A chitosan-based coating with or without clove oil extends the shelf life of cooked pork sausages in refrigerated storage

    A chitosan-based coating with or without clove oil extends the shelf life of cooked pork sausages in refrigerated storage Somwang Lekjing PII: DOI: Reference:

S0309-1740(15)30099-1 doi: 10.1016/j.meatsci.2015.10.003 MESC 6803

To appear in:

Meat Science

Received date: Revised date: Accepted date:

26 January 2015 3 July 2015 7 October 2015

Please cite this article as: Lekjing, S., A chitosan-based coating with or without clove oil extends the shelf life of cooked pork sausages in refrigerated storage, Meat Science (2015), doi: 10.1016/j.meatsci.2015.10.003

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A chitosan-based coating with or without clove oil extends the shelf life of cooked pork sausages in refrigerated storage

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Somwang Lekjing*

Surat Thani campus, Muang, Surat Thani 84000, Thailand

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*Corresponding author. Tel.: +66 7735 5453; Fax: +66 7735 5453.

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Department of Food Technology, Faculty of Science and Industrial Technology, Prince of Songkla University,

E-mail address: [email protected] (S. Lekjing).

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Abstract

Chitosan coatings, with and without clove oil, were investigated for effects on quality

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and shelf life of cooked pork sausages stored at a refrigerated temperature (4±2°C). The various treatments of cooked pork sausages were: untreated (control), coating with 2%

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chitosan (CS), and coating with a mixture having 2% chitosan and 1.5% clove oil (CS+CO).

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Various microbiological, physical, chemical and sensory properties were monitored over 25 days of storage. The total viable count, the psychrotrophic bacteria count, the L* value,

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peroxide value and the thiobarbituric acid reactive substances increased, while the a* value, the b* value, the pH and the sensory scores decreased with storage time, across all treatments.

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However, these changes were slowest with the CS+CO treatment. Based on sensory evaluation and microbiological quality, the shelf lives were 14 d for control, 20 d for CS, and 20 d for CS+CO treated samples, under refrigerated storage. Keywords: Chitosan, Clove oil, Cooked pork sausage, Shelf life extension, Refrigerated storage 1. Introduction Pork sausages are among the oldest processed meat products enjoyed by millions of consumers all over the world. Traditionally, cooked pork sausages are made of fresh pork meat and fat, chopped and mixed thoroughly with seasonings. Typical seasonings include pepper powder, spicy paprika, salt, rice wine, sugar, monosodium glutamate and ginger. After

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filling this meaty mixture in natural casings from the cleaned small intestine of pigs, the sausages are fully cooked (Qiu, Zhao, Sun, Zhou, & Cui, 2013; Sebranek, Sewalt, Robbins, &

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Houser, 2005). However, cooked pork sausage products typically have a short shelf life

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restricted by poor color retention, rancidity, and other quality losses.

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Chitosan (ß-(1,4)-2-amino-2-deoxy-D- glucopyranose), mainly manufactured from crustacean shells (from crab, shrimp, crayfish, etc.), is derived by deacetylation of chitin (Knorr, 1984; Rinaudo, 2006; Sandford, 2003). This nontoxic, biodegradable and

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biocompatible substance has antimicrobial and antioxidant properties, wound-healing

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properties, as well as hemostatic activity, and it is receiving increased attention as a promising renewable polymeric material (Knorr, 1984; Yen, Yang, & Mau, 2009). Several

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reports indicate that chitosan has potential in food packaging, especially as edible films and

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coatings (Fan et al., 2009; Petrou, Tsiraki, Giatrakou, & Savvaidis, 2012; Suman et al., 2010). The incorporation of chitosan and essential oils into food coatings is concurrently

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emerging as a promising technology, to inhibit the growth of microorganisms, to retard the oxidation at the surface, to improve the sensory quality, and to prolong the shelf life of the

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samples. Such studies demonstrating shelf life extension include the following. Chitosan with rosemary extract in pork sausages (Georgantelis, Ambrosiadis, Katikou, Blekas, & Georgakis, 2007) and in beef burgers (Georgantelis, Blekas, Katikou, Ambrosiadis, & Fletouris, 2007); chitosan and thyme oil in ready to cook poultry products (Giatrakou, Ntzimani, & Savvaidis, 2010; Giatrakou, Ntzimani, Zwietering, & Savvaidis, 2010); chitosan and cinnamon oil in refrigerated rainbow trout (Ojagh, Rezaei, Razavi, & Hosseini, 2010); chitosan and sunflower oil in pork meat hamburgers (Vargas, Albors, & Chiralt, 2011); chitosan and oregano oil in chicken breast meat (Petrou et al., 2012); and, recently, chitosan and Zataria multiflora essential oil in chicken breast meat ((Bazargani-Gilani, Aliakbarlu, & Tajik, 2015).

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Clove oil (Syzygium aromaticum, Lin) is a natural essential oil with antimicrobial and antioxidant activities (Burt, 2004; Gülçin, Elmastaş, & Aboul-Enein, 2012; Gülçin, Güngör

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Şat, Beydemir, Elmastaş, & İrfan Küfrevioǧlu, 2004; Lee & Shibamoto, 2001; Matan et al.,

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2006; Oskoueian, Maroufyan, Goh, Ramezani-Fard, & Ebrahimi, 2013), its active ingredient

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being eugenol (Lee & Shibamoto, 2001; Ordóñez, Llopis, & Peñalver, 2008). Eugenol has antifungal activity (Martini, Weidenbörner, Adams, & Kunz, 1996), and inhibits malonaldehyde formation from cod liver oil, as well as formation of hexanal (Lee &

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Shibamoto, 2001).

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The effects of chitosan-based coatings, with or without clove oil, on the shelf life of a meat product under refrigerated storage are poorly understood. Therefore, the objective of

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this study was to evaluate such effects on the quality and shelf life of pork sausage. If the

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shelf life of sausages in retail stores could be extended, this would reduce losses from having

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to dispose potentially spoiled product. This provides the economic motivation of the current

2. Materials and Methods

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2.1 Chemicals and media

Chitosan powder from shrimp shells (food grade), with a 200 mesh particle size, 8.97 x 105 molecular weight, and 80% degree of deacetylation, was purchased from Sinudom Agriculture Products Co., Ltd. (Surat Thani, Thailand). Pure clove oil of the genus Syzygium aromaticum, Lin was purchased from Thai China Flavors and Fragrances Industry Co., Ltd. (Bangkok, Thailand). The major component of this oil was eugenol at 70-80% (manufacturer’s data). The chemicals used were analytical grade, and included chloroform, methanol, trichloroacetic acid, potassium iodide, anhydrous sodium sulfate, hydrochloric acid, sodium hydroxide, sodium thiosulfate, thiobarbituric acid, potassium dihydrogen phosphate (Merck, Damstadt, Germany), acetic acid (Lab-Scan, Bangkok, Thailand), and

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1,1,3,3-tetramethoxypropane (Sigma–Aldrich, St. Louis., MO, USA). The medium for microbiological analyses was analytical grade plate count agar (Merck, Darmstadt,

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Germany).

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2.2 Preparation of chitosan and clove oil solutions

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A stock solution of chitosan was prepared by dissolving 2 g in 100 ml of 1% (w/v) glacial acetic acid, and stirring overnight at room temperature (final chitosan concentration = 2% w/v). To prepare chitosan solution incorporating clove oil, 0.5 ml of glycerol/g chitosan

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and 0.1%w/v of Tween 60 were added in the chitosan solution, then stirring for 30 min. The

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glycerol concentration gave suitable viscosity to the chitosan solution for use in manual dip coating. After that, 1.5 g clove oil was added to 100 ml of the chitosan mixture with glycerol

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and Tween 60, and stirred for 6 h at room temperature (final clove oil concentration = 1.5%

2.3 Sample preparation

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w/v). All stirring was done with a magnetic stirrer in a glass beaker at room temperature.

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Cooked pork sausages, approximately 500±10 g for each treatment, were purchased from Charoen Pokphand Foods PCL (Bangkok, Thailand). The samples were given a 3 min

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dip treatment in the 2% chitosan solution (CS); or the 2% chitosan solution incorporating 1.5% clove oil (CS+CO). Then the samples were dried for 60 min in aseptic laminar airflow conditions, and packed into PE ziplock bags holding 35 g per bag. The control samples (without coatings) were treated following the same procedure. The packed samples were kept in a refrigerator at 4±2°C for up to 25 days. Intermediate samples were taken randomly every 5 days to evaluate physical, chemical, microbiological and sensory properties. The optimal concentration of clove oil used was 1.5% by volume, based on preliminary experiments including microbiological analyses, physical analyses and sensory evaluations (Songsaeng, 2014). The sensory evaluations of the cooked pork sausage samples were investigated for effects of chitosan-based coating at 2.0% v/v incorporated with clove oil at 0.5, 1.0 and 1.5%

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v/v. The 1.5% v/v concentration of clove oil was the best among these treatments, giving the highest overall acceptability.

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2.4 Microbiological analysis

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Total viable counts (TVC) and psychrotrophic bacteria counts were determined as

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follows. Twenty-five grams of a sausage sample were blended for 2 min with 225 ml of sterile Butterfield’s phosphate-buffered water using a sterile blender jar (Waring, Torrington, CT), to obtain sample concentration 0.1 g/ml. By sequential 10-fold dilutions, sample

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concentrations from 10-2 to 10-8 g/ml were obtained in sterile Butterfield’s phosphate diluent.

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The TVC and psychrotrophic counts were determined by the pour plate method, using plate count agar. The diluted samples were incubated at 35°C for 48 h, or at 7°C for 10 days

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(BAM, 2001). The counts are expressed as log CFU/g.

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2.5 Physical and chemical analyses

2.5.1 Surface color measurement

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The surface color of each type of sample was measured for the L*, a* and b* values, using a Hunter Lab color analyzer (Color Flex, USA) calibrated with a standard white plate.

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The L* value represents lightness (L* = 0 for black and L* = 100 for white), while the a* value represents the red/green scale, with positive values for red and negative for green. The b* value represents the yellow/blue scale, with positive for yellow and negative for blue. The illuminant used was *C (D65), and the standard observer angle was 10°. The measured area was 1.25 inches in diameter. 2.5.2 Determination of pH Ten grams of a sausage sample were homogenized thoroughly with 20 ml distilled water. A high-shear homogenizer (IKA homogenizer, Model T25 digital ULTRA-TURRAX, Germany) was applied at 12,000 rpm for 1 min. The pH of the homogenate was measured

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using a pH meter (Mettler Toledo, SevenEasy, USA) (Songsaeng, Sophanodora, Kaewsrithong, & Ohshima, 2010).

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2.5.3 Lipid extraction

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Lipid was extracted following Bligh and Dyer (1959). The sample (30 g) was

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homogenized in 210 ml of a chloroform:methanol:distilled water (60:120:30) mixture, at a speed of 10,000 rpm for 1 min, at 4°C (IKA homogenizer, Model T25 digital ULTRATURRAX, Germany). The homogenate was diluted with 60 ml of chloroform, and

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homogenized at 10,000 rpm for 30 s. Then, 60 ml distilled water was added, and the mixture

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was homogenized again for 30 s. This homogenate was centrifuged at 5,000g for 10 min, at 4°C (Sorvall centrifuge, Model RC 55 Plus, USA), and the supernatant was transferred into a

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separating flask. The chloroform phase was drained off into a 250 ml Erlenmeyer flask

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containing about 2–5 g of anhydrous sodium sulfate, shaken well, and decanted into a roundbottom flask through Whatman No. 4 filter paper. The solvent was evaporated at 40°C using

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a rotary evaporator (Eyela, Model N-100, Tokyo, Japan), and the residual solvent was removed by flushing with nitrogen. The extracted lipid was subjected to an analysis of PV.

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2.5.3 Determination of peroxide value (PV) The PV was determined according to the method of Low and Ng (1978). The lipid sample (1.0 g) was treated with 30 ml of an organic solvent mixture (chloroform:acetic acid, 2:3). The mixture was shaken vigorously, followed by the addition of 0.5 ml of saturated potassium iodide solution. This mixture was kept in the dark for 1 min, and then 30 ml of distilled water were added and the mixture was shaken. To the mixture, 0.5 ml of starch solution (1% w/v) was added as an indicator. The PV was determined by titrating the iodine liberated from potassium iodide with standardized 0.01 N sodium thiosulfate solution. The PV is expressed as milliequivalents of free iodine per kg of lipid.

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2.5.4 Determination of thiobarbituric acid reactive substances (TBARS) The TBARS assay was performed as described by Buege and Aust (1978). A ground

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sample (0.5 g) was homogenized with 2.5 ml of a solution containing 0.375% (w/v)

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thiobarbituric acid, 15% (w/v) trichloroacetic acid and 0.25 N HCl. The mixture was heated

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in a boiling water bath (95–100°C) for 10 min to develop a pink color, then cooled with running tap water and centrifuged at 3,600g for 20 min, at 25°C. The absorbance of the supernatant was measured at 532 nm. A standard curve was prepared using 1,1,3,3-

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tetramethoxypropane at concentrations ranging from 0 to 10 ppm. The TBARS was

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calculated and is expressed as equivalents of mg malonaldehyde per kg sample. 2.6 Sensory evaluation

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Sensory attributes of the cooked pork sausage samples were determined after

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steaming with boiling water for 5 min, by a ten trained member panel from the staff of Department of Food technology, Faculty of Science and Industrial Technology, Prince of

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Songkla University, Surat Thani campus. The panelists gave scores for various sensory characteristics, such as appearance, odor, flavor, texture and overall acceptability, using a

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nine point hedonic scale (from 1 = dislike extremely to 9 = like extremely). It is noted that the panelists did not taste those samples that exceeded the 6 log CFU/g acceptability limit for TVC (Department of Medical Sciences, 2010). 2.7 Statistical analysis The experiments were repeated twice on different occasions, with different batches of cooked pork sausage samples. All the analyses were run in triplicates for each repetition (n = 2 x 3). All the data were subjected to analysis of variance (ANOVA), and comparisons of means were carried out by Duncan’s multiple range test, considering differences significant when P<0.05.

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3. Results and Discussion 3.1 Changes in TVC and psychrotrophic counts

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The TVC and psychrotrophs counts of untreated (control) and treated samples,

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observed during refrigerated storage, are shown in Figs. 1 and 2. The initial TVC counts

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ranged from 1.35 to 1.43 log CFU/g, and gradually increased throughout the duration of storage with each treatment. The TVC for sausage is acceptable when below 6 log CFU/g (Department of Medical Sciences, 2010), and this limit was exceeded on day 15 in the control

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samples, between day 20 and 25 with CS treatment, and on day 25 with CS+CO treatment.

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The CS and CS+CO samples had significantly (p<0.05) lower TVC than the control samples, at any observation time across the entire storage period. Thus, 8 to 10 days extension of the

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microbiological shelf life was achieved with the actual treatments CS and CS+CO. Chitosan

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is believed to act on the cells of spoilage microorganisms and pathogens, by changing the permeability of the cytoplasmatic membrane, leading to the leakage of intracellular

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electrolytes and proteinaceous constituents, and finally to death of the cell (Helander, Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001; Prashanth & Tharanathan, 2007).

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Apparently the clove oil in CS+CO inhibited bacterial activity due its eugenol content (Della Porta, Taddeo, D' Urso, & Reverchon, 1998; Farag, Daw, Hewedi, & El-Baroty, 1989). Clove oil is believed to act as an antimicrobial agent, by inhibiting the production of amylase and proteases in the cell, inducing cell wall deterioration and a high degree of cell lysis, and preventing enzyme action by binding to proteins (Thoroski, Blank, & Biliaderis, 1989; Wendakoon & Sakaguchi, 1995). Of the treatments examined in the present study, CS+CO was the most effective in controlling the TVC from day 5 to day 25 of storage. In other related studies, Petrou et al. (2012) report that chicken breast meat samples treated with either chitosan (1.5 % w/v) or oregano oil (0.25 % v/w) reduce the TVC by ca. 12 log CFU/g, whereas the combination of chitosan and oregano oil reduce the TVC by ca. 3-4

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log CFU/g, compared to the control samples. This extended the shelf life at 4°C of a chicken breast meat product by 5-6 days for oregano oil treatment alone, and by 10 days for chitosan

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or its combination with oregano oil. A combination of rosemary extract and chitosan reduces

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TVC by ca. 1-2 log CFU/g, and extends the shelf life of fresh pork sausages in refrigerated

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storage (Georgantelis, Ambrosiadis, et al., 2007). Giatrakou et al. (2010) report that a combination of thyme extract (0.2 % v/w) and chitosan (1.5 % w/v) reduces the TVC by ca.12 log CFU/g, and extends the shelf life of a ready to cook chicken product by 6 days (at 4°C).

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Little prior work exists on the application of an essential oil (including clove oil) together

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with chitosan onto pork sausages.

The initial psychrotroph counts were in the range from 1.39 to 1.51 log CFU/g, and

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the counts consistently increased with storage time for each treatment. However, the rate of

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increase was slowest for CS+CO treatment, indicating an effect by chitosan and clove oil. Based on prior studies, chitosan effectively inhibits Listeria monocytogenes, E.scherichia

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coli, Salmonella spp., Bacillus cereus, Staphylococcus aureus, Pseudomonas, Vibrio sp. and total anaerobic bacteria in fishery and meat products (Fan et al., 2009 ; Fernandez-Saiz, Soler,

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Lagaron, & Ocio, 2010; Kanatt, Chander, & Sharma, 2008; No, Kim, Lee, Park, & Prinyawiwatkul, 2006; Tsai, Su, Chen, & Pan, 2002; Ye, Neetoo, & Chen, 2008; Zivanovic, Chi, & Draughon, 2005). On the other hand, clove oil effectively inhibits L. monocytogenes, Aeromonas hydrophila, E. coli, S. Typhimurium, S. aureus, and autochthonous spoilage flora in various meat products (Burt, 2004; Hao, Brackett, & Doyle, 1998a, 1998b; Hosseini, Razavi, & Mousavi, 2009; Stecchini, Sarais, & Giavedoni, 1993; Vrinda Menon & Garg, 2001). 3.2 Changes in color The L*, a* and b* values of pork sausage samples were observed during refrigerated storage (Table 1). The initial L* value with actual treatments were slightly lower than in

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control samples but without statistical significance (p>0.05). During storage, the L* value of control samples was not a significantly altered for up to 20 days of storage, after which as

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statistically significant change indicated that the samples were turning lighter in color. The

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corresponding changes with actual treatments lacked statistical significance throughout the

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storage time. This was probably due to chitosan and clove oil acting as preventive antioxidants on the meat product. The lightness of sample color may also relate to the increases in PV and TBARS, shown in Figs. 3 and 4. While the measured L* values show

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statistically significant differences between the actual treatments and the control samples

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during storage, these effects on lightness were subjectively negligible, i.e. the effect size was practically small. On the other hand, Jo, Lee, Lee, and Byun (2001) reported that sausages

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containing chitosan had higher L* values than the control samples during storage.

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Furthermore, Giatrakou et al. (2010) found that the addition of chitosan to a ready-to-eat chicken product increased the L* value.

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In terms of the a* and b* values, the surface color of samples was unaffected by treatment at any single storage time (p>0.05). Petrou et al. (2012) have observed similar lack

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of effect on the a* value during storage, for control and chitosan-treated meat samples. Jo et al. (2001) reported a chitosan effect on the a* value of sausages, causing a more red surface. Additionally, Georgantelis, Blekas, et al. (2007) noted that the combination of chitosan and rosemary extract acted synergistically to improve the redness of beef burgers, during frozen storage, while individually chitosan or rosemary extract improved the color stability relative to control. The a* and b* values consistently decreased (p<0.05) with storage time, across our treatments. A decrease in these parameters could be associated with oxidative changes related to increases in PV and TBARS, shown in Figs. 3 and 4, causing pale color of the samples. However, Petrou et al. (2012) reported increasing a* value for chicken breast fillets treated with chitosan, during chilled storage. Youn, Park, Kim, and Ahn (1999) and Jo et al.

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(2001) reported that the addition of chitosan increased the b* value of sausages during storage, suggesting that the natural color of chitosan affected the surface color of the sausage.

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3.3 Changes in pH

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The pH changes of pork sausage samples during refrigerated storage are shown in

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Table 2, across the various treatments. The initial pH values were in the range from 6.23 to 6.35. The pH consistently slightly decreased with storage time (p<0.05), regardless of the treatment, and with statistically significant differences (p<0.05) between the treatments

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throughout the storage time. This may have been an indication of microbial growth. During

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the first 5 days of storage, the actual treatments lowered the pH values (p<0.05), probably due to the presence of acidified chitosan. These results agree with those reported by Lin and

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Chao (2001) for reduced-fat sausage (containing chitosan), and by Giatrakou et al. (2010) for

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ready to cook chicken (containing chitosan). Korkeala, Alanko, Mäkelä, and Lindroth (1990) also found that the pH of cooked ring sausages decreased from ca. 6.3 to about 5.8–5.9 with

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storage time under 2, 4 or 12°C, and the pH was indicative of spoilage. 3.4 Changes in PV and TBARS

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The primary oxidation products in the lipid fraction are indicated by the measured PV, and the PV values are shown along with the TBARS values during 25 days of storage at 4°C for the sausage samples, in Figs. 3 and 4. The initial PV and TBARS values were in the range from 1.57 to 2.18 meq/kg lipid and 1.97 to 2.24 mg malonaldehyde/kg sample, respectively. The actual treatments lowered (p<0.05) both these oxidation parameters relative to control after 5 days of storage, and the best antioxidative effect (p<0.05) was obtained with the combination of chitosan and clove oil (CS+CO). With this treatment, both PV and TBARS concentrations were consistently the lowest among the treatments at any observation time. The PV and TBARS of CS treated samples were significantly (p<0.05) higher than those of CS+CO treated samples, at 10 and 15 days of storage. Chitosan may retard oxidative

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rancidity in muscle foods by acting as a chelator of transition metal ions that initiate lipid peroxidation and start chain reactions, which in turn lead to the deterioration of flavor and

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taste in foods (Yen, Yang, & Mau, 2008). To the knowledge, few studies have examined the

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combined effects of chitosan and essential oils on the lipid oxidation of meats. In the study of

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Georgantelis, Ambrosiadis, et al. (2007), fresh pork sausages containing chitosan and its combination with rosemary show stronger antioxidative effects than the control. Moreover, the combination of chitosan and rosemary extract gives lower PV and MDA values than the

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individual antioxidants (chitosan, rosemary extract), throughout 20 days of storage at 4°C,

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indicating synergistic effects. Georgantelis, Blekas, et al. (2007) report that chitosan alone and in combination with rosemary used in frozen (-18°C) beef burgers stored for 180 days

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have stronger antioxidative effects than rosemary alone, and the best results are obtained with

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the combination of chitosan and rosemary. Kanatt et al. (2008), demonstrated that mint extract alone has good antioxidant activity but poor antimicrobial activity, whereas chitosan

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alone has poor antioxidant activity with excellent antimicrobial properties. Therefore, the potential as a preservative for meat and meat products was investigated for of chitosan and its

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combination with mint extract. Giatrakou, Ntzimani, Zwietering, et al. (2010), reported that the lipid oxidation of a ready-to-cook poultry meat product was more retarded by the combination of chitosan (1.5 % v/w) and thyme oil (0.2 % v/w) than by either as individual treatment, during storage at 4°C under modified atmosphere packaging. The lipid oxidation in chicken breast meat samples produced lower MDA values when treated with an individual antioxidant (chitosan, oregano oil) or their combination than those of the control samples. The combination of chitosan and oregano oil resulted in the lowest MDA values on day 12 of storage, leading to final values of approximately 0.1 mg MDA/kg (day 21) when stored under modified atmosphere packaging at 4°C (Petrou et al., 2012).

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3.5 Changes in sensory quality The results of sensory evaluations (odor, appearance, taste, texture and overall

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acceptability) of the pork sausage samples are presented in Table 3. All these attributes

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showed similarly decreasing acceptance (p<0.05) with storage time.

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On the initial 0-day of storage, the odor and taste attributes of CS+CO were the poorest, probably due to the distinct flavor of clove oil, while untreated pork sausage control samples had pleasant odor and taste. Appearance, texture and overall acceptability scores did

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not initially differ significantly between the treatments. In a prior published study, Songsaeng

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(2014) investigated the effects of chitosan-based coating at 2.0% v/v incorporated with clove oil at 0.0, 0.5, 1.0 and 1.5% v/v, on cooked pork sausage samples. It was found that the odor

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score decreased whereas the appearance score increased with the concentration of clove oil,

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without significant effects on the color score. The highest overall acceptability score of sausages was without clove oil (0.0% v/v) followed in rank order by 1.5, 1.0 and 0.5% v/v.

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Considering the score 5.0 as the threshold for acceptability, the shelf life was 15 days for the control samples, while with the actual treatments it was 20 days. Although the appearance,

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the taste and the texture scores decreased during storage, they remained acceptable with scores better than 5.0, whereas the odor and the overall acceptability scores limited the shelf lives using the same threshold criterion. The results of this study indicate that pork sausages’ shelf life can be extended by approximately 5 days, using chitosan alone (CS) or in combination with clove oil (CS+CO). Aside from the odor, the CS+CO treatment consistently performed better than CS in all other sensory attributes, throughout the entire storage period. The specific odor of clove oil clearly had a negative influence on the odor scores of pork sausages. However, this specific odor did not influence negatively at long storage times, because the odor due to spoilage dominates in the acceptability perceptions.

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According to Soultos, Tzikas, Abranhim, Georgantelis, and Amvrosiadis (2008), and also Roller et al. (2002), the addition of chitosan to sausage improved the acceptability of

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odor and flavor. Giatrakou, Ntzimani, Zwietering, et al. (2010) reported that a combination of

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chitosan and thyme oil extended a poultry product’s shelf life by 14 days with modified

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atmosphere packaging. A combination of chitosan and oregano oil also extended chicken breast meat’s shelf life by 14 days, with modified atmosphere packaging (Petrou et al., 2012). Shelf lives have also been extended by a combination of chitosan and rosemary in fresh pork

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sausage and beef burger (Georgantelis, Ambrosiadis, et al., 2007; Georgantelis, Blekas, et al.,

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2007), and by chitosan and caraway in dry fermented sausage (Krkić et al., 2013). 4. Conclusions

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Experiments demonstrated that the combination of chitosan and clove oil inhibited

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microbial growth, retarded lipid oxidation, and extended the shelf life of cooked pork sausages in refrigerated storage. However, there were some initially negative impacts on odor

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and taste qualities, at the start of storage. Acknowledgements

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The author gratefully acknowledges financial support by the Research Office of Prince of Songkla University, Surat Thani campus. Invaluable assistance by Dr. Seppo Karrila in the manuscript preparation is also sincerely appreciated.

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List of Tables Table 1. Effects of various treatments on the color parameters L*, a* and b* of cooked pork

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sausage samples. Observations were done every 5 days during refrigerated storage for up to

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Table 2. Effects of the various treatments on the pH of cooked pork sausages during refrigerated storage for up to 25 days.

Table 3. Effects of the various treatments on the sensory quality attributes of cooked pork

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sausages during refrigerated storage for up to 25 days.

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List of Figures Figure 1. Effects of the various treatments on the TVC count of cooked pork sausages during

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refrigerated storage for up to 25 days.

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pork sausages during refrigerated storage for up to 25 days.

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Figure 2. Effects of the various treatments on the psychrotrophic bacteria count of cooked

Figure 3. Effects of the various treatments on the PV of cooked pork sausages during refrigerated storage for up to 25 days.

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Figure 4. Effects of the various treatments on the TBARS of cooked pork sausages during

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refrigerated storage for up to 25 days.

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Table 1.

Treatment

Color Parameter

Storage time (days)

L*

0

66.50±0.59aA

65.12±1.06aA

65.59±0.41aA

5

66.62±0.32abB

65.57±0.37aA

65.27±0.68aA

10

66.75±0.15abcB

65.40±0.97aAB

65.12±0.82aA

15

66.94±0.44abcA

65.75±0.83aA

66.33±0.91aA

20

67.69±1.10bcB

66.54±0.39aAB

66.21±0.02aA

25

67.87±0.51cB

66.33±0.66aA

66.26±0.83aA

0

23.07±0.24bA

22.99±0.41cA

22.66±0.55dA

5

22.05±0.83abA

22.97±0.46cA

22.35±0.37cdA

21.74±0.39aA

22.79±0.37cB

21.64±0.31bcA

21.54±0.84aA

22.44±1.02bcA

21.63±0.27bcA

21.36±0.35aA

21.47±0.40abA

21.37±0.66abA

20.89±0.72aA

20.72±1.03aA

20.56±0.63aA

41.74±0.51cA

41.80±0.32bA

41.89±0.55cA

5

41.42±0.66cA

41.61±0.80bA

40.79±1.12bcA

10

39.75±0.97bA

41.43±0.60bB

40.70±0.79bcAB

15

39.09±0.26abA

41.14±0.57bB

40.26±1.12bAB

20

39.14±0.63abA

38.49±0.75aA

39.51±0.54abA

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a*

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b*

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25

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CS

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Control

CS+CO

25 38.12±0.34aA 38.04±0.48aA 38.45±0.22aA L* = lightness, a* = redness, b* = yellowness a-d Different superscripts within a column indicate significant differences (p<0.05). A-B Different superscripts within a row indicate significant differences (p<0.05). Control = untreated; CS = dipped in 2% w/v chitosan; CS+CO = dipped in 2% w/v chitosan with 1.5% w/v clove oil.

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Table 2.

pH value

Storage time (days)

CS

CS+CO

0

6.35±0.01eC

6.27±0.01dB

6.23±0.01bA

5

6.32±0.02dB

6.26±0.04dA

10

6.23±0.01cB

6.19±0.01cA

15

6.20±0.01cB

6.18±0.01bcA

6.18±0.01aA

20

6.16±0.03bA

6.15±0.01bA

6.18±0.01aA

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Control

6.22±0.02bA

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6.22±0.01bB

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25 6.09±0.01aA 6.12±0.01aA 6.19±0.03aB Different superscripts within a column indicate significant differences (p<0.05). A-B Different superscripts within a row indicate significant differences (p<0.05). Control = untreated; CS = dipped in 2% w/v chitosan; CS+CO = dipped in 2% w/v chitosan with 1.5% w/v clove oil.

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a-d

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Table 3.

CS

±0.52cD 7.50

±0.53bE

Appe arance

7.00 ±0.47aD

Co

8.50 ±0.53aD

ntrol CS

8.60 ±0.52

aD

CS Taste

8.60 ±0.52aD

+CO Co

9.00 ±0.00bD

ntrol CS

8.60 ±0.52

abC

CS Text ure

8.40 ±0.52aC

+CO Co

9.00 ±0.00aD

ntrol CS

9.00

CS

all acceptability

Co ntrol

9.00 ±0.00

CS

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Over

9.00 ±0.00aD

8.80

±0.42aE

8.90

±0.32aE

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CS +CO

5.90 ±0.74aC

6.00

5.80

±0.94aC

±0.63aC

8.10 ±0.32aCD 8.40 aD ±0.52 8.50 ±0.53aCD 8.50 ±0.53aC 8.50 aC ±0.53 8.20 ±0.42aC 8.50 ±0.53aD 8.60 ±0.52aD 8.60 ±0.52aD 8.60 aD ±0.52

7.90 ±0.32aBC 8.10 aCD ±0.32 8.20 ±0.42aCD 6.90 ±0.57aB 7.60 bB ±0.52 8.00 ±0.67bC 7.60 ±0.52aC 7.70 ±0.67aC 7.80 ±0.42aC 7.00 aC ±0.00

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±0.00aD +CO

6.70 ±0.67bD

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CS +CO

±0.74aC

8.40 ±0.52aDE 8.50 ±0.53aDE

Storage time (days) 15 20 5.54±1 3.90 .00aB ±0.57aA 5.80±0 5.30 .79aBC ±0.48bAB 5.60±0 5.10 .70aBC ±0.57bAB 7.60±0 6.70 .52aB ±0.48aA 7.80±0 7.40 abBC bAB .42 ±0.84 8.00±0 7.60 .00bBC ±0.70bB 6.00±0 NT .47aA 7.30±0 6.30 bB aA .48 ±0.48 7.60±0 6.40 .52bB ±0.52aA 6.10±0 5.30 .88aB ±0.67aA 6.30±0 5.70 .48aB ±0.48aA 7.40±0 6.80 .52bC ±0.92bB 5.80±0 4.60 aB aA .42 ±0.52

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±0.00cD

10 6.10

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Odor

5 8.60

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0 9.00

tment

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Trea

Attribute

8.10 ±0.32bD

7.50±0 .53bC

8.20 ±0.42bD

±0.70aA 4.90 ±0.32bA 4.60 ±0.52bA 6.40 ±0.52aA 6.90 ±0.57

bA

7.00 ±0.82bA NT NT NT 5.20 ±1.03aA 5.40 ±0.52aA 5.50 ±0.71aA 4.30 ±0.48

aA

6.20 ±0.42bB

7.60±0 .52bC

25 3.40

4.80 ±0.63aA

6.90 ±0.74cB

NT = No Test a-c Different superscripts within a column indicate significant differences (p<0.05). A-E Different superscripts within a row indicate significant differences (p<0.05). Control = untreated; CS = dipped in 2% w/v chitosan; CS+CO = dipped in 2% w/v chitosan with 1.5% w/v clove oil.

4.90 ±0.57aA

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Highlights Cooked pork sausages were dip coated with a mixture of chitosan and clove oil.

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The shelf life was assessed with microbiological and sensory quality tests.

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The coating improved the shelf life by about one third, from 15 to 20 days.

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