<0.05) depending on ozonation dose and storage time. Populations of spoilage flora
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along with sensory properties, were affected by packaging technology and storage time (P<0.05).
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Conclusively, the combination of gaseous ozone of 10 mg/L and vacuum packaging, under
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refrigeration, resulted to a 6 days shelf life extension of chicken legs, as compared to single vacuum
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packaging. The modeling of microbial growth, resulted in the collection of positive results (P<0.05).
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Keywords Chicken legs; vacuum packaging; ozonation; shelf-life extension; sensory properties
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ACCEPTED MANUSCRIPT Introduction
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Sixteen billion domesticated fowl, such as chickens, turkeys, ducks, and geese, are raised annually,
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more than half of these in industrialized factory-like production units, to cover consumers’ demand
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for meat or eggs. Poultry meat is a favorable food commodity worldwide. The largest producers are
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the United States (20%), China (16.6%), Brazil (15.1%) and the European Union (11.3%) (USDA
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Livestock and Poultry, 2014). It is a nutritional and qualitative type of food due to its low fat content,
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a relatively high concentration of polyunsaturated fatty acids, its excellent quality protein, and
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essential amino acids content needed by humans (Zhang et al., 2016).
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Considering the fact that poultry meat belongs to perishable foods, the main concern of food
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industries is to extend the shelf life of the product, by ensuring at the same time consumers’
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protection from spoilage and pathogenic bacteria, respectively (Kim and Yousef, 2000; Zeynep,
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2003; USDA Livestock and Poultry, 2014).
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Pseudomonas spp. (Gram positive, aerobe), Enterobacteriaceae (Gram negative, facultative
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anaerobe), and Brochothrix thermosphacta (Gram positive, facultative anaerobe) are commonly
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found in poultry meat, along with Lactic acid bacteria (Gram positive, aerotolerant anaerobe), and
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Shewanella putrefaciens (Gram negative, facultative anaerobe) representing the principle spoilage
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bacteria of fresh meats (Whitfield, 1998; Totosaus and Kuri, 2012).
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Market differentiation of fresh poultry products (i.e. legs, thighs, breasts or deboned breasts) is
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achieved through novel vacuum packaging development and product innovation. One of the main
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challenges of the food industry is the application of multifunctional packaging (Taylor, 1977;
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Spaulding, 1994; Kartika et al., 2003; James, 2008; Totosaus and Kuri, 2012).
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Plastic materials to develop such a novel packaging system include: Saran or polyvinyl chloride
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(PVC), an absorbent pad in conventional foam trays with PVC overwrap, polypropylene (PP),
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polystyrene (PS), polyvinyl chloride (PVC), and various grades of polyethylene (HDPE, LDPE, etc.)
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ACCEPTED MANUSCRIPT (Duncan, 2011). Other thermoforming film materials that can be applied for meat packaging include
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ethylene-vinyl alcohol (EVOH), polypropylene-oriented polypropylene (OPP), ethylene vinyl acetate
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(EVA), and polyvinylidene chloride (PVdC), each having different mechanical and barrier properties
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(Crippa et al.,2007).
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New trends in meat and poultry packaging using aforementioned plastic materials alone or in
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combination, involve the combined use of modified atmosphere packaging with essential oils, freeze
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chilling, irradiation, absorbent pads containing essential oils, and vacuum packaging, have been
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shown to delay spoilage in poultry meat (Rao and Sachindra, 2002; Patsias et al., 2006; Chouliara et
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al., 2007; Balamatsia et al., 2007; Chouliara et al., 2008; Oral et al., 2009).
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On the other hand, ozonation is an innovative non-thermal method with potential applications in the
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modern food industry (Jeong et al., 2007; Cullen et al., 2010; Piachin and Trachoo, 2011; Cárdenas
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et al., 2011; Shah et al., 2011; Blogoslawski and Stewart, 2011; Fratamico et al., 2012).
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Ozonation is a safe way to oxidize contaminants while leaving no residues and without affecting the
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quality of food. Ozone-based treatments are eco-friendly, applicable to sanitizing a wide range of
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materials and replace alternative chemical sanitizers such as, chlorine, acids, salts, etc. (Trindade et
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al., 2012). It not only acts on microorganisms (Muthukumarappan et al., 2008), but also on pests,
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serving thus, as a fumigant. Several researchers have suggested that ozonized water effectively
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improved the chemical properties and safety of refrigerated meat (Bhattacharya, 2015). Since ozone
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treatment meets all the requirements of the food consumer, it can be regarded as a ‘’greener’’
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additive (Da-Wen Sun, 2014). However, due to its strong oxidizing power, ozone may be toxic for
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humans above certain levels of concentration (0.1-0.3 ppm) and length of exposure (Perry and
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Yousef, 2011; Bhattacharya, 2015).
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accordance with current industry standards of good manufacturing practice (FSIS Directive 2016).
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The excessive use of ozone is not universally beneficial and in some cases may promote oxidative
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spoilage in foods, such as meat, as well as discoloration or even development of undesirable odours
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(Khadre et al., 2001).
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Given the limited data available in the literature on the application of ozone and vacuum packaging
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to poultry meat products (Yang and Chen, 1979; Nieto et al.,1984; Sheldon and Brown, 1986; Al-
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Haddad et al., 2005; Muthukumar and Muthuchamy, 2013) and ozone’s properties considered
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aforementioned, the aim of the present study was to investigate the effectiveness of different dose
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ozone treatments (2, 5, and 10 mg/L) in combination with vacuum packaging on the shelf life
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extension of fresh chicken legs, stored at 4 °C, as assessed by microbiological, physicochemical, and
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sensory attributes.
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2.1. Chicken samples
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Fresh chicken legs meat in chunks weighing ca.300 g, were provided by a local poultry processing
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industry (Pindos S.A., Ioannina, Greece) within one hour after slaughter in insulated polystyrene
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boxes on ice. Chicken legs were subjected to cooling with cold water after slaughtering.
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2.2. Vacuum packaging and ozonation conditions
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All chicken samples were exposed for 1h under gaseous ozone treatment (concentrations of 2, 5 and
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10 mg/L) at room temperature. System of ozonation was C-Lasky L010 supplied from Air tree
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company (Taiwan).
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consumption (180 W) and stable ozone production. Detectable concentrations are in the range of
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This ozone generator is designed to provide high efficiency, low energy
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ozone concentration [in our case 2, 5, and 10 mg/L (or ppm)] was accurate and constant, the OS-4
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(Eco-Sensors, Santa Fe, New Mexico, USA) probe was used.
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Chicken leg samples were then placed in polyamide/polyethylene (PA/PE) barrier pouches 29.5 x
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29.5 cm, 90 µm in thickness having an oxygen permeability < 15 cm3 m-2 d−1 atm−1, nitrogen
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permeability < 15 cm3 m-2 d−1 atm−1, carbon dioxide permeability < 200 cm3 m-2 d−1 atm−1 at 75%
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relative humidity (RH), 23°C (Method DIN 53380-2),
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<1 g m−2 d−1 at 85% RH, 23°C (Method DIN 53122-2). Finally, pouches were heat-sealed using a
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BOSS model N48 vacuum sealer (BOSS, Bad Homburg, Germany) and kept at 4±1 °C. Sampling,
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following all aforementioned technical criteria, was carried out on 0, 2, 4, 6, 8, 10, 12, 14, and 16
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days of storage.
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2.3.Microbiological analysis
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The following groups of microflora were monitored: Total viable counts (TVC), Pseudomonas spp.,
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Yeasts and moulds, Lactic acid bacteria (LAB), as well as Enterobacteriaceae. Ten grams of fresh
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chicken leg meat were removed aseptically from the package using a spoon, transferred to a
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stomacher bag (Seward Medical, Worthing, West Sussex , UK), containing 90 mL of sterile buffered
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peptone water (LAB 204, LAB M), and homogenized using a stomacher (LAB Blender 400, Seward
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Medical) for 60 s at room temperature.
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For microbial enumeration, 0.1 mL samples of serial dilutions (1:10 diluents, buffered peptone
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water) of chicken leg meat homogenates were spread on the surface of the following agar plates
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prepared. TVC were determined using plate count agar (PCA, LAB 010, LAB M, Lancashire, UK),
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after incubation for 2 days at 30 oC. Pseudomonads
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cephaloridine agar (LAB 108, LAB M, supplemented with X 108, Lancashire, UK) after incubation
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and a water vapor permeability of
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were determined on cetrimide fusidin
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036, LAB M, Lancashire, UK) after incubation at 30 oC for 3-5 days.
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For members of the Enterobacteriaceae spp., 1.0 mL sample was inoculated into 10 mL of molten
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(45 °C) violet red bile glucose agar (LAB 088, LAB M, Lancashire, UK) after incubation for 24 h at
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37 oC. The large colonies with purple haloes were counted. Lactic acid bacteria were determined on
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de Man, Rogosa and Sharpe medium (LAM 093, LAB M, Lancashire, UK) after incubation at 30 °C
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for 3 days. All plates were examined visually for typical colony types and morphological
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characteristics associated with each medium.
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2.4.Physicochemical analysis
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pH values of fresh chicken leg meat were measured using a Delta OHM, model HD 3456.2, pH-
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meter (Padova, Italy) with a precision of ±0.002. Chicken leg samples were thoroughly homogenized
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with 10 mL of distilled water and the homogenate used for pH determination. Colour determination
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was carried out on the surface of chicken legs, which were placed into a cylindrical optical cell,
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using a Hunter Lab model DP-9000 colorimeter coupled to a D25 L optical sensor (Hunter
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Associates Laboratory, Reston VA, USA). Reflectance values were obtained using a 45 mm viewing
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aperture. Each determination was run in triplicate (n=3).
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2.5.Sensory evaluation
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Chicken leg samples (ca. 100 g) after defrosting, were cooked in a microwave oven set at high power
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(700 W) for 4 min. A panel of seven judges experienced in chicken evaluation was used for sensory
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analysis. Panelists were asked to evaluate odour, texture, appearance, and taste intensities of cooked
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samples. Along with the test samples, the panelists were presented with a freshly thawed chicken leg,
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stored at −30 °C throughout the experiment, this serving as the reference sample. Acceptability of
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odour, texture, appearance, and taste was estimated using an acceptability scale ranging from 5 to 0,
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3 was taken as the lower limit of acceptability. Each evaluation was run in triplicate (n=3) and every
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sample was evaluated individually.
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2.6.Statistical analysis
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Physicochemical and microbiological data were subjected to analysis of variance (ANOVA) in order
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to investigate whether vacuum packaging or the combination of vacuum packaging and ozonation,
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could affect the aforementioned parameters throughout storage time. In addition, in order to develop
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a microbial growth model, linear regression analysis was also conducted. All statistical treatment
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was performed using the SPSS v.20.0 statistics software. Experiments were replicated three times on
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different occasions with different chicken leg samples. Analyses were run in triplicate (three
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different packaged samples) for each replicate (n=3×3). Microbiological data were transformed into
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logarithms of the number of colony forming units (log CFU/g).
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2.7. Theory
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2.7.1.
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Ozone (O3) is generated by the reaction of oxygen free radicals with diatomic oxygen. It is an
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allotropic form of oxygen, with very good antimicrobial properties. It was in 1840 when Christian
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Friedrich Schönbein a German-Swiss chemist named the substance ozone, based on the Greek word
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ozein for ‘’smell’’ (Kogelschatz, 1988). The chemical reactions of ozone are either indirect (chain-
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reactions mechanism leading to the production of hydroxyl free radicals) or direct (with substances
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present in the matrix) (Cullen et al., 2010; O’Donell et al., 2012).
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It has been previously reported that, ozone chemical reactions are highly selective against a wide
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range of bacteria, fungi, viruses, protozoa, bacterial and fungal spores (Khadre et al., 2001;
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Muthukumarappan et al., 2008; Cullen et al., 2010).
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is the primary target site for antimicrobial action. Inactivation by ozone is a complex procedure that
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attacks cell membrane, cell wall constituents, and intracellular constituents (Khadre et al., 2001). In
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the present study we applied different gaseous ozone concentrations. Depending on the
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concentrations required, ozone gas can be generated using coronal discharges.
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2.7.2. Modeling of microbial growth
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Modeling of microbial growth in food is a mandatory tool for food safety prediction and shelf-life of
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products in the real market world. Over the past 25 years numerous growth models have been
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proposed, depending on different parameters (i.e. elevated temperatures, pH, salt, etc.) that may
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affect the kinetics of microbial growth (Bratchell et al.,1989; Zwietering et al., 1990; Fujikawa et al.,
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2004).
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The growth of a homogenous microbial population can be described by a curve with three phases if
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the death phase is excluded: a lag phase (adaptation period of microbial cells to their new
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environment) followed by an exponential growth phase (multiplication of cells exponentially) and
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finally a stationary phase (reaching to the maximum population density) (Buzrul, 2009). Equation 1
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has been proposed to describe the microbial growth in different matrices:
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where N(t) and N0 are the momentary and initial number of microbial population, respectively. A is
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the asymptotic value which is reached at time tA, and tm is the time where maximum growth rate is
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achieved (Buzrul, 2009).
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to be considered as constant. In that sense, two were the main parameters that could affect the
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specific microorganisms’ growth: a) packaging media and b) storage time.
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3. Results and discussion
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3.1 Microbiological changes
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TVC values for the four different chicken leg treatments are shown in Figure 1a as a function of
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storage time. TVC reached the value of 7 log CFU/g, which is considered as the microbiological
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upper limit for good quality poultry meat (Chouliara et al., 2007), on day 10 for vacuum packaged
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samples, day 14 for samples treated with 2 mg/L of ozone, and day 16 for samples treated with 5 and
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10 mg/L of ozone, respectively.
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The use of a higher ozone dose, i.e. 5 or 10 mg/L, resulted in extending the shelf life of fresh chicken
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legs by ca. 6 days as compared to vacuum packaged samples (P<0.001). On the other hand, a shelf
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life extension by ca. 4 days was observed for chicken leg samples packaged in vacuum and treated
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with 2 mg/L of gaseous ozone.
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Present results are in agreement with those reported previously, in which ozonation proved to be
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effective against a wide range of natural poultry microflora, providing thus, a substantial increase in
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shelf life of poultry products investigated (Yang and Chen 1979; Nieto et al., 1984; Sheldon and
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Brown, 1986; Fabrizio et al., 2002).
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Pseudomonads reached the value of 7 log CFU/g on day 10 (6.93 log CFU/g) for the vacuum
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packaged samples, while vacuum packaged chicken legs treated with 2, 5, and 10 mg/L of gaseous
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ozone never reached that upper limit value. The combined use of vacuum packaging and ozonation,
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ca. 16 days (P<0.001) (Figure 1b).
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Al-Haddad et al. (2005) applied gaseous ozone of >2000 mg/L for 30 min to control populations of
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Salmonella infantis and Pseudomonas aeruginosa inoculated on the skin of chicken portions (chilled
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breasts). Such a high concentration of ozone resulted in reducing the counts of salmonellae by 97%
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and pseudomonads by 95%.
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Lactic acid bacteria showed fluctuations in their counts depending on storage time. They reached 7
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log CFU/g on day 12-14 for the vacuum packaged samples, and day 14-16 for chicken samples
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treated with 2 mg/L. The combined use of vacuum packaging and ozonation at 5 or 10 mg/L
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resulted in a decrease in LAB counts during the 16 days of storage, since their populations never
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reached 7 log CFU/g (Figure 1c).
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Enterobacteriaceae, are considered a hygiene indicator (Chouliara et al., 2007). Their initial counts
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were ca. 2.83 log CFU/g, indicative of good quality chicken meat. Enterobacteriaceae were increased
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(P<0.001) by storage time in all treatments, and reached 7 log CFU/g on day 12-14 for the vacuum
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packaged samples, day 14-16
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Enterobacteriaceae never reached 7 log CFU/g using the combination of vacuum packaging and
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ozonation dose of 5 and 10 mg/L for 16 days storage period (Figure 1d).
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Trindade et al. (2012), compared ozone and chlorine solutions as sanitizing agents on chicken
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carcasses. They concluded that, in general, ozone was as effective as chlorine in the disinfection of
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chicken carcasses, and can be a potential substitute of chlorine in poultry slaughterhouses, resulting
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thus, to more hygiene conditions (i.e. inhibition or low counts of Enterobacteriaceae among other
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microorganisms).
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Finally, with respect to yeasts and moulds, their initial population was increased by storage time
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(P<0.001) in vacuum and ozone treated samples. Vacuum packaging in combination with ozonation
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throughout the experimental procedure (Figure 1e). As far we know, information regarding
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variations in the population of yeasts and moulds in poultry meat, after application of different ozone
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dose, is scarce. Data available involve grapes, apple cider, dried figs, date fruits (Jaiswal, 2017), and
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mineral water (Watanabe et al., 2010).
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In the present study, the use of a much lower concentration of ozone (5 or 10 mg/L) for 1h in
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combination with vacuum packaging resulted in 0.5 to 1.0 log cycles reduction to the counts of TVC
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and pseudomonads, whereas 10 mg/L of gaseous ozone resulted to >1.0 log cycles to the population
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of Enterobacteriaceae, yeast and molds and lactic acid bacteria.
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Finally, Muthukumar and Muthuchamy (2013) reported that ozone at specific doses of ca. 33 mg/L
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for 9 min in gaseous phase could be used as an effective method for inactivating 2×106 CFU/g of L.
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monocytogenes on chicken samples before they reach the market.
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Based on the obtained results, the combination of ozonation and vacuum packaging had a
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preservative effect on chicken leg meat, since it kept the populations of spoilage microorganisms at
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lower levels as compared to the vacuum packaged samples (P<0.001). To the best of our knowledge,
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data on the specific spoilage microorganisms studied in chicken leg meat, after the application of
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ozonation in combination with vacuum packaging has not been previously published, this
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constituting the novelty of the present work.
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3.2 Physicochemical changes
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pH values showed significant variations (P<0.001 and P<0.005) in vacuum packaging and vacuum
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packaging plus 2 and 5 mg/L of ozone, respectively, for a storage period of 16 days. This was not the
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case (P>0.005) for the higher ozone dose applied (10 mg/L) in the vacuum packaging system. It
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should be noted that pH is considered as an intrinsic factor in the present packaging technology
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investigated.
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decrease substantially the ozone decomposition rate. This hypothesis is supported by the kinetics of
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inactivation of E. coli, where the inactivation was much faster in an acidic medium, than in a basic
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one. For example, the inactivation of E. coli in water (108 CFU/mL) at a flow rate of 2 L/min and
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an ozone concentration of 0.90 mg/L at room temperature, resulted in a higher rate constant at pH=
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4.93 than at pH= 9.16 (Zuma et al.,2009).
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In the present study, pH was decreased in the vacuum packaging containing 5 and 10 mg/L of ozone
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as compared to the control samples, by 0.1-0.2 units (Table 1). Present pH variation is a slight
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decrease, compared to that of Zuma et al. (2009).
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Colour values of all chicken leg treatments at selected sampling days are given in Table 1. The L*
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value which refers to the lightness, decreased up to day 16 of storage, indicative of the fact that the
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colour of the product became more obscure.
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Sheldon and Brown (1986) reported that poultry carcasses were not affected by ozone treatment,
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since no skin colour loss was observed. Chouliara et al. (2007), in contrast to present results, reported
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an increase in L* values in chicken breast meat after using oregano essential oil and modified
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atmosphere packaging.
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Regarding parameter a*, which corresponds to degree of redness when positive and to degree of
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greenness when negative, as it shown in Table 1 such variations were observed, depending on
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storage period (P<0.001) and packaging treatment. Variations in colour parameter a* were also
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reported by Chouliara et al. (2007), in a study involving fresh chicken breast meat stored at 4 °C and
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treated with MAP and oregano essential oil.
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In a similar study, carried out by Chouliara et al. (2008), colour parameters L*, a* and b* values
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were not considerably affected by MAP, whereas applied irradiation resulted to a small increase only
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in parameter a*.
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storage and treatments. Parameter b* corresponds to yellowness of colour (when positive) and to
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blueness of colour (when negative) (Commission Internationale de l' Eclairage, CIE). b* values were
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not affected (P>0.05) by the gaseous ozone dose, whereas fluctuations (P<0.001) depending on
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storage time were observed in all samples. Such fluctuations are in agreement with the results
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reported by Chouliara et al. (2007).
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We may then conclude that above recent novel applications in food packaging, including ozonation
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such in our case, retain an acceptable chicken meat colour. Yes, they might be observed some
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variations depending on the use of a different packaging technology, but the main goal for food
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scientists is to increase shelf life of a favorable food commodity, such as poultry meat, without
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downgrading its physicochemical or sensory properties.
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3.3 Sensory changes
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In Table 2 full data are listed full data regarding sensory properties (odour, texture, appearance, and
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taste) of cooked chicken leg meat with respect to storage period and packaging technology. The
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lower acceptability score of 3 was reached for odour, texture, appearance, and taste after 10 days, for
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the vacuum packaged samples. Vacuum packaged samples and ozone treated with 2 mg/L, retained
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an acceptable score of 3 for 12 days regarding taste and texture, and for 14 days regarding
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appearance. However, odour retained an acceptable score for 10 days.
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By increasing the ozone dose to 5 mg/L, the results were positively affected since odour, texture, and
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taste retained an acceptable score for 14 days, whereas that of appearance for 16 days. By further
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increasing the ozone dose to 10 mg/L, the best results were obtained since all sensory properties
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investigated, retained an acceptable score for 16 days.
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quality of the pullet (Gallus gallus) for a 10 days storage time. Based on the collected sensory results
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(P<0.05), we propose the value of ≥3 to be considered as an overall sensory index (OSI) in similar
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studies.
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3.4. Comparison of sensory and microbiological data for estimating shelf life extension of fresh
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chicken legs based on the combination of vacuum packaging and ozonation
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Sensory data are in very good agreement with microbiological data, based on the TVC populations,
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after the application of vacuum packaging and ozonation at a dose of 10 mg/L. For example, odour,
322
taste, texture, and appearance acceptability scores (16 days) are in excellent agreement with TVC
323
results. Such an observation, is in agreement with the aerobic plate count and sensory (odour and
324
taste evaluation) data reported by other researchers (Nieto et al., 1984; Patsias et al., 2006; Ntzimani
325
et al. 2010), involving sensory evaluation of chicken meat after cooking.
326
On the other hand, vacuum packaged chicken legs treated with 2 and 5 mg/L of gaseous ozone
327
resulted into an agreement between microbiological and sensory data for 12-14 days of storage
328
period. As it can be observed, the application of a higher dose of ozone did not affect sensory
329
properties of fresh chicken legs packaged under vacuum. Analytical details regarding sensory and
330
microbiological data are given in supplementary Table 2.
331
3.5. Mathematical modeling for predicting the microbial growth of spoilage microorganisms in
332
chicken legs
333
Microbial growth modeling was estimated by constructing a linear regression analysis model as
334
mentioned in the work of Zwietering et al.(1990), using log CFU/g populations of the specific
335
microflora in a given treatment = f (storage time) (Table 3).
336
R-squared that is a measure of the amount of variation around the mean, and explained by the model,
337
can be defined as follows:
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ACCEPTED MANUSCRIPT R2 = 1 – SSresid/SSmodel + SSresid= 1 + SSerror/SStotal
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(Eq.2)
339
In this equation, SSresid is residual sum of squares and SSmodel is model sum of squares. Another
341
important parameter for evaluating the constructed model is adjusted R-squared (Radj2). This
342
parameter can be considered as a measure of the amount of variation around the mean explained by
343
the model adjusted for the number of terms in the model. In other words, the Radj2 decreases as the
344
number of terms in the model increases.
345
In addition, predicted R-square (Rpred2) which is a measure of the amount of variation in new data
346
explained by the model can be applied for the evaluation of the model. The Rpred2 and the Radj2 have
347
been obtained using Eqs. (3) and (4).
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R2adj= 1 – (n – 1/n – p) (SSerror/SStotal) = 1 − (n − 1)/(n = p)(1 − R2)
348 349
R2pred= (1 – PESS) / (SStotal − SSblock)
350
(Eq.4)
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(Eq.3)
where n is the number of experiments, p is the number of model parameters including intercept and
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any block coefficient, and PESS is the prediction error of sum of squares:
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PESS= ∑ !
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, − 2 , e i, -i = yi- ȳ i, -i
(Eq.5)
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where ei,−i is residual, yi is the experimental value, and ȳi,−1 is the predicted value.
357
358
Another important parameter that it should be considered is the Durbin–Watson statistic (d) test. This
359
statistic test is used to detect the presence of auto-correlation (a relationship between values
360
separated from each other by a given time lag) in the residuals (prediction errors) from a regression
361
analysis.
16
ACCEPTED MANUSCRIPT If the Durbin–Watson statistic is less than 2, there is evidence of positive serial correlation. On the
363
other hand, if Durbin–Watson is less than 1, there may be cause of concern. Small values of d
364
indicate successive error terms are, on average, close in value to one another, or positively
365
correlated. If d > 2, successive error terms are, on average, much different in value from one another,
366
i.e., negatively correlated. In regression analysis, this can imply an underestimation of the level of
367
statistical significance.
368
Goodness of fit of the obtained microbial growth model, was statistically significant affected
369
(P<0.01) by the type of the microorganism investigated. This is in general agreement with previous
370
work in the literature involving the role of different temperatures on the development of a microbial
371
growth model, regarding specific bacteria (Zwietering et al., 1990; Buzrul, 2009) (Table 3).
372
However, Durbin-Watson test gave d values< 1 for lactic acid bacteria in vacuum packaging, and in
373
vacuum packaging containing 2 mg/L of ozone. In addition, the adjusted R-squared values were
374
negative (-0.140) in the case of Enterobacteriaceae in the vacuum packaging plus 10 mg/L of ozone,
375
whereas low predicted R–squared values were also observed. In that sense, there was not any
376
predictive ability for Enterobacteriaceae.
377
Thus, considering all aforementioned criteria, the best modeling bacterial growth results, were
378
obtained for TVC, Pseudomonas spp., yeasts and moulds, and lactic acid bacteria (involving the
379
vacuum packaging containing 5 and 10 mg/L of ozone), as shown in Table 3.
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4. Conclusions
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Based on microbiological and sensory attributes, the shelf-life of vacuum packaged fresh chicken
383
legs was ca. 10 days. Addition of gaseous ozone of 10 mg/L for 1 hour, for chicken legs packaged in
384
plastic containers of PA/PE under refrigeration, proved to be an appropriate method for maintaining
385
freshness and quality of chicken leg meat, since its shelf life was extended by 6 days, as compared to
386
vacuum packaged samples. Vacuum packaging in combination with ozonation has the potential to
17
ACCEPTED MANUSCRIPT aid in the shelf life extension of fresh chicken legs as an innovative, simple, and economic
388
technology. The innovative non-thermal ozonation method has the following benefits: a) low
389
operation cost, b) ecological and chemical-free, c) simple to use. The overall cost of such an
390
application is low, definitely lower than the losses that could cause natural spoilage, if we consider
391
that poultry meat has a short shelf-life. A prospective shelf life extension of poultry meat with the
392
application of a novel technology, as supported by a microbial growth model, would increase its
393
market distribution at an international level.
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References
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ACCEPTED MANUSCRIPT Table 1. Variations in pH and colour parameters (mean±SD) according to packaging treatment and storage time Day/Parameter
Cv
Cv+2 mg/L
0 2 4 6 8 10 12 14 16 F df p
6.85 ± 0.11 6.79 ± 0.07 6.80 ± 0.03 6.90 ± 0.05 6.73 ± 0.05 7.14 ± 0.04 7.07 ± 0.06 6.96 ± 0.08 7.12 ± 0.05 17.329 26 <0.001
6.85 ± 0.11 6.81 ± 0.07 6.76 ± 0.05 6.76 ± 0.04 6.86 ± 0.06 7.08 ± 0.04 6.99 ± 0.07 7.00 ± 0.08 6.90 ± 0.05 8.457 26 <0.001
0 2 4 6 8 10 12 14 16 F df p
60.90 ± 0.33 58.94 ± 0.06 60.45 ± 0.12 55.85 ± 0.18 58.48 ± 0.14 54.88 ± 0.80 57.11 ± 0.31 61.57 ± 0.18 58.65 ± 0.13 140.498 26 <0.001
60.90 ± 0.33 58.72 ± 0.41 59.62 ± 0.04 59.62 ± 0.04 56.62 ± 0.42 56.96 ± 0.71 56.32 ± 0.44 60.52 ± 0.23 58.01 ± 0.15 59.595 26 <0.001
0 2 4 6 8 10 12 14 16 F df p
0.91 ± 0.29 1.35 ± 0.56 0.82 ± 0.61 0.49 ± 0.30 0.18 ± 0.57 1.27 ± 1.29 0.08 ± 0.75 0.57 ± 0.16 0.51 ± 0.38 1.655 26 0.178
Cv+5 mg/L
Cv+10 mg/L
pH
L*
0 2 4 6 8 10 12 14 16 F df p
9.68 ± 0.39 11.76 ± 0.48 10.26 ± 0.19 10.37 ± 0.15 10.32 ± 0.23 8.82 ± 0.28 8.19 ± 0.33 8.77 ± 0.13 8.37 ± 0.06 53.484 26 <0.001
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b*
60.90 ± 0.33 51.07 ± 0.18 53.44 ± 0.13 50.78 ± 0.27 54.55 ± 0.56 53.42 ± 0.25 57.11 ± 0.63 57.85 ± 0.30 52.59 ± 0.15 261.590 26 <0.001
0.91 ± 0.29 0.63 ± 0.28 0.43 ± 0.27 0.54 ± 0.43 0.52 ± 0.53 0.11 ± 0.49 0.14 ± 0.81 0.22 ± 1.57 0.29 ± 1.04 1.038 26 0.445
0.91 ± 0.29 0.03 ± 0.19 1.48 ± 0.79 1.27 ± 0.70 0.61 ± 0.32 0.51 ± 0.99 0.78 ± 1.15 0.80 ± 0.81 0.17 ± 0.84 1.191 26 0.357
0.91 ± 0.29 2.12 ± 1.08 2.26 ± 0.16 0.05 ± 0.65 0.48 ± 0.34 2.92 ± 0.90 2.11 ± 1.78 0.13 ± 0.80 0.60 ± 0.22 4.405 26 0.004
9.68 ± 0.39 9.79 ± 0.11 10.00 ± 0.13 9.22 ± 0.15 11.31 ± 0.30 8.67 ± 0.25 8.78 ± 0.46 7.82 ± 0.21 9.91 ± 0.11 43.169 26 <0.001
9.68 ± 0.39 6.74 ± 0.23 10.48 ± 0.19 7.64 ± 0.20 7.67 ± 0.23 7.98 ± 0.27 9.70 ± 0.15 7.70 ± 0.11 10.88 ± 0.29 112.379 26 <0.001
9.68 ± 0.39 8.23 ± 0.33 9.07 ± 0.20 7.60 ± 0.17 7.54 ± 0.76 9.54 ± 0.12 12.89 ± 0.63 7.56 ± 0.16 9.19 ± 0.62 44.382 26 <0.001
EP
a*
60.90 ± 0.33 57.32 ± 0.03 51.41 ± 0.29 57.14 ± 0.15 58.38 ± 0.13 50.85 ± 0.29 53.65 ± 0.07 56.21 ± 0.11 57.68 ± 0.14 833.283 26 <0.001
6.85 ± 0.11 6.77 ± 0.05 6.68 ± 0.03 6.89 ± 0.12 6.76 ± 0.06 6.81 ± 0.04 6.81 ± 0.17 6.92 ± 0.03 6.83 ± 0.06 2.104 26 0.091
SC
6.85 ± 0.11 6.85 ± 0.04 6.95 ± 0.09 7.04 ± 0.08 6.86 ± 0.06 6.78 ± 0.06 6.83 ± 0.09 6.82 ± 0.02 6.82 ± 0.05 3.645 26 0.011
ANOVA in comparison of means (p<0.001), mean±SD: average ±standard deviation values of three replicates (n=3). Cv: control packaging (vacuum). F: variation between sample means / variation within the samples, df: degrees of freedom, p:probability
ACCEPTED MANUSCRIPT Table 2. Variations in sensory attributes (mean±SD) according to packaging treatment and storage time Cv
Cv+2 mg/L
Cv+5 mg/L
Cv+10 mg/L
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.33 ± 0.29 4.00 ± 0.00 3.00 ± 0.00 2.67 ± 0.58 2.57 ± 0.00 1.00 ± 0.00 138.600
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.53 ± 0.06 4.23 ± 0.06 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 2.67 ± 0.58 22.300
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.73 ± 0.06 4.53 ± 0.06 4.00 ± 0.00 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 24.094
26 <0.001
26 <0.001
26 <0.001
26 <0.001
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.47 ± 0.25 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 1.33 ± 0.58
EP
5.00 ± 0.00 5.00 ± 0.00 4.67 ± 0.58 4.00 ± 0.00 3.33 ± 0.58 3.00 ± 0.00 2.67 ± 0.58 2.00 ± 0.00 1.00 ± 0.00
803.726 26 <0.001
52.333 26 <0.001
5.00 ± 0.00 5.00 ± 0.00 4.67 ± 0.58 4.33 ± 0.29 3.33 ± 0.29 3.00 ± 0.00 2.00 ± 0.00 ND ND
55.556 20 <0.001
27.384 26 <0.001
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67.000 26 <0.001
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.57 ± 0.12 4.27 ± 0.12 3.97 ± 0.25 3.67 ± 0.58 3.00 ± 0.00 2.67 ± 0.58
40.881 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.63 ± 0.06 4.00 ± 0.20 3.40 ± 0.17 3.03 ± 0.25 ND ND
104.626 20 <0.001
SC
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.53 ± 0.06 4.17 ± 0.15 3.57 ± 0.21 3.00 ± 0.00 2.00 ± 0.00 1.00 ± 0.00
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.73 ± 0.06 4.50 ± 0.10 4.37 ± 0.15 4.17 ± 0.15 3.33 ± 0.58 3.00 ± 0.00
M AN U
5.00 ± 0.00 5.00 ± 0.00 4.67 ± 0.29 4.00 ± 0.00 3.33 ± 0.29 3.00 ± 0.00 2.67 ± 0.58 1.67 ± 0.58 1.00 ± 0.00
RI PT
5.00 ± 0.00 5.00 ± 0.00 4.00 ± 0.00 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 1.67 ± 0.58 1.00 ± 0.00 1.00 ± 0.00 65.083
AC C
Days/Sensory attributes Odour 0 2 4 6 8 10 12 14 16 F df P Texture 0 2 4 6 8 10 12 14 16 F df P Appearance 0 2 4 6 8 10 12 14 16 F df P Taste 0 2 4 6 8 10 12 14 16 F df P
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.60 ± 0.17 4.00 ± 0.00 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00
25.019 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.70 ± 0.00 4.37 ± 0.15 4.00 ± 0.00 3.33 ± 0.58 3.00 ± 0.00 2.33 ± 0.58
38.088 26 <0.001
37.125 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.73 ± 0.06 4.33 ± 0.29 4.00 ± 0.00 3.67 ± 0.58 3.00 ± 0.00
33.841 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.80 ± 0.10 4.63 ± 0.12 4.33 ± 0.29 3.67 ± 0.58 3.33 ± 0.29 3.00 ± 0.00
31.325 26 <0.001
ANOVA in comparison of means (p<0.001), mean±SD: average ±standard deviation values of three replicates (n=3). Cv: control packaging (vacuum). ND: not determined. F: variation between sample means / variation within the samples, df: degrees of freedom, P:probability
ACCEPTED MANUSCRIPT Table 3. Microbial growth modeling using linear regression analysis with respect to storage time and packaging treatment Microbial growth
Cv
Cv+2 ppm
Cv+5 ppm
Cv+10 ppm
0.918 0.316 3.744 0.57 0.904 0.958 10.425 7 66.925 <0.001 0.50 1.86
0.856 0.263 3.425 0.52 0.845 0.925 10.647 8 41.596 <0.001 0.63 1.40
0.787 0.155 3.701 0.38 0.806 0.911 14.069 8 34.329 <0.001 0.49 1.58
0.831 0.187 3.782 0.32 0.757 0.887 15.476 8 25.860 <0.001 0.47 1.47
0.918 0.333 3.487 0.40 0.904 0.958 9.656 7 66.805 <0.001 0.53 1.61
0.773 0.212 3.401 0.35 0.816 0.916 10.841 8 36.498 <0.001 0.62 1.03
0.884 0.308 2.961 0.33 0.864 0.940 9.024 7 45.618 <0.001 0.59 1.35 0.855 0.396 1.288 0.33 0.831 0.925 5.476 7 35.459
Slope Intercept SEM Radj 2 Rpred 2 t df F P PESS Durbin-Watson Yeasts and Moulds R2
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0.803 0.166 3.590 0.25 0.774 0.896 14.559 8 28.442 <0.001 0.48 2.0
0.820 0.133 3.746 0.20 0.794 0.906 17.893 8 31.874 <0.001 0.37 1.62
0.838 0.283 2.596 0.29 0.815 0.916 8.611 8 36.284 <0.001 0.73 1.10
0.774 0.21 2.641 0.23 0.742 0.880 9.915 8 23.961 <0.001 0.66 1.22
0.637 0.151 2.728 0.19 0.585 0.798 11.377 8 12.260 <0.001 0.67 1.54
0.841 0.402 0.71 0.28 0.435 0.711 6.635 8 7.149
0.804 0.322 1.016 0.22 0.490 0.744 7.659 8 8.697
0.712 0.249 0.946 0.22 neg 0.650 6.533 8 0.017
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0.898 0.342 1.599 0.26 0.883 0.948 6.577 8 61.644 <0.001 0.68 0.96
0.847 0.286 1.494 0.21 0.825 0.920 6.668 8 38.748 <0.001 0.71 1.52
0.771 0.245 1.473 0.21 0.739 0.878 6.742 8 23.627 <0.001 0.78 1.48
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Cv: vacuum packaging (control), Cv+2ppm: vacuum packaging plus 2 ppm of ozone, Cv+5ppm: vacuum packaging plus 5 ppm of ozone, Cv+10ppm: vacuum packaging plus 10ppm of ozone. TVC: total viable count, LAB: lactic acid bacteria, SEM: standard error of mean, t: T-test, df: degrees of freedom, P: probability at the confidence level P<0.05. Radj2: adjusted R-squared values, Rpred2 : predicted R-squared values. neg: negative value, not considered. F: variation between sample means / variation within the samples. PESS: predicted error of sum of squares.
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Vacuum packaging (VP) plus Ozonation (O) comprises an innovative
technology Sensory properties of chicken legs were not affected negatively by ozonation
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6 days shelf life extension using 10 mg/L of ozone and vacuum packaging
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VP plus O exhibited an additive preservation effect on fresh chicken legs
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S0260-8774(17)30272-8
DOI:
10.1016/j.jfoodeng.2017.06.026
Reference:
JFOE 8931
To appear in:
Journal of Food Engineering
Received Date: 29 October 2016 Revised Date:
20 June 2017
Accepted Date: 22 June 2017
Please cite this article as: Gertzou, I.N., Karabagias, I.K., Drosos, P.E., Riganakos, K.A., Effect of combination of ozonation and vacuum packaging on shelf life extension of fresh chicken legs during storage under refrigeration, Journal of Food Engineering (2017), doi: 10.1016/j.jfoodeng.2017.06.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of combination of ozonation and vacuum packaging on shelf life extension
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of fresh chicken legs during storage under refrigeration
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by
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Ioanna N. Gertzou, Ioannis K. Karabagias, Panagiotis E. Drosos, and Kyriakos A. Riganakos*
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Laboratory of Food Chemistry, Department of Chemistry, University of Ioannina, 45110-Ioannina,
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*Corresponding author: Tel.: +302651008341; Fax: +302651008795 E-mail address: [email protected] (K.A. Riganakos)
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ACCEPTED MANUSCRIPT Abstract
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The objective of the present study was to investigate the combined effect of different ozone dose (2,
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5, and 10 mg/L) and of vacuum packaging on shelf life extension of fresh chicken legs, packaged
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with polyamide/polyethylene bags and stored at 4±1 °C, for 16 days. Parameters considered were:
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microbiological (TVC, Pseudomonas spp., LAB, Yeasts and moulds, and Enterobacteriaceae),
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physicochemical (pH, colour parameters) and sensory (odour, appearance, texture, and taste)
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attributes. The results obtained in this study showed that physicochemical parameters varied
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significantly (P<0.05) depending on ozonation dose and storage time. Populations of spoilage flora
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along with sensory properties, were affected by packaging technology and storage time (P<0.05).
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Conclusively, the combination of gaseous ozone of 10 mg/L and vacuum packaging, under
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refrigeration, resulted to a 6 days shelf life extension of chicken legs, as compared to single vacuum
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packaging. The modeling of microbial growth, resulted in the collection of positive results (P<0.05).
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Keywords Chicken legs; vacuum packaging; ozonation; shelf-life extension; sensory properties
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ACCEPTED MANUSCRIPT Introduction
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Sixteen billion domesticated fowl, such as chickens, turkeys, ducks, and geese, are raised annually,
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more than half of these in industrialized factory-like production units, to cover consumers’ demand
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for meat or eggs. Poultry meat is a favorable food commodity worldwide. The largest producers are
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the United States (20%), China (16.6%), Brazil (15.1%) and the European Union (11.3%) (USDA
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Livestock and Poultry, 2014). It is a nutritional and qualitative type of food due to its low fat content,
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a relatively high concentration of polyunsaturated fatty acids, its excellent quality protein, and
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essential amino acids content needed by humans (Zhang et al., 2016).
48
Considering the fact that poultry meat belongs to perishable foods, the main concern of food
49
industries is to extend the shelf life of the product, by ensuring at the same time consumers’
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protection from spoilage and pathogenic bacteria, respectively (Kim and Yousef, 2000; Zeynep,
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2003; USDA Livestock and Poultry, 2014).
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Pseudomonas spp. (Gram positive, aerobe), Enterobacteriaceae (Gram negative, facultative
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anaerobe), and Brochothrix thermosphacta (Gram positive, facultative anaerobe) are commonly
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found in poultry meat, along with Lactic acid bacteria (Gram positive, aerotolerant anaerobe), and
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Shewanella putrefaciens (Gram negative, facultative anaerobe) representing the principle spoilage
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bacteria of fresh meats (Whitfield, 1998; Totosaus and Kuri, 2012).
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Market differentiation of fresh poultry products (i.e. legs, thighs, breasts or deboned breasts) is
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achieved through novel vacuum packaging development and product innovation. One of the main
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challenges of the food industry is the application of multifunctional packaging (Taylor, 1977;
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Spaulding, 1994; Kartika et al., 2003; James, 2008; Totosaus and Kuri, 2012).
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Plastic materials to develop such a novel packaging system include: Saran or polyvinyl chloride
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(PVC), an absorbent pad in conventional foam trays with PVC overwrap, polypropylene (PP),
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polystyrene (PS), polyvinyl chloride (PVC), and various grades of polyethylene (HDPE, LDPE, etc.)
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ACCEPTED MANUSCRIPT (Duncan, 2011). Other thermoforming film materials that can be applied for meat packaging include
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ethylene-vinyl alcohol (EVOH), polypropylene-oriented polypropylene (OPP), ethylene vinyl acetate
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(EVA), and polyvinylidene chloride (PVdC), each having different mechanical and barrier properties
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(Crippa et al.,2007).
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New trends in meat and poultry packaging using aforementioned plastic materials alone or in
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combination, involve the combined use of modified atmosphere packaging with essential oils, freeze
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chilling, irradiation, absorbent pads containing essential oils, and vacuum packaging, have been
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shown to delay spoilage in poultry meat (Rao and Sachindra, 2002; Patsias et al., 2006; Chouliara et
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al., 2007; Balamatsia et al., 2007; Chouliara et al., 2008; Oral et al., 2009).
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On the other hand, ozonation is an innovative non-thermal method with potential applications in the
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modern food industry (Jeong et al., 2007; Cullen et al., 2010; Piachin and Trachoo, 2011; Cárdenas
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et al., 2011; Shah et al., 2011; Blogoslawski and Stewart, 2011; Fratamico et al., 2012).
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Ozonation is a safe way to oxidize contaminants while leaving no residues and without affecting the
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quality of food. Ozone-based treatments are eco-friendly, applicable to sanitizing a wide range of
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materials and replace alternative chemical sanitizers such as, chlorine, acids, salts, etc. (Trindade et
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al., 2012). It not only acts on microorganisms (Muthukumarappan et al., 2008), but also on pests,
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serving thus, as a fumigant. Several researchers have suggested that ozonized water effectively
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improved the chemical properties and safety of refrigerated meat (Bhattacharya, 2015). Since ozone
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treatment meets all the requirements of the food consumer, it can be regarded as a ‘’greener’’
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additive (Da-Wen Sun, 2014). However, due to its strong oxidizing power, ozone may be toxic for
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humans above certain levels of concentration (0.1-0.3 ppm) and length of exposure (Perry and
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Yousef, 2011; Bhattacharya, 2015).
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accordance with current industry standards of good manufacturing practice (FSIS Directive 2016).
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The excessive use of ozone is not universally beneficial and in some cases may promote oxidative
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spoilage in foods, such as meat, as well as discoloration or even development of undesirable odours
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(Khadre et al., 2001).
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Given the limited data available in the literature on the application of ozone and vacuum packaging
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to poultry meat products (Yang and Chen, 1979; Nieto et al.,1984; Sheldon and Brown, 1986; Al-
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Haddad et al., 2005; Muthukumar and Muthuchamy, 2013) and ozone’s properties considered
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aforementioned, the aim of the present study was to investigate the effectiveness of different dose
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ozone treatments (2, 5, and 10 mg/L) in combination with vacuum packaging on the shelf life
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extension of fresh chicken legs, stored at 4 °C, as assessed by microbiological, physicochemical, and
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sensory attributes.
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Fresh chicken legs meat in chunks weighing ca.300 g, were provided by a local poultry processing
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industry (Pindos S.A., Ioannina, Greece) within one hour after slaughter in insulated polystyrene
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boxes on ice. Chicken legs were subjected to cooling with cold water after slaughtering.
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2.2. Vacuum packaging and ozonation conditions
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All chicken samples were exposed for 1h under gaseous ozone treatment (concentrations of 2, 5 and
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10 mg/L) at room temperature. System of ozonation was C-Lasky L010 supplied from Air tree
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company (Taiwan).
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consumption (180 W) and stable ozone production. Detectable concentrations are in the range of
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This ozone generator is designed to provide high efficiency, low energy
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ozone concentration [in our case 2, 5, and 10 mg/L (or ppm)] was accurate and constant, the OS-4
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(Eco-Sensors, Santa Fe, New Mexico, USA) probe was used.
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Chicken leg samples were then placed in polyamide/polyethylene (PA/PE) barrier pouches 29.5 x
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29.5 cm, 90 µm in thickness having an oxygen permeability < 15 cm3 m-2 d−1 atm−1, nitrogen
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permeability < 15 cm3 m-2 d−1 atm−1, carbon dioxide permeability < 200 cm3 m-2 d−1 atm−1 at 75%
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relative humidity (RH), 23°C (Method DIN 53380-2),
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<1 g m−2 d−1 at 85% RH, 23°C (Method DIN 53122-2). Finally, pouches were heat-sealed using a
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BOSS model N48 vacuum sealer (BOSS, Bad Homburg, Germany) and kept at 4±1 °C. Sampling,
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following all aforementioned technical criteria, was carried out on 0, 2, 4, 6, 8, 10, 12, 14, and 16
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days of storage.
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2.3.Microbiological analysis
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The following groups of microflora were monitored: Total viable counts (TVC), Pseudomonas spp.,
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Yeasts and moulds, Lactic acid bacteria (LAB), as well as Enterobacteriaceae. Ten grams of fresh
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chicken leg meat were removed aseptically from the package using a spoon, transferred to a
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stomacher bag (Seward Medical, Worthing, West Sussex , UK), containing 90 mL of sterile buffered
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peptone water (LAB 204, LAB M), and homogenized using a stomacher (LAB Blender 400, Seward
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Medical) for 60 s at room temperature.
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For microbial enumeration, 0.1 mL samples of serial dilutions (1:10 diluents, buffered peptone
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water) of chicken leg meat homogenates were spread on the surface of the following agar plates
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prepared. TVC were determined using plate count agar (PCA, LAB 010, LAB M, Lancashire, UK),
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after incubation for 2 days at 30 oC. Pseudomonads
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cephaloridine agar (LAB 108, LAB M, supplemented with X 108, Lancashire, UK) after incubation
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036, LAB M, Lancashire, UK) after incubation at 30 oC for 3-5 days.
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For members of the Enterobacteriaceae spp., 1.0 mL sample was inoculated into 10 mL of molten
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(45 °C) violet red bile glucose agar (LAB 088, LAB M, Lancashire, UK) after incubation for 24 h at
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37 oC. The large colonies with purple haloes were counted. Lactic acid bacteria were determined on
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de Man, Rogosa and Sharpe medium (LAM 093, LAB M, Lancashire, UK) after incubation at 30 °C
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for 3 days. All plates were examined visually for typical colony types and morphological
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characteristics associated with each medium.
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2.4.Physicochemical analysis
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pH values of fresh chicken leg meat were measured using a Delta OHM, model HD 3456.2, pH-
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meter (Padova, Italy) with a precision of ±0.002. Chicken leg samples were thoroughly homogenized
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with 10 mL of distilled water and the homogenate used for pH determination. Colour determination
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was carried out on the surface of chicken legs, which were placed into a cylindrical optical cell,
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using a Hunter Lab model DP-9000 colorimeter coupled to a D25 L optical sensor (Hunter
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Associates Laboratory, Reston VA, USA). Reflectance values were obtained using a 45 mm viewing
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aperture. Each determination was run in triplicate (n=3).
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2.5.Sensory evaluation
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Chicken leg samples (ca. 100 g) after defrosting, were cooked in a microwave oven set at high power
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(700 W) for 4 min. A panel of seven judges experienced in chicken evaluation was used for sensory
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analysis. Panelists were asked to evaluate odour, texture, appearance, and taste intensities of cooked
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samples. Along with the test samples, the panelists were presented with a freshly thawed chicken leg,
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stored at −30 °C throughout the experiment, this serving as the reference sample. Acceptability of
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odour, texture, appearance, and taste was estimated using an acceptability scale ranging from 5 to 0,
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3 was taken as the lower limit of acceptability. Each evaluation was run in triplicate (n=3) and every
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sample was evaluated individually.
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2.6.Statistical analysis
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Physicochemical and microbiological data were subjected to analysis of variance (ANOVA) in order
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to investigate whether vacuum packaging or the combination of vacuum packaging and ozonation,
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could affect the aforementioned parameters throughout storage time. In addition, in order to develop
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a microbial growth model, linear regression analysis was also conducted. All statistical treatment
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was performed using the SPSS v.20.0 statistics software. Experiments were replicated three times on
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different occasions with different chicken leg samples. Analyses were run in triplicate (three
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different packaged samples) for each replicate (n=3×3). Microbiological data were transformed into
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logarithms of the number of colony forming units (log CFU/g).
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2.7. Theory
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2.7.1.
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Ozone (O3) is generated by the reaction of oxygen free radicals with diatomic oxygen. It is an
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allotropic form of oxygen, with very good antimicrobial properties. It was in 1840 when Christian
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Friedrich Schönbein a German-Swiss chemist named the substance ozone, based on the Greek word
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ozein for ‘’smell’’ (Kogelschatz, 1988). The chemical reactions of ozone are either indirect (chain-
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reactions mechanism leading to the production of hydroxyl free radicals) or direct (with substances
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present in the matrix) (Cullen et al., 2010; O’Donell et al., 2012).
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It has been previously reported that, ozone chemical reactions are highly selective against a wide
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range of bacteria, fungi, viruses, protozoa, bacterial and fungal spores (Khadre et al., 2001;
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Muthukumarappan et al., 2008; Cullen et al., 2010).
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is the primary target site for antimicrobial action. Inactivation by ozone is a complex procedure that
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attacks cell membrane, cell wall constituents, and intracellular constituents (Khadre et al., 2001). In
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the present study we applied different gaseous ozone concentrations. Depending on the
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concentrations required, ozone gas can be generated using coronal discharges.
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2.7.2. Modeling of microbial growth
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Modeling of microbial growth in food is a mandatory tool for food safety prediction and shelf-life of
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products in the real market world. Over the past 25 years numerous growth models have been
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proposed, depending on different parameters (i.e. elevated temperatures, pH, salt, etc.) that may
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affect the kinetics of microbial growth (Bratchell et al.,1989; Zwietering et al., 1990; Fujikawa et al.,
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2004).
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The growth of a homogenous microbial population can be described by a curve with three phases if
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the death phase is excluded: a lag phase (adaptation period of microbial cells to their new
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environment) followed by an exponential growth phase (multiplication of cells exponentially) and
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finally a stationary phase (reaching to the maximum population density) (Buzrul, 2009). Equation 1
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the asymptotic value which is reached at time tA, and tm is the time where maximum growth rate is
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specific microorganisms’ growth: a) packaging media and b) storage time.
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3. Results and discussion
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3.1 Microbiological changes
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TVC values for the four different chicken leg treatments are shown in Figure 1a as a function of
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storage time. TVC reached the value of 7 log CFU/g, which is considered as the microbiological
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upper limit for good quality poultry meat (Chouliara et al., 2007), on day 10 for vacuum packaged
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samples, day 14 for samples treated with 2 mg/L of ozone, and day 16 for samples treated with 5 and
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10 mg/L of ozone, respectively.
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The use of a higher ozone dose, i.e. 5 or 10 mg/L, resulted in extending the shelf life of fresh chicken
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legs by ca. 6 days as compared to vacuum packaged samples (P<0.001). On the other hand, a shelf
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life extension by ca. 4 days was observed for chicken leg samples packaged in vacuum and treated
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with 2 mg/L of gaseous ozone.
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Present results are in agreement with those reported previously, in which ozonation proved to be
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effective against a wide range of natural poultry microflora, providing thus, a substantial increase in
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shelf life of poultry products investigated (Yang and Chen 1979; Nieto et al., 1984; Sheldon and
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Brown, 1986; Fabrizio et al., 2002).
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Pseudomonads reached the value of 7 log CFU/g on day 10 (6.93 log CFU/g) for the vacuum
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packaged samples, while vacuum packaged chicken legs treated with 2, 5, and 10 mg/L of gaseous
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ozone never reached that upper limit value. The combined use of vacuum packaging and ozonation,
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ca. 16 days (P<0.001) (Figure 1b).
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Al-Haddad et al. (2005) applied gaseous ozone of >2000 mg/L for 30 min to control populations of
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Salmonella infantis and Pseudomonas aeruginosa inoculated on the skin of chicken portions (chilled
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breasts). Such a high concentration of ozone resulted in reducing the counts of salmonellae by 97%
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and pseudomonads by 95%.
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Lactic acid bacteria showed fluctuations in their counts depending on storage time. They reached 7
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log CFU/g on day 12-14 for the vacuum packaged samples, and day 14-16 for chicken samples
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treated with 2 mg/L. The combined use of vacuum packaging and ozonation at 5 or 10 mg/L
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resulted in a decrease in LAB counts during the 16 days of storage, since their populations never
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reached 7 log CFU/g (Figure 1c).
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Enterobacteriaceae, are considered a hygiene indicator (Chouliara et al., 2007). Their initial counts
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were ca. 2.83 log CFU/g, indicative of good quality chicken meat. Enterobacteriaceae were increased
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(P<0.001) by storage time in all treatments, and reached 7 log CFU/g on day 12-14 for the vacuum
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packaged samples, day 14-16
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Enterobacteriaceae never reached 7 log CFU/g using the combination of vacuum packaging and
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ozonation dose of 5 and 10 mg/L for 16 days storage period (Figure 1d).
237
Trindade et al. (2012), compared ozone and chlorine solutions as sanitizing agents on chicken
238
carcasses. They concluded that, in general, ozone was as effective as chlorine in the disinfection of
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chicken carcasses, and can be a potential substitute of chlorine in poultry slaughterhouses, resulting
240
thus, to more hygiene conditions (i.e. inhibition or low counts of Enterobacteriaceae among other
241
microorganisms).
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Finally, with respect to yeasts and moulds, their initial population was increased by storage time
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(P<0.001) in vacuum and ozone treated samples. Vacuum packaging in combination with ozonation
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ACCEPTED MANUSCRIPT at 5 or 10 mg/L, had a strong effect on yeasts population, since they never reached 7 log CFU/g
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throughout the experimental procedure (Figure 1e). As far we know, information regarding
246
variations in the population of yeasts and moulds in poultry meat, after application of different ozone
247
dose, is scarce. Data available involve grapes, apple cider, dried figs, date fruits (Jaiswal, 2017), and
248
mineral water (Watanabe et al., 2010).
249
In the present study, the use of a much lower concentration of ozone (5 or 10 mg/L) for 1h in
250
combination with vacuum packaging resulted in 0.5 to 1.0 log cycles reduction to the counts of TVC
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and pseudomonads, whereas 10 mg/L of gaseous ozone resulted to >1.0 log cycles to the population
252
of Enterobacteriaceae, yeast and molds and lactic acid bacteria.
253
Finally, Muthukumar and Muthuchamy (2013) reported that ozone at specific doses of ca. 33 mg/L
254
for 9 min in gaseous phase could be used as an effective method for inactivating 2×106 CFU/g of L.
255
monocytogenes on chicken samples before they reach the market.
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Based on the obtained results, the combination of ozonation and vacuum packaging had a
257
preservative effect on chicken leg meat, since it kept the populations of spoilage microorganisms at
258
lower levels as compared to the vacuum packaged samples (P<0.001). To the best of our knowledge,
259
data on the specific spoilage microorganisms studied in chicken leg meat, after the application of
260
ozonation in combination with vacuum packaging has not been previously published, this
261
constituting the novelty of the present work.
262
3.2 Physicochemical changes
263
pH values showed significant variations (P<0.001 and P<0.005) in vacuum packaging and vacuum
264
packaging plus 2 and 5 mg/L of ozone, respectively, for a storage period of 16 days. This was not the
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case (P>0.005) for the higher ozone dose applied (10 mg/L) in the vacuum packaging system. It
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should be noted that pH is considered as an intrinsic factor in the present packaging technology
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investigated.
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decrease substantially the ozone decomposition rate. This hypothesis is supported by the kinetics of
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inactivation of E. coli, where the inactivation was much faster in an acidic medium, than in a basic
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one. For example, the inactivation of E. coli in water (108 CFU/mL) at a flow rate of 2 L/min and
272
an ozone concentration of 0.90 mg/L at room temperature, resulted in a higher rate constant at pH=
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4.93 than at pH= 9.16 (Zuma et al.,2009).
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In the present study, pH was decreased in the vacuum packaging containing 5 and 10 mg/L of ozone
275
as compared to the control samples, by 0.1-0.2 units (Table 1). Present pH variation is a slight
276
decrease, compared to that of Zuma et al. (2009).
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Colour values of all chicken leg treatments at selected sampling days are given in Table 1. The L*
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value which refers to the lightness, decreased up to day 16 of storage, indicative of the fact that the
279
colour of the product became more obscure.
280
Sheldon and Brown (1986) reported that poultry carcasses were not affected by ozone treatment,
281
since no skin colour loss was observed. Chouliara et al. (2007), in contrast to present results, reported
282
an increase in L* values in chicken breast meat after using oregano essential oil and modified
283
atmosphere packaging.
284
Regarding parameter a*, which corresponds to degree of redness when positive and to degree of
285
greenness when negative, as it shown in Table 1 such variations were observed, depending on
286
storage period (P<0.001) and packaging treatment. Variations in colour parameter a* were also
287
reported by Chouliara et al. (2007), in a study involving fresh chicken breast meat stored at 4 °C and
288
treated with MAP and oregano essential oil.
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In a similar study, carried out by Chouliara et al. (2008), colour parameters L*, a* and b* values
290
were not considerably affected by MAP, whereas applied irradiation resulted to a small increase only
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in parameter a*.
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storage and treatments. Parameter b* corresponds to yellowness of colour (when positive) and to
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blueness of colour (when negative) (Commission Internationale de l' Eclairage, CIE). b* values were
295
not affected (P>0.05) by the gaseous ozone dose, whereas fluctuations (P<0.001) depending on
296
storage time were observed in all samples. Such fluctuations are in agreement with the results
297
reported by Chouliara et al. (2007).
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We may then conclude that above recent novel applications in food packaging, including ozonation
299
such in our case, retain an acceptable chicken meat colour. Yes, they might be observed some
300
variations depending on the use of a different packaging technology, but the main goal for food
301
scientists is to increase shelf life of a favorable food commodity, such as poultry meat, without
302
downgrading its physicochemical or sensory properties.
303
3.3 Sensory changes
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In Table 2 full data are listed full data regarding sensory properties (odour, texture, appearance, and
305
taste) of cooked chicken leg meat with respect to storage period and packaging technology. The
306
lower acceptability score of 3 was reached for odour, texture, appearance, and taste after 10 days, for
307
the vacuum packaged samples. Vacuum packaged samples and ozone treated with 2 mg/L, retained
308
an acceptable score of 3 for 12 days regarding taste and texture, and for 14 days regarding
309
appearance. However, odour retained an acceptable score for 10 days.
310
By increasing the ozone dose to 5 mg/L, the results were positively affected since odour, texture, and
311
taste retained an acceptable score for 14 days, whereas that of appearance for 16 days. By further
312
increasing the ozone dose to 10 mg/L, the best results were obtained since all sensory properties
313
investigated, retained an acceptable score for 16 days.
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315
quality of the pullet (Gallus gallus) for a 10 days storage time. Based on the collected sensory results
316
(P<0.05), we propose the value of ≥3 to be considered as an overall sensory index (OSI) in similar
317
studies.
318
3.4. Comparison of sensory and microbiological data for estimating shelf life extension of fresh
319
chicken legs based on the combination of vacuum packaging and ozonation
320
Sensory data are in very good agreement with microbiological data, based on the TVC populations,
321
after the application of vacuum packaging and ozonation at a dose of 10 mg/L. For example, odour,
322
taste, texture, and appearance acceptability scores (16 days) are in excellent agreement with TVC
323
results. Such an observation, is in agreement with the aerobic plate count and sensory (odour and
324
taste evaluation) data reported by other researchers (Nieto et al., 1984; Patsias et al., 2006; Ntzimani
325
et al. 2010), involving sensory evaluation of chicken meat after cooking.
326
On the other hand, vacuum packaged chicken legs treated with 2 and 5 mg/L of gaseous ozone
327
resulted into an agreement between microbiological and sensory data for 12-14 days of storage
328
period. As it can be observed, the application of a higher dose of ozone did not affect sensory
329
properties of fresh chicken legs packaged under vacuum. Analytical details regarding sensory and
330
microbiological data are given in supplementary Table 2.
331
3.5. Mathematical modeling for predicting the microbial growth of spoilage microorganisms in
332
chicken legs
333
Microbial growth modeling was estimated by constructing a linear regression analysis model as
334
mentioned in the work of Zwietering et al.(1990), using log CFU/g populations of the specific
335
microflora in a given treatment = f (storage time) (Table 3).
336
R-squared that is a measure of the amount of variation around the mean, and explained by the model,
337
can be defined as follows:
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(Eq.2)
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In this equation, SSresid is residual sum of squares and SSmodel is model sum of squares. Another
341
important parameter for evaluating the constructed model is adjusted R-squared (Radj2). This
342
parameter can be considered as a measure of the amount of variation around the mean explained by
343
the model adjusted for the number of terms in the model. In other words, the Radj2 decreases as the
344
number of terms in the model increases.
345
In addition, predicted R-square (Rpred2) which is a measure of the amount of variation in new data
346
explained by the model can be applied for the evaluation of the model. The Rpred2 and the Radj2 have
347
been obtained using Eqs. (3) and (4).
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R2adj= 1 – (n – 1/n – p) (SSerror/SStotal) = 1 − (n − 1)/(n = p)(1 − R2)
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R2pred= (1 – PESS) / (SStotal − SSblock)
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(Eq.4)
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(Eq.3)
where n is the number of experiments, p is the number of model parameters including intercept and
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any block coefficient, and PESS is the prediction error of sum of squares:
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PESS= ∑ !
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, − 2 , e i, -i = yi- ȳ i, -i
(Eq.5)
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where ei,−i is residual, yi is the experimental value, and ȳi,−1 is the predicted value.
357
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Another important parameter that it should be considered is the Durbin–Watson statistic (d) test. This
359
statistic test is used to detect the presence of auto-correlation (a relationship between values
360
separated from each other by a given time lag) in the residuals (prediction errors) from a regression
361
analysis.
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ACCEPTED MANUSCRIPT If the Durbin–Watson statistic is less than 2, there is evidence of positive serial correlation. On the
363
other hand, if Durbin–Watson is less than 1, there may be cause of concern. Small values of d
364
indicate successive error terms are, on average, close in value to one another, or positively
365
correlated. If d > 2, successive error terms are, on average, much different in value from one another,
366
i.e., negatively correlated. In regression analysis, this can imply an underestimation of the level of
367
statistical significance.
368
Goodness of fit of the obtained microbial growth model, was statistically significant affected
369
(P<0.01) by the type of the microorganism investigated. This is in general agreement with previous
370
work in the literature involving the role of different temperatures on the development of a microbial
371
growth model, regarding specific bacteria (Zwietering et al., 1990; Buzrul, 2009) (Table 3).
372
However, Durbin-Watson test gave d values< 1 for lactic acid bacteria in vacuum packaging, and in
373
vacuum packaging containing 2 mg/L of ozone. In addition, the adjusted R-squared values were
374
negative (-0.140) in the case of Enterobacteriaceae in the vacuum packaging plus 10 mg/L of ozone,
375
whereas low predicted R–squared values were also observed. In that sense, there was not any
376
predictive ability for Enterobacteriaceae.
377
Thus, considering all aforementioned criteria, the best modeling bacterial growth results, were
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obtained for TVC, Pseudomonas spp., yeasts and moulds, and lactic acid bacteria (involving the
379
vacuum packaging containing 5 and 10 mg/L of ozone), as shown in Table 3.
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4. Conclusions
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Based on microbiological and sensory attributes, the shelf-life of vacuum packaged fresh chicken
383
legs was ca. 10 days. Addition of gaseous ozone of 10 mg/L for 1 hour, for chicken legs packaged in
384
plastic containers of PA/PE under refrigeration, proved to be an appropriate method for maintaining
385
freshness and quality of chicken leg meat, since its shelf life was extended by 6 days, as compared to
386
vacuum packaged samples. Vacuum packaging in combination with ozonation has the potential to
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ACCEPTED MANUSCRIPT aid in the shelf life extension of fresh chicken legs as an innovative, simple, and economic
388
technology. The innovative non-thermal ozonation method has the following benefits: a) low
389
operation cost, b) ecological and chemical-free, c) simple to use. The overall cost of such an
390
application is low, definitely lower than the losses that could cause natural spoilage, if we consider
391
that poultry meat has a short shelf-life. A prospective shelf life extension of poultry meat with the
392
application of a novel technology, as supported by a microbial growth model, would increase its
393
market distribution at an international level.
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ACCEPTED MANUSCRIPT Table 1. Variations in pH and colour parameters (mean±SD) according to packaging treatment and storage time Day/Parameter
Cv
Cv+2 mg/L
0 2 4 6 8 10 12 14 16 F df p
6.85 ± 0.11 6.79 ± 0.07 6.80 ± 0.03 6.90 ± 0.05 6.73 ± 0.05 7.14 ± 0.04 7.07 ± 0.06 6.96 ± 0.08 7.12 ± 0.05 17.329 26 <0.001
6.85 ± 0.11 6.81 ± 0.07 6.76 ± 0.05 6.76 ± 0.04 6.86 ± 0.06 7.08 ± 0.04 6.99 ± 0.07 7.00 ± 0.08 6.90 ± 0.05 8.457 26 <0.001
0 2 4 6 8 10 12 14 16 F df p
60.90 ± 0.33 58.94 ± 0.06 60.45 ± 0.12 55.85 ± 0.18 58.48 ± 0.14 54.88 ± 0.80 57.11 ± 0.31 61.57 ± 0.18 58.65 ± 0.13 140.498 26 <0.001
60.90 ± 0.33 58.72 ± 0.41 59.62 ± 0.04 59.62 ± 0.04 56.62 ± 0.42 56.96 ± 0.71 56.32 ± 0.44 60.52 ± 0.23 58.01 ± 0.15 59.595 26 <0.001
0 2 4 6 8 10 12 14 16 F df p
0.91 ± 0.29 1.35 ± 0.56 0.82 ± 0.61 0.49 ± 0.30 0.18 ± 0.57 1.27 ± 1.29 0.08 ± 0.75 0.57 ± 0.16 0.51 ± 0.38 1.655 26 0.178
Cv+5 mg/L
Cv+10 mg/L
pH
L*
0 2 4 6 8 10 12 14 16 F df p
9.68 ± 0.39 11.76 ± 0.48 10.26 ± 0.19 10.37 ± 0.15 10.32 ± 0.23 8.82 ± 0.28 8.19 ± 0.33 8.77 ± 0.13 8.37 ± 0.06 53.484 26 <0.001
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AC C
b*
60.90 ± 0.33 51.07 ± 0.18 53.44 ± 0.13 50.78 ± 0.27 54.55 ± 0.56 53.42 ± 0.25 57.11 ± 0.63 57.85 ± 0.30 52.59 ± 0.15 261.590 26 <0.001
0.91 ± 0.29 0.63 ± 0.28 0.43 ± 0.27 0.54 ± 0.43 0.52 ± 0.53 0.11 ± 0.49 0.14 ± 0.81 0.22 ± 1.57 0.29 ± 1.04 1.038 26 0.445
0.91 ± 0.29 0.03 ± 0.19 1.48 ± 0.79 1.27 ± 0.70 0.61 ± 0.32 0.51 ± 0.99 0.78 ± 1.15 0.80 ± 0.81 0.17 ± 0.84 1.191 26 0.357
0.91 ± 0.29 2.12 ± 1.08 2.26 ± 0.16 0.05 ± 0.65 0.48 ± 0.34 2.92 ± 0.90 2.11 ± 1.78 0.13 ± 0.80 0.60 ± 0.22 4.405 26 0.004
9.68 ± 0.39 9.79 ± 0.11 10.00 ± 0.13 9.22 ± 0.15 11.31 ± 0.30 8.67 ± 0.25 8.78 ± 0.46 7.82 ± 0.21 9.91 ± 0.11 43.169 26 <0.001
9.68 ± 0.39 6.74 ± 0.23 10.48 ± 0.19 7.64 ± 0.20 7.67 ± 0.23 7.98 ± 0.27 9.70 ± 0.15 7.70 ± 0.11 10.88 ± 0.29 112.379 26 <0.001
9.68 ± 0.39 8.23 ± 0.33 9.07 ± 0.20 7.60 ± 0.17 7.54 ± 0.76 9.54 ± 0.12 12.89 ± 0.63 7.56 ± 0.16 9.19 ± 0.62 44.382 26 <0.001
EP
a*
60.90 ± 0.33 57.32 ± 0.03 51.41 ± 0.29 57.14 ± 0.15 58.38 ± 0.13 50.85 ± 0.29 53.65 ± 0.07 56.21 ± 0.11 57.68 ± 0.14 833.283 26 <0.001
6.85 ± 0.11 6.77 ± 0.05 6.68 ± 0.03 6.89 ± 0.12 6.76 ± 0.06 6.81 ± 0.04 6.81 ± 0.17 6.92 ± 0.03 6.83 ± 0.06 2.104 26 0.091
SC
6.85 ± 0.11 6.85 ± 0.04 6.95 ± 0.09 7.04 ± 0.08 6.86 ± 0.06 6.78 ± 0.06 6.83 ± 0.09 6.82 ± 0.02 6.82 ± 0.05 3.645 26 0.011
ANOVA in comparison of means (p<0.001), mean±SD: average ±standard deviation values of three replicates (n=3). Cv: control packaging (vacuum). F: variation between sample means / variation within the samples, df: degrees of freedom, p:probability
ACCEPTED MANUSCRIPT Table 2. Variations in sensory attributes (mean±SD) according to packaging treatment and storage time Cv
Cv+2 mg/L
Cv+5 mg/L
Cv+10 mg/L
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.33 ± 0.29 4.00 ± 0.00 3.00 ± 0.00 2.67 ± 0.58 2.57 ± 0.00 1.00 ± 0.00 138.600
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.53 ± 0.06 4.23 ± 0.06 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 2.67 ± 0.58 22.300
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.73 ± 0.06 4.53 ± 0.06 4.00 ± 0.00 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 24.094
26 <0.001
26 <0.001
26 <0.001
26 <0.001
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.47 ± 0.25 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 1.33 ± 0.58
EP
5.00 ± 0.00 5.00 ± 0.00 4.67 ± 0.58 4.00 ± 0.00 3.33 ± 0.58 3.00 ± 0.00 2.67 ± 0.58 2.00 ± 0.00 1.00 ± 0.00
803.726 26 <0.001
52.333 26 <0.001
5.00 ± 0.00 5.00 ± 0.00 4.67 ± 0.58 4.33 ± 0.29 3.33 ± 0.29 3.00 ± 0.00 2.00 ± 0.00 ND ND
55.556 20 <0.001
27.384 26 <0.001
TE D
67.000 26 <0.001
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.57 ± 0.12 4.27 ± 0.12 3.97 ± 0.25 3.67 ± 0.58 3.00 ± 0.00 2.67 ± 0.58
40.881 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.63 ± 0.06 4.00 ± 0.20 3.40 ± 0.17 3.03 ± 0.25 ND ND
104.626 20 <0.001
SC
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.53 ± 0.06 4.17 ± 0.15 3.57 ± 0.21 3.00 ± 0.00 2.00 ± 0.00 1.00 ± 0.00
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.73 ± 0.06 4.50 ± 0.10 4.37 ± 0.15 4.17 ± 0.15 3.33 ± 0.58 3.00 ± 0.00
M AN U
5.00 ± 0.00 5.00 ± 0.00 4.67 ± 0.29 4.00 ± 0.00 3.33 ± 0.29 3.00 ± 0.00 2.67 ± 0.58 1.67 ± 0.58 1.00 ± 0.00
RI PT
5.00 ± 0.00 5.00 ± 0.00 4.00 ± 0.00 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00 1.67 ± 0.58 1.00 ± 0.00 1.00 ± 0.00 65.083
AC C
Days/Sensory attributes Odour 0 2 4 6 8 10 12 14 16 F df P Texture 0 2 4 6 8 10 12 14 16 F df P Appearance 0 2 4 6 8 10 12 14 16 F df P Taste 0 2 4 6 8 10 12 14 16 F df P
5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.60 ± 0.17 4.00 ± 0.00 3.67 ± 0.58 3.33 ± 0.58 3.00 ± 0.00
25.019 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.70 ± 0.00 4.37 ± 0.15 4.00 ± 0.00 3.33 ± 0.58 3.00 ± 0.00 2.33 ± 0.58
38.088 26 <0.001
37.125 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.73 ± 0.06 4.33 ± 0.29 4.00 ± 0.00 3.67 ± 0.58 3.00 ± 0.00
33.841 26 <0.001 5.00 ± 0.00 5.00 ± 0.00 5.00 ± 0.00 4.80 ± 0.10 4.63 ± 0.12 4.33 ± 0.29 3.67 ± 0.58 3.33 ± 0.29 3.00 ± 0.00
31.325 26 <0.001
ANOVA in comparison of means (p<0.001), mean±SD: average ±standard deviation values of three replicates (n=3). Cv: control packaging (vacuum). ND: not determined. F: variation between sample means / variation within the samples, df: degrees of freedom, P:probability
ACCEPTED MANUSCRIPT Table 3. Microbial growth modeling using linear regression analysis with respect to storage time and packaging treatment Microbial growth
Cv
Cv+2 ppm
Cv+5 ppm
Cv+10 ppm
0.918 0.316 3.744 0.57 0.904 0.958 10.425 7 66.925 <0.001 0.50 1.86
0.856 0.263 3.425 0.52 0.845 0.925 10.647 8 41.596 <0.001 0.63 1.40
0.787 0.155 3.701 0.38 0.806 0.911 14.069 8 34.329 <0.001 0.49 1.58
0.831 0.187 3.782 0.32 0.757 0.887 15.476 8 25.860 <0.001 0.47 1.47
0.918 0.333 3.487 0.40 0.904 0.958 9.656 7 66.805 <0.001 0.53 1.61
0.773 0.212 3.401 0.35 0.816 0.916 10.841 8 36.498 <0.001 0.62 1.03
0.884 0.308 2.961 0.33 0.864 0.940 9.024 7 45.618 <0.001 0.59 1.35 0.855 0.396 1.288 0.33 0.831 0.925 5.476 7 35.459
Slope Intercept SEM Radj 2 Rpred 2 t df F P PESS Durbin-Watson Yeasts and Moulds R2
Enterobacteriaceae R2 Slope Intercept SEM Radj 2 Rpred 2 t df F
SC
0.803 0.166 3.590 0.25 0.774 0.896 14.559 8 28.442 <0.001 0.48 2.0
0.820 0.133 3.746 0.20 0.794 0.906 17.893 8 31.874 <0.001 0.37 1.62
0.838 0.283 2.596 0.29 0.815 0.916 8.611 8 36.284 <0.001 0.73 1.10
0.774 0.21 2.641 0.23 0.742 0.880 9.915 8 23.961 <0.001 0.66 1.22
0.637 0.151 2.728 0.19 0.585 0.798 11.377 8 12.260 <0.001 0.67 1.54
0.841 0.402 0.71 0.28 0.435 0.711 6.635 8 7.149
0.804 0.322 1.016 0.22 0.490 0.744 7.659 8 8.697
0.712 0.249 0.946 0.22 neg 0.650 6.533 8 0.017
AC C
Slope Intercept SEM Radj 2 Rpred 2 t df F P PESS Durbin-Watson
M AN U
Pseudomonas spp. R2
TE D
Slope Intercept SEM Radj 2 Rpred 2 t df F P PESS Durbin-Watson
EP
R2
RI PT
TVC
P PESS Durbin-Watson LAB R2
<0.001 1.48 1.91
<0.001 1.14 1.72
<0.001 2.16 1.90
0.905 0.343 2.295 0.29 0.889 0.951 7.520 7 57.328 <0.001 0.59 0.72
0.898 0.342 1.599 0.26 0.883 0.948 6.577 8 61.644 <0.001 0.68 0.96
0.847 0.286 1.494 0.21 0.825 0.920 6.668 8 38.748 <0.001 0.71 1.52
0.771 0.245 1.473 0.21 0.739 0.878 6.742 8 23.627 <0.001 0.78 1.48
SC
Slope Intercept SEM Radj 2 Rpred 2 t df F P PESS Durbin-Watson
0.001 0.86 1.44
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
Cv: vacuum packaging (control), Cv+2ppm: vacuum packaging plus 2 ppm of ozone, Cv+5ppm: vacuum packaging plus 5 ppm of ozone, Cv+10ppm: vacuum packaging plus 10ppm of ozone. TVC: total viable count, LAB: lactic acid bacteria, SEM: standard error of mean, t: T-test, df: degrees of freedom, P: probability at the confidence level P<0.05. Radj2: adjusted R-squared values, Rpred2 : predicted R-squared values. neg: negative value, not considered. F: variation between sample means / variation within the samples. PESS: predicted error of sum of squares.
ACCEPTED MANUSCRIPT
TVC 10 9
6
RI PT
log CFU/g
8 7 Cv (control)
5 4
Cv+2ppm
3
Cv+5ppm
2 1
SC
Cv+10ppm
0 2
4
6
8 Days
10
12
14
16
M AN U
0
Figure 1a.
Pseudomonas spp. 9
TE D
8
6 5 4 3 2
2
4
AC C
0
EP
log CFU/g
7
Figure 1b.
6
Cv (control) Cv+2ppm Cv+5ppm Cv+10ppm
8
10 Days
12
14
16
ACCEPTED MANUSCRIPT
9
Yeasts and moulds
8
6
Cv (control)
5
Cv+2ppm
RI PT
log CFU/g
7
Cv+5ppm
4
Cv+10ppm
3 2 0
2
4
6
8
10
12
16
SC
Days
14
M AN U
Figure 1c.
Enterobacteriaceae 9 8 6 5 4 3 2 1 0
4
AC C
Figure 1d.
2
6
EP
0
TE D
log CFU/g
7
8
Cv (control) Cv+2ppm Cv+5ppm Cv+10ppm
10
Days
12
14
16
ACCEPTED MANUSCRIPT LAB 9 8 6 5
Cv (control)
4
Cv+2ppm
3
RI PT
log CFU/g
7
Cv+5ppm
2
Cv+10ppm
1 0 0
2
4
6
8
10
12
16
SC
Days
14
M AN U
Figure 1e.
L*
70 65
TE D
L*
60 55 50
40
2
4
AC C
0
EP
45
Fig.2a
6
8
Cv (control) Cv+2ppm Cv+5ppm Cv+10ppm
10 Days
12
14
16
ACCEPTED MANUSCRIPT a* 5 4 3
Cv (control) Cv+2ppm
a*
2
RI PT
Cv+5ppm 1
Cv+10ppm
0 0
2
4
6
8
10
12
14
-1 -2
SC
Days
16
M AN U
Fig.2b b*
16 14 12
8 6 4 2 0
4
AC C
Fig.2c
2
6
EP
0
TE D
b*
10
8
Days
Cv (control) Cv+2ppm Cv+5ppm Cv+10ppm
10
12
14
16
ACCEPTED MANUSCRIPT Odour 6
Score
5 4 Cv (control) 3
RI PT
Cv+2ppm Cv+5ppm
2
Cv+10ppm
1 0 0
2
4
6
8
10
14
16
SC
Days
12
M AN U
Fig.3a Texture
6
4
Cv (control)
3 2 1 0 4
AC C
Fig.3b
2
6
EP
0
TE D
Score
5
8
Days
Cv+2ppm Cv+5ppm Cv+10ppm
10
12
14
16
ACCEPTED MANUSCRIPT Appearance 6
Score
5 4 Cv (control)
3
RI PT
Cv+2ppm
2
Cv+5ppm
Cv+10ppm
1 0 2
4
6
8
10
12
14
16
SC
0
Days
M AN U
Fig.3c
Taste
6
TE D
5
Score
4 3
Cv+2ppm Cv+5ppm
EP
2
Cv (control)
1 0 2
4
AC C
0
Fig.3d
6
Cv+10ppm
8
10 Days
12
14
16
ACCEPTED MANUSCRIPT
•
Vacuum packaging (VP) plus Ozonation (O) comprises an innovative
technology Sensory properties of chicken legs were not affected negatively by ozonation
•
6 days shelf life extension using 10 mg/L of ozone and vacuum packaging
•
VP plus O exhibited an additive preservation effect on fresh chicken legs
AC C
EP
TE D
M AN U
SC
RI PT
•