Relationship between antioxidative activity and oxidative stability of various types of poultry meat during chill storage

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1 Relationship between antioxidative activity and oxidative stability of various types of poultry meat during chill storage M. B. MIELNIK* 1, A. RZESZUTEK 1 and A. VEBERG 1, 2 1 Matforsk AS, Norwegian Food Research Institute, Osloveien 1, N-143 Aas, Norway 2 Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, P. O. Box 54, N-1432 Ås, Norway *Corresponding author: maria.mielnik@matforsk.no Antioxidative activity and its impact on the oxidative stability of meat derived from two different muscles (breast and thigh) and two species (chicken and turkey) was studied in raw and cooked meat, chill stored in air for, 2, 4, 7, and 9 days. Development of lipid oxidation in minced raw and cooked meat during storage was evaluated by TBARS and fluorescence spectroscopy. Antioxidant activity of meat was measured the DPPH free radical scavenging method. The antioxidative activity, expressed as antiradical power (ARP), varied appreciably between muscles from different species. Generally, turkey meat had significantly (P =.1) lower ARP values than chicken meat. The lowest activity in raw meat was found for turkey breasts and the highest for chicken thighs, respectively.26 and.62 mg DPPH /g meat. No changes for ARP values were noticed in raw meat stored for 3 days at 2 C. The cooking process significantly decreased ARP in turkey thighs, but in the other samples the ARP was only reduced slightly. The ARP in cooked samples showed the greatest decline after 2 days chill storage from.36 to.21 mg DPPH /g meat. The ARP was influenced mainly by species and to a lesser extent by storage time and muscle type. Although the raw thigh had higher antioxidant activity then raw breast, the thigh meat showed lower oxidative stability during the chill storage because of a higher content of fat and hem. Lipid oxidation increased during storage time in all samples, but the highest increase in TBARS values over time were observed in the turkey thigh meat. ANOVA revealed that oxidative stability of cooked meat was mostly dependent on species, but also on muscle type and storage time. Differences in fluorescence spectra were found for breast and thigh meat. The fluorescence intensities for cooked samples increased continuously with storage time and showed high correlation with TBARS (r =.99). Keywords: poultry; antioxidative activity; DPPH ; TBARS; fluorescence Introduction Modern trends towards convenient foods have resulted in the production of pre-cooked meat products, which are very susceptible to lipid oxidation. Poultry meat, because of its relatively high content of unsaturated fatty acids, oxidises more rapidly than pork, beef, or lamb (Wilson et al., 1976). Turkey

2 meat is more prone to oxidative rancidity than chicken meat due to higher phospholipids) and lower natural tocopherols content ( Pearson, et al. 1977; Dawson and Gartner, 1983, ). Temperature-depended acceleration of lipid oxidation in cooked meat could be partially due to heat inactivation of endogenous antioxidants (Mei et al., 1994). However, not all antioxidant, among others phenols, undergo denaturation. To follow the development of lipid oxidation in the raw and cooked meat, two methods has been chosen. The TBA assay, specific for malondialdehyde and other TBA reactive substances, is the most common method, but is destructive and time consuming. On other hand, front face fluorescence spectroscopy is a rapid and non-destructive method (Wold and Mielnik 2). This method is very sensitive, but not specific, because the signals arise from different oxidation products among them from reaction of oxidized lipids, unsaturated aldehyde and malondialdehyde with proteins, peptides, amino acids, phospholipids, DNA, and nucleic acid (Frankel, 1998). The signals from complex mixtures of the various oxidation products are similar and overlapping (Veberg et al. 26 b). It has been shown that fluorescence spectroscopy can determine the level of lipid oxidation in freeze stored poultry meat (Wold and Mielnik, 2, Veberg et al. 26 c), chill stored minced turkey and pork meat (Veberg et al.26 a), and meat loaf made from freeze stored turkey and pork meat (Wold et al., 22). The aim of the present study was to compare the antioxidative activity of two different muscles (breast and thigh) from two species (chicken and turkey) and their impact on the oxidative stability, after cooking and chill storage (2 C) in air atmosphere. The usefulness of front face fluorescence spectroscopy as a rapid method for detection of lipid oxidation in cooked poultry meat was also tested. Materials and methods Poultry meat obtained from a commercial processing plant 3 days after slaughter was ground through a.4 cm plate in an industrial meat grinder in the pilot plant at Matforsk AS and stored at 2 C for 3 days. Four experimental batches: chicken breast (CB), chicken thigh (CT), turkey breast (TB), and turkey thigh (TT) were divided into smaller portions. The samples were vacuum-packed, cooked in a water bath at 8 C for 3 min, and cooled in tap water for 2 min. Then, the meat samples were homogenised, pre-packaged into transparent boxes with air access, and stored for, 2, 4, 7, or 9 days in a dark room at 2 C before analysis. Development of lipid oxidation in minced raw and cooked poultry meat during refrigerated storage was evaluated by Thiobarbituric Acid Reactive Substances (TBARS) and fluorescence spectroscopy. Antioxidant activity of meat was measured the DPPH free radical scavenging method. Antiradical power (ARP) - The antioxidant activity of meat were determined by using the 2,2-diphenyl-1-picrylhydrazyl, DPPH according to the procedure described by Brand-Williams et al. (1995) with some modifications. DPPH (25 mg/l) and samples were dissolved in absolute ethanol instead of methanol. Muscle sample (2.5 g) were homogenized with 1 ml of absolute ethanol for 3 s at 82 rpm and then centrifuged for 2 min at 137 rpm, at 4 C. The supernatant was filtrated using a filter paper (white band 5892). Muscle extract (.8 ml) was mixed with DPPH solution (3.2 ml). Samples were prepared in duplicate for each of at least three concentrations used (.8,.7,.6). Blank samples contained.8 ml absolute ethanol and 3.2 ml DPPH solution. The reaction mixtures were covered and left in the dark at room temperature. The reduction of the DPPH free radical was measured by reading the absorbence at 515 nm after 6, 9, and 12 min of incubation. The percentage of remaining DPPH at steady state was calculated and plotted against the sample concentration to obtain the amount of sample required to decrease the initial DPPH concentration by 5% (EC5). Antioxidant activity is given as the reciprocal of EC5, the antiradical power (ARP) in units of mg of DPPH per g meat. Thiobarbituric acid reactive substances (TBARS) - The TBARS values were determined in duplicate with the method of Sørensen and Jørgensen (1996). For extraction, 1 g ground meat was homogenized (15 rpm, 3 s, 2 C) together with 3 ml of a 7.5% aqueous solution of trichloroacetic acid in a Bühler homogenizer Type H4 (74 Tübingen, Germany). After filtration, 5. ml extract was mixed with 5. ml.2 M aqueous solution of TBA in a

3 stoppered test tube, kept at 1 C for 35 min in a water bath, and cooled for 1 min in cold water. Absorbance was measured at 532 nm by Ultraspec 3 (Pharmacia Biotech, Cambridge, U.K.) against a blank containing 5. ml distilled water and 5. ml TBA reagent. Results expressed as mg malondialdehyde (MDA) per kg meat were calculated from the 1,1,3,3-tetraethyoxypropane (TEP) based standard curve. Front face fluorescence measurements. Fluorescence emission spectra were measured directly on the poultry meat. The samples were placed into sample cuvettes, which exposed a flat circular surface with a diameter of 5 cm for the measurements. The fluorescence emission spectra in the range of 41 nm to 75 nm were measured for excitation at 382 nm, using an optical bench system, suitable for solutions and solid samples. The excitation light was generated by a 3 W Xenon light source (Oriel 6258, Oriel Corporation, Stratford, CT) and passed through a 1 nm bandwidth interference filter (Oriel 5992). The light was directed onto the sample at an angle of about 45. The spectra were collected by an imaging spectrograph (Acton SP-15, Acton Research Corporation, Acton, MA) connected to a sensitive charge coupled device (CCD-camera) (Roper Scientific NTE/CCD-134/4-EMB, Roper Scientific, Trenton, NJ). A cut-off filter at 4 nm (Melles Griot 3FCG49) was positioned in front of the spectrograph slit to suppress excitation light reflected from the sample. Exposure time was 1 sec for each sample. All samples were measured twice and an average was used in the analysis. The field of illumination was not perfectly homogenous, so the samples were rotated 9 between each measurement to even out sample heterogeneity. The spectra were not subjected to any kind of pre-processing before analysis. Statistical Analyses. Two replications were carried out with all samples. Effects of muscle type, species, and storage time on TBARS and ARP values were analysed by ANOVA. Relationships between fluorescence spectra and TBARS values were analysed by Partial Least Squares Regression (PLSR) with full cross validation using The Unscrambler (version 9.2., Camo AS, Trondheim, Norway). Results and discussion ANOVA results (mean and F values) for TBARS and ARP values assessed in raw and newly cooked poultry meat are presented in Table 1. The oxidation rate measured as TBARS values was significantly affected by poultry species, type of meat and heat treatment. The heating process had greatest effect, resulting in seven folds increase of TBARS values in cooked meat in comparison to the raw meat analysed the same day. Poultry species appeared as a second most important factor, influencing the oxidative stability of the meat. The turkey samples had on average 1.48 mg more MDA per kg meat than the chicken meat samples. According to Dawson and Gartner (1983), turkey meat is more prone to oxidative rancidity than chicken meat due to higher phospholipids and lower natural tocopherol content. The oxidation process was influenced by muscle type. Breast muscles had significantly lower TBARS values than thigh muscles, apparently due to the lower content of fat and haemoglobin. The most extensive interactions for TBARS were noticed for poultry species and heat treatment. The increase in TBARS values after cooking was five folds for chicken meat while for the turkey meat it was seven folds. Interactions between muscle type and heating process revealed that the oxidation developed more rapid in thigh meat than in breast meat. The differences in the initial TBARS values between breast and thigh measured in raw meat were considerable smaller than the differences between cooked samples, respectively.15 mg/kg versus 1.1 mg/kg. The variation of TBARS for cooked and chill stored poultry meat was highly dependent on storage time (Table 2). The TBARS values increased in all samples continually, however the highest rises were noticed after the first two days of storage. The rapid accumulation of MDA in cooked samples confirms that the development of rancidity in cooked, refrigerated meat products occurs much faster than lipid oxidation in frozen raw meat as observed by others (Tims & Watts, 1958; Pearson, Love, & Shorland, 1977). The poultry species and muscle types affected also the development of oxidation. Turkey meat oxidized faster than chicken meat and thigh meat oxidized faster than breast meat. The chicken breast meat had the lowest TBARS values throughout the storage period, while the turkey thigh meat had the highest values.

4 Table 1. Analysis of variance for TBARS and ARP values assessed in raw and newly cooked poultry meat Source of Factor TBARS (mg MDA/kg) ARP (mg/g) variance Mean F-value 1 Mean F-value Species Chicken *** *** Turkey Type Breast *** ** Thigh Treatment Raw *** ** Cooked Species x Type Chicken breast ** NS Chicken thigh.5.57 Turkey breast Turkey thigh Species x Chicken raw *** ** Treatment Chicken cooked Turkey raw Turkey cooked Type x Treatment Breast raw *** NS Breast cooked Thigh raw Thigh cooked Table 2. Analysis of variance for TBARS and ARP values assessed in cooked and chill stored poultry meat in air Source of Factor TBARS (mg MDA/kg) ARP (mg/g) variance Mean F-value Mean F-value Species Chicken *** *** Turkey Type Breast ***.25.7 NS Thigh Day *** ** Species x type Chicken breast NS NS Chicken thigh Turkey breast Turkey thigh *** significant at P <.1; ** significant at P <.1; NS not significant 1 F-values from Fisher significance test The variation of the DPPH radical scavenger capacities in poultry meat are shown as antiradical power (ARP) in Table 1 and 2. A high ARP value indicates high antioxidant activity. Antiradical power of raw and newly cooked poultry meat was affected first of all by poultry species. Chicken meat had twofold higher ARP values than turkey meat. Besides, muscle type and heat treatment influenced the variation between samples. Thigh meat scavenged more free radicals than breast meat and raw meat showed higher ARP values than cooked meat. The lowest antioxidant activity was found for turkey breast and highest for chicken thigh. Significant interaction was observed between poultry species and heat treatment. The ARP in cooked turkey meat decreased considerable in comparison with raw turkey meat, while in chicken meat there was not any change. Although the thigh muscles, both from chicken and turkey, showed higher antioxidant activity than breast muscles within the same species, the thigh muscles had lower oxidative stability during the chill storage. This was apparently caused by higher content of fat, myoglobin, and hemoglobin. Typical emission spectra from chicken and turkey meat, excited at 382 nm, are shown in Figure 1 and 2, respectively. As a result of thermal processing, an increase in fluorescence intensity and shift in the fluorescence spectra were observed. Emission spectra from chicken and turkey thighs had similar shape, but differed considerably in fluorescence intensity after heating and chill storage in air, already from second day. For turkey thigh meat, the fluorescence intensity increased more rapidly after cooking than in chicken thighs. The highest peak in the emission spectra from cooked chicken thigh and both type of turkey meat, had a maximum intensity at nm.

5 Fluorescence intensity a CB raw CB cooked d CB cooked 2 d CB cooked 4 d CB cooked 7 d Fluorescence intensity a TB raw TB cooked d TB cooked 2 d TB cooked 4 d TB cooked 7 d Fluorescence intensity b CT raw CT cooked d CT cooked 2 d CT cooked 4 d CT cooked 7 d Fluorescence intensity b TT raw TT cooked d TT cooked 2 d TT cooked 4 d TT cooked 7 d Figure 1. Fluorescence emission spectra of raw and cooked (a) chicken breast (CB) Figure 2. Fluorescence emission spectra of raw and cooked (a) turkey breast and (b) chicken thigh (CT), chill stored in air for 7 days. (TB) and (b) turkey thigh (TT), chill stored in air for 7 days.

6 Veberg et al. (26 b) showed that unsaturated aldehydes formed fluorophores with lysine and glycin in model systems as well as in meat systems (turkey, pork, cod), with maximum intensity in the same region. Probably, we measured the same type of oxidation products in our experiment. For cooked chicken breast, the shape and maximum intensity of the spectra changed with storage time, moving towards shorter wavelengths (42-45 nm). This shift to shorter wavelengths could indicate the formation of other fluorphores in this meat. A small peak at 59 nm, probably zinc protoporphyrin (Veberg et al. 26 a) was observed only in raw meat and disappeared after cooking. The TBARS values for the meat samples ranged from.8 to 8.22 for chicken meat and.1 to 1.17 for turkey meat. The results clearly showed that fluorescence spectra correspond well with TBARS values. The correlation coefficients (r) obtained from PLSR models for cooked thigh and breast were.99 (2 and 5 Principal Components (PCs, respectively). This is higher than for all samples modelled together (r =.96, 5 PCs). This confirms the dissimilarity of the cooked meat spectra. References BRAND-WILLIAMS, W., CUVELIER, M.E. and BERSET, C. (1995) Use of free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie 28: DAWSON, L.E. and GARTNER, R. (1983) Lipid oxidation in mechanically deboned poultry. Food Technology 37: FRANKEL, E.N. (1998) Lipid oxidation. Dundee: The Oily Press LDT. 33. MEI, L., CRUM, A.D., and DECKER, E.A. (1994) Development of lipid oxidation and inactivation antioxidant enzymes in cooked pork and beef. Journal of Food Lipids, 1: PEARSON, A.M., LOVE, J.D., and SHORLAND, F.B. (1977) Warmed over flavor in meat, poultry and fish. Advances in Food Research, 23: SØRENSEN, G. and JØRGENSEN, S.S. (1996) A critical examination of some experimental variables in the 2-thiobarbituric acid (TBA) test for lipid oxidation in meat products. Zeitschrift für LebensmittelUntersuchung und Forschung 22: TIMMS, M.J. and WATTS, B.M.(1958) Protection of cooked meats with phosphates. Food Technology 12: WILSON, B.R., PEARSON, A.M., and SHORLAND, F.B. (1976) Effect of total lipids and phospholipids on warmed-over flavor in red and white muscle from several species as measured by thiobarbituric acid analysis. Journal of Agricultural and Food Chemistry, 2: VEBERG, A., SØRHEIM, O., MOAN, J., IANI, V., JUZENAS, P., NILSEN, A.N., and WOLD J.P. (26 a) Mapping of lipid oxidation and porphyrins in high oxygen modified atmosphere and vacuum packed ground turkey and pork by fluorescence spectra and images. Meat Science, 73: VEBERG, A., VOGT, G., and WOLD, J.P. (26 b) Fluorescence in aldehyde model systems related to lipid oxidation. Lebensmittel-Wissenschaft Und- Technologie-Food Science and Technology, 39: VEBERG, A., OLSEN, E., VOGT, G., MIELNIK, M., NILSEN, A.N., and WOLD, J.P.(26 c) Front face fluorescence spectroscopy- a rapid method to detect lipid oxidation in freeze stored minced turkey meat. Journal of Food Science, 71: WOLD, J.P. and MIELNIK, M. (2). Nondestructive assessment of lipid oxidation in minced poultry meat by autofluorescence spectroscopy. Journal of Food Science, 65: WOLD, J.P., MIELNIK, M., PETTERSEN, M.K., AABY, K., and BAARDSETH P. (22). Rapid assessment of rancidity in complex meat products by front face fluorescence spectroscopy. Journal of Food Science 67: