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Effect of grazing pastures of different botanical composition on antioxidant enzyme activities and oxidative stability of lamb meat
MEAT SCIENCE Meat Science 75 (2007) 737–745 www.elsevier.com/locate/meatsci
Effect of grazing pastures of different botanical composition on antioxidant enzyme activities and oxidative stability of lamb meat M.J. Petron a, K. Raes b, E. Claeys b, M. Lourenc¸o b, D. Fremaut c, S. De Smet a
b,*
Food Technology and Biochemistry, Escuela de Ingenierı´as Agrarias, Universidad de Extremadura, Carretera de Ca´ceres s/n 06071, Badajoz, Spain b Laboratory of Animal Nutrition and Animal Product Quality, Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium c Faculty of Biosciences and Landscape Architecture, University College Gent, Voskenslaan 270, 9000 Gent, Belgium Received 22 February 2006; received in revised form 2 October 2006; accepted 6 October 2006
Abstract The aim of this work was to study the influence of different pastures (Intensive ryegrass, Botanically diverse and Leguminosa rich pastures) on the antioxidant status and oxidative stability of meat from lambs that had been exclusively grazing for three months. Lipid, colour and protein oxidation, a-tocopherol content and activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GSH-Px)) were measured in Longisimus thoracis et lumborum muscle samples taken 1 day after slaughter. Pasture type significantly affected protein oxidation and the activity of GSH-Px, but no significant differences were found for the a-tocopherol content, colour and lipid oxidation, and the activities of SOD and Cat. Grazing a Botanically diverse pasture induced significantly higher protein oxidation in meat, as measured by the free thiol and carbonyl contents, compared to a Leguminosa rich or Intensive ryegrass pasture (P < 0.05). The GSH-Px activity was significantly higher in meat from lambs on the Leguminosa rich pasture compared to the other pasture groups (P < 0.01). 2006 Elsevier Ltd. All rights reserved. Keywords: Pasture; Lamb meat; Superoxide dismutase; Glutathione peroxidase; Catalase; Protein oxidation; Lipid oxidation; Colour oxidation
1. Introduction Oxidative processes in meat are the most important factors responsible for quality deterioration including flavour, colour and nutritive value. The oxidative stability of meat depends on the balance between antioxidants and pro-oxidants and the content of oxidation substrates including polyunsaturated fatty acids (PUFA), cholesterol, proteins and pigments (Bertelsen et al., 2000; Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998). Living cells have several mechanisms of protection against oxidative processes which include the endogenous enzymes superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GSH-Px). SOD and Cat are cou*
Corresponding author. E-mail address: [email protected] (S. De Smet).
0309-1740/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2006.10.010
pled enzymes. SOD scavenges superoxide anions by forming hydrogen peroxide and catalase safely decomposes hydrogen peroxide to H2O and O2 . GSH-Px can decompose both hydrogen peroxide and lipoperoxides formed during lipid oxidation. It has been postulated that the activity of these antioxidant enzymes might be induced in animals exposed to oxidative stress, reflected in an increased production of free radicals (De Haan, Newman, & Kola, 1992). It has also been shown that these enzymes exhibit residual activity in muscle postmortem (De Vore & Greene, 1982; Renerre, Dumont, & Gatellier, 1996). Besides the contribution of endogenous enzymes, the oxidative stability of meat is determined by the presence of antioxidants of dietary origin, e.g., the role of vitamin E in retarding lipid oxidation and improving colour stability (Liu, Lanari, & Schaefer, 1995; Morrisey, Buckley, Sheehy, & Monahan, 1994; Morrissey et al., 1998) is well
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recognized. The effects of other dietary compounds with antioxidative properties, such as carotenoids or flavonoids, and the contribution of endogenous enzymes on the oxidative status of meat is less well known but is being increasingly investigated. In addition, the endogenous antioxidant enzyme activity system may be modulated by nutritional factors (Huang, Chen, Osio, & Cohen, 1994), e.g., some trace elements are essential for the normal function of endogenous antioxidant systems. The minerals Cu, Mn, Zn, Se and Fe are important cofactors of the antioxidant enzyme activities (Papas, 1999). Also other antioxidants could interfere, e.g., the regeneration of a-tocopherol by the oxidation/ reduction of glutathione (Budowski & Sklan, 1989). Apart from the many studies that have examined the supplementation of diets with antioxidants on the antioxidant status of meat, few studies have been performed on the effects of the basal diet, e.g., the effect of pasture varying in botanical composition and hence in the supply of minor compounds. The objective of the present study was to examine the effect of grazing pastures with different botanical composition by lambs on the oxidative stability of their meat. 2. Material and methods 2.1. Animals and experimental setup A total of 21 male lambs (mean age 86 ± 9 days, mean live weight 22.3 ± 3.1 kg) of similar genetic background (‘Vlaams Kuddeschaap’, a ‘herding’ sheep breed), all born from yearling ewes, and originating from an organic farm were used for this experiment. At weaning, they were assigned to one of three pasture types, i.e. an Intensive ryegrass (IR) pasture, a Leguminosa rich (L) pasture and a Botanically diverse (BD) pasture. Lambs had been exclusively grazing with their mother before the trial. The L and BD pastures were located on the farm of origin (Berendrecht; near Antwerp), whereas the IR pasture was located on the experimental farm of Ghent University (Melle; near Ghent). No supplementary feeding was provided, except for a licking mineral block (Timac Potasco, Belgium) with the following composition: sodium (270 g/kg), calcium (60 g/kg), phosphor (2 g/kg), magnesium (1 g/kg), zinc (18 000 mg/kg), manganese (2000 mg/kg), iodine (100 mg/kg), cobalt (40 mg/kg) and selenium (10 mg/kg). The trial lasted for 83 days from 1 July 2004 until 22 September 2004, when the lambs were slaughtered (mean live weight at slaughter 32.3 ± 6.5 kg) and sampled. The lambs were pastured in a larger group, but the experimental subgroups were matched for similar average live weight at the onset of the trial. On the IR pasture, some heifers were intermittently introduced to manage the sward height. The stocking density was low and on average less than 850 kg live weight ha 1 for the BD pasture and less than 1200 kg live weight ha 1 for the IR and L pasture. The L and BD pasture were not fertilized
at all. The IR pasture received 59 kg N ha 1 and 3 kg P2O5 ha 1 on 4 May and an additional 35 kg N ha 1 on 18 August. All pastures were mowed once in the first week of August. After slaughter, carcasses were cooled for 24 h at 2 C. One day after slaughter, the Longissimus thoracis et lumborum (LTL) muscle was dissected. Sub-samples were used immediately for measuring colour, lipid and protein oxidation after chilled illuminated storage (until 8 days postmortem), whereas other sub-samples were vacuum packaged and stored immediately at 18 C until analysis (a-tocopherol content and enzyme activities). 2.2. Pasture botanical and chemical composition The botanical composition of the pastures was assessed according to the method of De Vries (1933) on nine occasions during the trial (three times per month). At these times, samples were also taken for chemical analyses, dried at 50 C for 48 h, finely (0.5–1 mm) ground (Grindomix GM 200, Retsch, Germany) and pooled per month. Crude protein was determined according to the Kjeldhal method (European Community, 1993), Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) using the Van Soest method (Van Soest, Robertson, & Lewis, 1991), lignin according to the method described by Van Soest and Wine (1968) and crude fat by the Soxhlet method (International Standards Organisation, ISO-1444, 1973). The following mineral and trace elements were determined by plasma emission spectrometry (ICP-AES Iris Intrepid II XSP, Thermo Electron corporation) following dry incineration at 500 C for 4 h and dissolving in 6 N HCl: B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S. The content of Se was determined by ICP-AES with hydride generation, but with a different preparation. The sample is first destroyed with HNO3 and H2O2 in a microwave, pre-reduced with HCl to convert Se6+ to Se4+ and further reduced and converted to H2Se using NaBH4. 2.3. Colour stability Steaks (1.5 cm thickness) were over-wrapped in an O2permeable PVC film and stored at 4 C for 8 days under constant illumination with white fluorescent lights (900 lux). Colour and colour stability measurements were performed using a HunterLab Miniscan spectrocolorimeter (D65 light source, 10 standard, 45/0 geometry, 1 in. light surface, white standard). The colour coordinates, expressed as CIE L*, a*, b* values and the reflectance values to calculate metmyoglobin (MetMb%) (Krzywicki, 1979) were measured daily for 8 days. 2.4. Lipid and protein oxidation Lipid and protein oxidation were measured on day 4 and 8 for the muscle samples. Lipid oxidation was measured by the TBARS (2-thiobarbituric acid reactive sub-
M.J. Petron et al. / Meat Science 75 (2007) 737–745
stances) method described by Tarladgis, Watts, and Younathan (1960). The results were expressed as lg MDA (malondialdehyde) g 1 muscle. Protein oxidation was estimated by measuring the free thiol content following the method described by Batifoulier, Mercier, Gatellier, and Renerre (2002) and the carbonyl content according to Mercier, Gatellier, Viau, Remignon, and Renerre (1997). The free thiol concentration was measured spectrophotometrically at 412 nm and was calculated using an absorption coefficient of 13.6 mM 1 cm 1. Results were expressed as nmol free thiols mg 1 protein. The total carbonyl content was quantified by a spectrophotometric assay at 370 nm, and was expressed as nmol DNPH (2,4-dinitrophenylhydrazine) incorporated mg 1 protein. The protein content was not measured, but was assumed to be constant at 0.22 g protein g 1 meat.
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Total superoxide dismutase (SOD) activity (Cu–Zn SOD + Mn SOD) was determined according to the procedure of Marklund and Marklund (1974), by measuring the inhibition of pyrogallol autoxidation. One unit was taken as the activity that inhibits the reaction by 50%. 2.7. Statistical analysis A one-way analysis of variance was performed to test for the effect of pasture type, followed by the Tukey post-hoc comparison means test in case of significance. Principal component analysis (PCA) was carried out to evaluate relationships among the various parameters of oxidative stability and to further distinguish the effect of pasture type. The statistical analyses were performed by SPSS version 11.5 (SPSS Inc. Chicago, IL, USA). 3. Results and discussion
2.5. a-Tocopherol content The a-tocopherol content was determined according to the method of Desai (1984). After saponification and hexane extraction, the samples were analysed by HPLC on a Supelcosil LC18 column (25 mm · 4.6 mm · 5 lm) equipped with a UV-detector (k = 292 nm). The results are expressed as lg a-tocopherol g 1 muscle. 2.6. Antioxidant enzyme activities A 5 g muscle sample was homogenized in 25 ml of 0.005 M phosphate buffer (pH 7.0) and centrifuged at 4 C for 20 min at 7000g. The supernatant fraction was filtered through glass wool and used to determine Cat, GSHPx and SOD activities. The catalase activity assay was performed as described by Aebi (1974). The supernatant (2 ml) was reacted at room temperature (22 C) with 1 ml of 30 mM H2O2 in 0.05 M phosphate buffer (pH 7.0), and the reaction (H2O2 decomposition) was monitored by measuring the absorbance at 240 nm during the initial 30 s. An extinction coefficient of 0.040 cm2 lmol 1 was used for calculation of H2O2 splitting. One unit (U) of catalase activity was defined as the amount of extract needed to decompose 1 lmol of H2O2 per min. GSH-Px activity was determined by measuring the oxidation of NADPH at 22 C (De Vore & Greene, 1982; Flohe´ & Gu¨nzler, 1984). The assay medium (3 ml) consisted of 1 mM reduced glutathione, 0.15 mM NADPH, 0.15 mM H2O2, 40 mM potassium phosphate buffer (pH 7.0), 0.5 mM EDTA, 1 mM NaN3, 1.5 units of glutathione reductase, and 300 ll of muscle extract. Absorbance at 340 nm was recorded over 3 min. An extinction coefficient of 6300 M 1 cm 1 was used for calculation of NADPH concentration. One unit of GSH-Px activity was defined as the amount of extract required to oxidize 1 lmol of NADPH per min at 22 C.
3.1. Feed and animal data Perennial ryegrass (Lolium perenne) was the predominant plant species (60–80%) on the IR pasture, followed by lower proportions of soft brome (Bromus hordeaceus) and Italian ryegrass (Lolium multiflorum). The L pasture harboured as predominant plants white clover (Trifolium repens) (approximately 40%), lucerne (Medicago sativa), perennial ryegrass and timothee (Phleum pratense). On the BD pasture, the predominant plant species were creeping bentgrass (Agrostis stolonifera) (approximately 40%), soft brome, thistle species and timothee, along with various herb species. Pastures did not only differ in botanical composition but also in chemical composition and contents of minerals and trace elements (Table 1). The crude protein content was significantly higher (P < 0.01) and the NDF content was significantly lower (P < 0.05) for the L pasture than for the BD and IR pastures. Pasture L had significantly higher contents of B, Ca, Cu, Na and Zn compared to the other pastures, whereas pasture IR was significantly higher in Mn (P < 0.05). The Se content could not be properly compared since most values were very low, but it appeared that the L pasture was also richer in this element. Mean values for live weight at the start and at slaughter and carcass weight per pasture group are given in Table 2. Lambs on the BD pasture group gained little weight during the trial and were significantly lighter at slaughter compared to the lambs from the two other pasture groups (P < 0.001). Carcass yield was also significantly lower for this group (P < 0.001). The very low average daily gain of the BD animals is very likely due to the low protein content of the BD pasture, that did not meet the protein requirements of growing lambs (CVB, 2004). The BD pasture group also displayed higher pH values in the LTL muscle at 1 h (P < 0.05) and 2 h (P < 0.001) postmortem compared to the two other groups, that did not differ. Pasture groups did not significantly differ in their pH values at
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Table 1 Mean values for chemical composition and content of minerals and trace elements of the pastures (n = 3) Botanically diverse
Leguminosa rich
SEM1
P
Chemical composition (% of dry matter) Crude Protein 15.9b Crude fat 3.99 Crude ash 11.3 NDF 60.8a ADF 35.0 Lignin 4.68
10.6b 3.86 10.9 61.3a 36.7 5.79
25.4a 4.03 10.5 42.8b 31.7 7.64
2.23 0.504 0.648 3.60 2.63 0.938
0.009 0.965 0.714 0.018 0.443 0.159
Mineral and trace element composition B (ppm) 4.87b Ca (ppm) 4427b Cu (ppm) 8.03b Fe (ppm) 230 K (ppm) 29100a Mg (ppm) 1557b Mn (ppm) 59.8a Na (ppm) 1720b P (ppm) 3680 S (ppm) 2340 Zn (ppm) 29.7b Se (ppb)2 0 · >4.0
6.79b 5973b 8.70b 283 18400b 1277b 23.5b 666c 2917 2363 29.1b 1 · >4.0
36.8a 14767a 17.1a 377 21633ab 2763a 25.0b 3770a 4327 1703 40.9a 2 · >4.0
2.76 1102 1.21 45.2 2410 132 6.06 86.5 357 212 2.84
<0.001 0.001 0.003 0.147 0.049 <0.001 0.009 <0.001 0.082 0.177 0.045
Intensive ryegrass
a,b
Mean values with different superscripts differ significantly (P < 0.05). Standard error of mean. 2 No mean values could be calculated for the content of Selenium, since most values were below the detection limit (4 ppb). The number of samples above the detection limit are given as an indication. 1
Table 2 Mean values for live weight at slaughter, cold carcass weight, LTL muscle pH and colour L*, a*, b* values according to pasture group (n = 7)
Live weight at start (kg) Live weight at slaughter (kg) Cold carcass weight (kg) Carcass yield (%) pH 1 h postmortem pH 2 h postmortem pH 24 h postmortem L* 1 day postmortem a* 1 day postmortem b* 1 day postmortem a,b
Intensive ryegrass
Botanically diverse
Leguminosa rich
SEM
P
22.6 36.4a 15.8a 43.3a 6.51a 5.96a 5.63 40.7 11.6 12.8
23.1 24.8b 8.9b 35.7b 6.76b 6.41b 5.80 38.8 11.3 12.6
21.1 35.6a 15.8a 44.3a 6.68ab 6.07a 5.63 37.7 11.3 12.2
1.67 2.06 1.07 1.13 0.209 0.269 0.165 0.62 0.18 0.34
0.503 <0.001 <0.001 <0.001 0.056 <0.001 0.093 0.076 0.672 0.428
Mean values with different superscripts differ significantly (P < 0.05).
24 h postmortem but there was a tendency for higher values in meat from BD lambs. Whether this slower rate of muscle pH fall in the BD animals is related to their lower growth rate is difficult to say, but it could be hypothesized that the lower growth rate resulted in a more oxidative muscle fibre type and/or lower glycogen levels at slaughter, and hence caused slightly higher muscle pH values postmortem. The effect of grazing these pastures differing in botanical composition on rumen fatty acid metabolism and on the intramuscular fatty acid composition was also studied but is not reported here. In summary the total polyunsaturated fatty acid (PUFA) proportion in the LTL intramuscular fat was significantly higher in the BD animals compared to the other groups. However, the total intramuscular fatty acid content differed between groups
(BD < IR < L), resulting in similar contents of PUFA (mg/g meat) in meat from the BD and L lambs and lower levels in meat from the IR lambs. The differences in the intramuscular fatty acid composition were not due to differences in the dietary fatty acid supply, but were mainly related to rumen metabolism. In view of the fact that no differences between pasture groups were observed in lipid oxidation (see further), differences in the intramuscular fatty composition were probably of little or no importance in the present study. These data are therefore not included. 3.2. Colour, lipid and protein oxidation There were no significant differences between the groups for the colour a* value and MetMb% at any time
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a
20
BD
IR
L
a* value
15 10 5 0 0
1
2
3
4
5
6
7
8
6
7
8
b
50
% metmyoglobin
Day of display
40
BD
IR
L
30 20 10 0
0
1
2
3
4
5
Day of display Fig. 1. Effect of type of pasture (L: Leguminosa rich; IR: Intensive ryegrass; BD: Botanically diverse) on changes in a* value (a) and % metmyoglobin (b) of LTL muscle samples during storage.
during chilled storage and display (Fig. 1). The mean a* value increased from day 0 to day 1 and thereafter decreased until day 8. These results agree with those from Guidera, Kerry, Buckley, Lynch, and Morrissey (1997) in lamb meat who found the a* value to decrease during chilled storage. As expected, the MetMb% increased during display. Results for lipid (TBARS values) and protein oxidation (free thiol and carbonyl content) on samples at day 4 and 8 of storage are given in Table 3. There was no significant effect of pasture type on TBARS values. Mean values were low (<0.7 lg MDA g 1 meat) for all pasture groups, even after 8 days of chilled storage, and are in agreement with findings from other authors on lamb (Macit, Aksakal, Emsen, Esenbuga, & Aksu, 2003; Macit, Aksakal, Emsen, Table 3 Mean values for TBARS, free thiol content and carbonyl content in LTL muscle according to pasture group (n = 7) Intensive ryegrass TBARS (lg MDA g Day 4 0.32 Day 8 0.68
Botanically diverse 1
Leguminosa rich
meat) 0.41 0.63
SEM
P
0.31 0.52
0.06 0.12
0.359 0.631
protein) 70.4 64.4a
2.71 1.97
0.106 <0.001
Carbonyl content (nmol DNPH incorporated mg 1 protein) Day 4 0.65a 0.85b 0.73a 0.04 a b 1.42 0.98a 0.13 Day 8 0.98
0.009 0.046
Free thiol content (nmol of free thiol mg Day 4 62.9 62.9 Day 8 61.0a 48.6b
a,b
1
Mean values with different superscripts differ significantly (P < 0.05).
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Aksu, et al., 2003). As expected, an increase in TBARS values was observed with storage. Protein oxidation measured by the carbonyl content was significantly affected by the type of pasture (Table 3). Samples from the BD group had significantly higher mean carbonyl contents than samples from the L and IR group on both day 4 (P < 0.01) and day 8 (P < 0.05) samples. A decrease in the free thiol content of muscle with time of storage is also indicative of protein oxidation (Batifoulier et al., 2002). Sista, Erickson, and Shewfelt (2000) suggested, using a chicken muscle model system, that sulphydryls are utilized to stabilize primary oxidation products. On samples at day 4, there was no significant difference between the pasture groups for the free thiol contents, but on samples at day 8, lamb meat of pasture BD had significantly lower values (P < 0.001) compared to the other groups. There was no significant difference at any time in the free thiol and carbonyl contents between the L and IR groups. Hence, for reasons that we cannot explain, the BD pasture appeared to induce the most pronounced protein oxidation in meat postmortem, which could be associated with increased protein degradation, fragmentation or aggregation (Martinaud et al., 1997). This effect of diet on protein oxidation was somewhat unexpected since no significant effect of diet on colour and lipid oxidation was found. In previous studies, protein oxidation was related to lipid oxidation in beef (Mercier, Gatellier, & Renerre, 1995), turkey meat (Mercier et al., 1997) and fish (Srinivasan & Hultin, 1995). Others studies on beef have shown an effect of diet on lipid oxidation but no significant dietary effect on protein oxidation (Mercier, Gatellier, & Renerre, 2004). The present study is the first report to our knowledge on protein oxidation in lamb meat. Our results show a significant effect of diet on protein oxidation, as measured by both the free thiol and carbonyl contents, that was not associated with a dietary effect on lipid oxidation. Whether the effect of the BD pasture on protein oxidation observed in this study is primarily due to a different balance in the dietary supply of pro-oxidants and antioxidants, or whether it is related to the lower growth rate of the lambs in this group and possible accompanied changes in muscle protein properties, remains to be investigated. Interference of the higher pH values in the LTL measured postmortem can also not be excluded, although it is difficult to hypothesize a causal relationship. 3.3. a-Tocopherol content and antioxidant enzyme activities Oxidation of muscle components postmortem can be retarded by antioxidants provided by the diet, of which a-tocopherol is the most important (Morrissey et al., 1998; lamb: Turner, McClure, Weiss, Borton, & Foster, 2002), and by the action of endogenous antioxidant enzymes, which seem to be relatively stable during refrigerated storage (Renerre et al., 1996). The muscle levels of atocopherol found in this study (1.1–1.7 lg g 1; Table 4) were close to those previously described in LTL muscle
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Table 4 Mean values for LTL muscle a-tocopherol content and activities of glutathione peroxidase (GSH-Px), catalase (Cat) and superoxide dismutase (SOD) according to pasture group (n = 7) a-tocopherol content (lg g 1) GSH-Px (U g 1) Cat (U g 1) SOD (U g 1) a,b
Intensive ryegrass
Botanically diverse
Leguminosa rich
SEM
P
1.72 0.090a 40.8 64.4
1.24 0.082a 30.8 68.5
1.09 0.181b 30.1 72.4
0.20 0.07 9.96 8.99
0.091 0.006 0.074 0.259
Within a row, mean values with different superscripts differ significantly (P < 0.05).
of lambs (Guidera et al., 1997; Salvatori et al., 2004). Pasture groups did not significantly differ in their muscle atocopherol contents in this study, but there was a tendency for higher levels in meat from the IR lambs compared to the BD and L lambs. Antioxidant enzymes activities are presented in Table 4. Diet did not modify muscle postmortem SOD activity, while a trend for a higher Cat activity was observed for the IR group compared to the other groups. Activities of SOD and Cat help to scavenge the highly reactive superoxide and hydroxyl ions respectively, and a decrease in the SOD and Cat activities has been associated with oxidative stress in vivo (Pigeolot et al., 1990). On the other hand, some authors considered an increase in Cat activity as a feedback response mechanism to oxidative stress (Venkatraman & Pinnavaia, 1998; Renerre, Poncet, Mercier, Gatellier, & Me´tro, 1999). The background and the impact of differences in the residual activity of these enzymes in meat remains to be further elucidated. As far as we are aware no other data are available in literature on the activity of these enzymes in lamb meat postmortem. In contrast to SOD and Cat, a significant effect of pasture type was found on the GSH-Px activity, that was higher for the L group compared to the IR and BD groups (P < 0.01). Although no clear explanation is available, the finding is in line with several studies on bovine meat that have shown an effect of finishing mode on the activity of GSH-Px (Descalzo, Insani, Eyherabide, Guidi, & Pensel, 2000; Mouty et al., 2002; Gatellier, Mercier, & Renerre, 2004; Mercier et al., 2004). GSH-Px plays a central role in the antioxidant defence system. An elevation of GSHPx activity has been associated with oxidative stress (Frank & Messaro, 1980). If residual postmortem activity of GSHPx is related to its activity in vivo, this would suggest that animals on the L pasture were subject to more oxidative stress. However, this L group showed similar values for lipid oxidation to the other groups and lower protein oxidation than the BD group. The results could therefore also suggest that the increase in GSH-Px activity on the L pasture enhanced the antioxidant defence capacity. The effect of feeding regimes on muscle antioxidant enzyme activities, and to what extent this is related to lipid and especially protein oxidation postmortem, is still largely unknown. Factors that might be responsible for this dietary effect include differences in the deposition of n-3 PUFA (e.g., white clover was shown to limit biohydrogenation of n-3 PUFA by Lee, Harris, Dewhurst, Merry, &
Scollan (2003)), but other antioxidants, trace elements and especially Se could also influence the activity of GSH-Px (Huang et al., 1994; Papas, 1999). Selenium is an essential cofactor for GSH-Px (Huang et al., 1994; Papas, 1999). Supplementation of selenium to feed can increase the selenium and GSH-Px contents of tissues in farm animals (Mahan & Parret, 1996). The L pasture showed a tendency for a higher content of Se and had clearly higher values for most trace elements, except for Mn, than the other pastures. Although levels of cofactors that are required in feed to modulate the enzyme activities in vivo are not known, these differences may have contributed to the difference in GSH-Px activity. On the other hand, the activities of SOD and Cat were not affected by the type of pasture in this study and the levels of cofactors for these enzymes (Mn, Zn, Cu) also differed between pastures. As argued by Papas (1999), not only the dietary supply of enzyme cofactors is important in this respect, interaction with other minerals and plant compounds that determines, e.g., the oxidation state of these cofactors and their absorption and bioavailability should be considered.
3.4. Relationships between measures of oxidation and antioxidant parameters In the present study, the correlations between a-tocopherol content on the one hand and lipid peroxidation, protein oxidation and endogenous enzyme activities on the other hand were not significant. However, significant correlations between GSH-Px activity and free thiol content (day 8) (r = 0.445; P < 0.05), and between Cat activity and TBARS content (day 4) (r = 0.581; P < 0.01) and carbonyl content (day 4) (r = 0.606; P < 0.01) were found. As mentioned above, the relationship between nutritional background and muscle a-tocopherol content and the effect on slowing down postmortem oxidative changes has been extensively described in literature for various species, but the relationship between a-tocopherol levels and endogenous antioxidant enzyme activities is unclear. Several studies have shown that a-tocopherol did not affect endogenous antioxidant activities in pork (Cadenas, Rojas, Perez-Campo, Lopez-Torres, & Barja, 1995), turkey (Renerre et al., 1999) and beef (Walsh, Kennedy, Goodall, & Kennedy, 1993; O’Grady, Monahan, Fallon, & Allen, 2001), but a positive significant correlation between SOD and a-tocopherol content has been reported by Gatellier
M.J. Petron et al. / Meat Science 75 (2007) 737–745
et al. (2004) in beef. Our data do not support a possible relationship. The relationship between Cat activity and lipid or protein oxidation is poorly documented. Cand and Verdetti (1989) found no correlation between SOD, Cat and GSH-Px activities and the TBARS content in rat organs.
Principal component 2 (19.44%)
a
1.0 cat carbonyl d4 tba d4
carbonyl d8
.5
tba d8
a*d4 a*d8
0.0
vit e sod
t hiol d4
m mb mmb d8d4
gsh-px
-.5 thiol d8
.0 -1.0
-.5
0.0
.5
1.0
Principal component 1 (38.28%)
b
3 GROUP L IR BD
Principal component 2
2
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However, in the present results on lamb meat, Cat activity was positively related to lipid oxidation and protein oxidation, when the latter was measured by carbonyl content. In a study in rabbits an increase in Cat activity in response to oxidative stress was insufficient to inhibit the increased free radical production, contributing to further lipid and protein oxidation (Gumieniczek, 2005). A possible explanation for the significant correlation between GSH-Px activity and protein oxidation, as measured by free thiol content, is that glutathione (GHS) is a major non-protein thiol in living organisms. It has been shown in the muscle of rats that increased activity of GSH-Px is associated with higher levels of GSH (Kumaran, Savitha, Anusuya Devi, & Panneerselvam, 2004). This finding is consistent with our results with the higher GSHPx activity corresponding to a higher free thiol content. Principal component analysis (PCA) was carried out to further elucidate relationships between the variables in this study (Fig. 2). The first and second PC explained 38% and 19% of the variation respectively. The relationships between antioxidant enzyme activities and oxidation variables as mentioned above are also apparent from this plot. It is possible to distinguish a group of variables composed of Cat activity, TBARS values and carbonyl content in the positive axis on the PC2, far from the origin and explaining an important part of the variation. GSH-Px, SOD and free thiol content are located on the negative axis on PC2. Explaining an independent cause of variation, a* value and MetMb% are located on PC1, far from the origin, and being opposite to each other. The regression factor scores show the discrimination between the pasture groups. Although few significant differences between individual variables were apparent, there is a rather good distinction between the groups based on this factor analysis. However, variability between animals within groups also appeared to be large.
1
4. Conclusions Finishing lambs for three months on pastures that differed in botanical composition (intensive ryegrass, botanically diverse or leguminosa rich) did affect protein oxidation and GSH-Px activity of muscle postmortem. However, it did not significantly affect colour and lipid oxidation and activities of SOD and Cat. More research is needed to elucidate dietary factors that affect the oxidative status and endogenous antioxidant enzyme activities of meat.
0
-1
-2 -3
-2
-1
0
1
2
Principal component 1 Fig. 2. (a) PCA loadings plot for the variables a-tocopherol content (vit e), lipid oxidation (tba), protein oxidation (carbonyl; thiol), colour oxidation (a*; metmb) and enzyme activities (cat, sod and gsh-px) of LTL muscle according to the first two factors. d4 and d8 denotes day 4 and day 8 stored samples. (b) Regression factor scores according to the pasture groups (L: Leguminosa rich; IR: Intensive ryegrass; BD: Botanically diverse).
Acknowledgements This work was financially supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), Brussels. Marı´a Jesu´s Petro´n is grateful for the financial support provided by the Secretarı´a de Estado de Educacio´n y Universidades and to the Fondo Social Europeo during the development of this
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Effect of grazing pastures of different botanical composition on antioxidant enzyme activities and oxidative stability of lamb meat M.J. Petron a, K. Raes b, E. Claeys b, M. Lourenc¸o b, D. Fremaut c, S. De Smet a
b,*
Food Technology and Biochemistry, Escuela de Ingenierı´as Agrarias, Universidad de Extremadura, Carretera de Ca´ceres s/n 06071, Badajoz, Spain b Laboratory of Animal Nutrition and Animal Product Quality, Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium c Faculty of Biosciences and Landscape Architecture, University College Gent, Voskenslaan 270, 9000 Gent, Belgium Received 22 February 2006; received in revised form 2 October 2006; accepted 6 October 2006
Abstract The aim of this work was to study the influence of different pastures (Intensive ryegrass, Botanically diverse and Leguminosa rich pastures) on the antioxidant status and oxidative stability of meat from lambs that had been exclusively grazing for three months. Lipid, colour and protein oxidation, a-tocopherol content and activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GSH-Px)) were measured in Longisimus thoracis et lumborum muscle samples taken 1 day after slaughter. Pasture type significantly affected protein oxidation and the activity of GSH-Px, but no significant differences were found for the a-tocopherol content, colour and lipid oxidation, and the activities of SOD and Cat. Grazing a Botanically diverse pasture induced significantly higher protein oxidation in meat, as measured by the free thiol and carbonyl contents, compared to a Leguminosa rich or Intensive ryegrass pasture (P < 0.05). The GSH-Px activity was significantly higher in meat from lambs on the Leguminosa rich pasture compared to the other pasture groups (P < 0.01). 2006 Elsevier Ltd. All rights reserved. Keywords: Pasture; Lamb meat; Superoxide dismutase; Glutathione peroxidase; Catalase; Protein oxidation; Lipid oxidation; Colour oxidation
1. Introduction Oxidative processes in meat are the most important factors responsible for quality deterioration including flavour, colour and nutritive value. The oxidative stability of meat depends on the balance between antioxidants and pro-oxidants and the content of oxidation substrates including polyunsaturated fatty acids (PUFA), cholesterol, proteins and pigments (Bertelsen et al., 2000; Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998). Living cells have several mechanisms of protection against oxidative processes which include the endogenous enzymes superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GSH-Px). SOD and Cat are cou*
Corresponding author. E-mail address: [email protected] (S. De Smet).
0309-1740/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2006.10.010
pled enzymes. SOD scavenges superoxide anions by forming hydrogen peroxide and catalase safely decomposes hydrogen peroxide to H2O and O2 . GSH-Px can decompose both hydrogen peroxide and lipoperoxides formed during lipid oxidation. It has been postulated that the activity of these antioxidant enzymes might be induced in animals exposed to oxidative stress, reflected in an increased production of free radicals (De Haan, Newman, & Kola, 1992). It has also been shown that these enzymes exhibit residual activity in muscle postmortem (De Vore & Greene, 1982; Renerre, Dumont, & Gatellier, 1996). Besides the contribution of endogenous enzymes, the oxidative stability of meat is determined by the presence of antioxidants of dietary origin, e.g., the role of vitamin E in retarding lipid oxidation and improving colour stability (Liu, Lanari, & Schaefer, 1995; Morrisey, Buckley, Sheehy, & Monahan, 1994; Morrissey et al., 1998) is well
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recognized. The effects of other dietary compounds with antioxidative properties, such as carotenoids or flavonoids, and the contribution of endogenous enzymes on the oxidative status of meat is less well known but is being increasingly investigated. In addition, the endogenous antioxidant enzyme activity system may be modulated by nutritional factors (Huang, Chen, Osio, & Cohen, 1994), e.g., some trace elements are essential for the normal function of endogenous antioxidant systems. The minerals Cu, Mn, Zn, Se and Fe are important cofactors of the antioxidant enzyme activities (Papas, 1999). Also other antioxidants could interfere, e.g., the regeneration of a-tocopherol by the oxidation/ reduction of glutathione (Budowski & Sklan, 1989). Apart from the many studies that have examined the supplementation of diets with antioxidants on the antioxidant status of meat, few studies have been performed on the effects of the basal diet, e.g., the effect of pasture varying in botanical composition and hence in the supply of minor compounds. The objective of the present study was to examine the effect of grazing pastures with different botanical composition by lambs on the oxidative stability of their meat. 2. Material and methods 2.1. Animals and experimental setup A total of 21 male lambs (mean age 86 ± 9 days, mean live weight 22.3 ± 3.1 kg) of similar genetic background (‘Vlaams Kuddeschaap’, a ‘herding’ sheep breed), all born from yearling ewes, and originating from an organic farm were used for this experiment. At weaning, they were assigned to one of three pasture types, i.e. an Intensive ryegrass (IR) pasture, a Leguminosa rich (L) pasture and a Botanically diverse (BD) pasture. Lambs had been exclusively grazing with their mother before the trial. The L and BD pastures were located on the farm of origin (Berendrecht; near Antwerp), whereas the IR pasture was located on the experimental farm of Ghent University (Melle; near Ghent). No supplementary feeding was provided, except for a licking mineral block (Timac Potasco, Belgium) with the following composition: sodium (270 g/kg), calcium (60 g/kg), phosphor (2 g/kg), magnesium (1 g/kg), zinc (18 000 mg/kg), manganese (2000 mg/kg), iodine (100 mg/kg), cobalt (40 mg/kg) and selenium (10 mg/kg). The trial lasted for 83 days from 1 July 2004 until 22 September 2004, when the lambs were slaughtered (mean live weight at slaughter 32.3 ± 6.5 kg) and sampled. The lambs were pastured in a larger group, but the experimental subgroups were matched for similar average live weight at the onset of the trial. On the IR pasture, some heifers were intermittently introduced to manage the sward height. The stocking density was low and on average less than 850 kg live weight ha 1 for the BD pasture and less than 1200 kg live weight ha 1 for the IR and L pasture. The L and BD pasture were not fertilized
at all. The IR pasture received 59 kg N ha 1 and 3 kg P2O5 ha 1 on 4 May and an additional 35 kg N ha 1 on 18 August. All pastures were mowed once in the first week of August. After slaughter, carcasses were cooled for 24 h at 2 C. One day after slaughter, the Longissimus thoracis et lumborum (LTL) muscle was dissected. Sub-samples were used immediately for measuring colour, lipid and protein oxidation after chilled illuminated storage (until 8 days postmortem), whereas other sub-samples were vacuum packaged and stored immediately at 18 C until analysis (a-tocopherol content and enzyme activities). 2.2. Pasture botanical and chemical composition The botanical composition of the pastures was assessed according to the method of De Vries (1933) on nine occasions during the trial (three times per month). At these times, samples were also taken for chemical analyses, dried at 50 C for 48 h, finely (0.5–1 mm) ground (Grindomix GM 200, Retsch, Germany) and pooled per month. Crude protein was determined according to the Kjeldhal method (European Community, 1993), Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) using the Van Soest method (Van Soest, Robertson, & Lewis, 1991), lignin according to the method described by Van Soest and Wine (1968) and crude fat by the Soxhlet method (International Standards Organisation, ISO-1444, 1973). The following mineral and trace elements were determined by plasma emission spectrometry (ICP-AES Iris Intrepid II XSP, Thermo Electron corporation) following dry incineration at 500 C for 4 h and dissolving in 6 N HCl: B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S. The content of Se was determined by ICP-AES with hydride generation, but with a different preparation. The sample is first destroyed with HNO3 and H2O2 in a microwave, pre-reduced with HCl to convert Se6+ to Se4+ and further reduced and converted to H2Se using NaBH4. 2.3. Colour stability Steaks (1.5 cm thickness) were over-wrapped in an O2permeable PVC film and stored at 4 C for 8 days under constant illumination with white fluorescent lights (900 lux). Colour and colour stability measurements were performed using a HunterLab Miniscan spectrocolorimeter (D65 light source, 10 standard, 45/0 geometry, 1 in. light surface, white standard). The colour coordinates, expressed as CIE L*, a*, b* values and the reflectance values to calculate metmyoglobin (MetMb%) (Krzywicki, 1979) were measured daily for 8 days. 2.4. Lipid and protein oxidation Lipid and protein oxidation were measured on day 4 and 8 for the muscle samples. Lipid oxidation was measured by the TBARS (2-thiobarbituric acid reactive sub-
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stances) method described by Tarladgis, Watts, and Younathan (1960). The results were expressed as lg MDA (malondialdehyde) g 1 muscle. Protein oxidation was estimated by measuring the free thiol content following the method described by Batifoulier, Mercier, Gatellier, and Renerre (2002) and the carbonyl content according to Mercier, Gatellier, Viau, Remignon, and Renerre (1997). The free thiol concentration was measured spectrophotometrically at 412 nm and was calculated using an absorption coefficient of 13.6 mM 1 cm 1. Results were expressed as nmol free thiols mg 1 protein. The total carbonyl content was quantified by a spectrophotometric assay at 370 nm, and was expressed as nmol DNPH (2,4-dinitrophenylhydrazine) incorporated mg 1 protein. The protein content was not measured, but was assumed to be constant at 0.22 g protein g 1 meat.
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Total superoxide dismutase (SOD) activity (Cu–Zn SOD + Mn SOD) was determined according to the procedure of Marklund and Marklund (1974), by measuring the inhibition of pyrogallol autoxidation. One unit was taken as the activity that inhibits the reaction by 50%. 2.7. Statistical analysis A one-way analysis of variance was performed to test for the effect of pasture type, followed by the Tukey post-hoc comparison means test in case of significance. Principal component analysis (PCA) was carried out to evaluate relationships among the various parameters of oxidative stability and to further distinguish the effect of pasture type. The statistical analyses were performed by SPSS version 11.5 (SPSS Inc. Chicago, IL, USA). 3. Results and discussion
2.5. a-Tocopherol content The a-tocopherol content was determined according to the method of Desai (1984). After saponification and hexane extraction, the samples were analysed by HPLC on a Supelcosil LC18 column (25 mm · 4.6 mm · 5 lm) equipped with a UV-detector (k = 292 nm). The results are expressed as lg a-tocopherol g 1 muscle. 2.6. Antioxidant enzyme activities A 5 g muscle sample was homogenized in 25 ml of 0.005 M phosphate buffer (pH 7.0) and centrifuged at 4 C for 20 min at 7000g. The supernatant fraction was filtered through glass wool and used to determine Cat, GSHPx and SOD activities. The catalase activity assay was performed as described by Aebi (1974). The supernatant (2 ml) was reacted at room temperature (22 C) with 1 ml of 30 mM H2O2 in 0.05 M phosphate buffer (pH 7.0), and the reaction (H2O2 decomposition) was monitored by measuring the absorbance at 240 nm during the initial 30 s. An extinction coefficient of 0.040 cm2 lmol 1 was used for calculation of H2O2 splitting. One unit (U) of catalase activity was defined as the amount of extract needed to decompose 1 lmol of H2O2 per min. GSH-Px activity was determined by measuring the oxidation of NADPH at 22 C (De Vore & Greene, 1982; Flohe´ & Gu¨nzler, 1984). The assay medium (3 ml) consisted of 1 mM reduced glutathione, 0.15 mM NADPH, 0.15 mM H2O2, 40 mM potassium phosphate buffer (pH 7.0), 0.5 mM EDTA, 1 mM NaN3, 1.5 units of glutathione reductase, and 300 ll of muscle extract. Absorbance at 340 nm was recorded over 3 min. An extinction coefficient of 6300 M 1 cm 1 was used for calculation of NADPH concentration. One unit of GSH-Px activity was defined as the amount of extract required to oxidize 1 lmol of NADPH per min at 22 C.
3.1. Feed and animal data Perennial ryegrass (Lolium perenne) was the predominant plant species (60–80%) on the IR pasture, followed by lower proportions of soft brome (Bromus hordeaceus) and Italian ryegrass (Lolium multiflorum). The L pasture harboured as predominant plants white clover (Trifolium repens) (approximately 40%), lucerne (Medicago sativa), perennial ryegrass and timothee (Phleum pratense). On the BD pasture, the predominant plant species were creeping bentgrass (Agrostis stolonifera) (approximately 40%), soft brome, thistle species and timothee, along with various herb species. Pastures did not only differ in botanical composition but also in chemical composition and contents of minerals and trace elements (Table 1). The crude protein content was significantly higher (P < 0.01) and the NDF content was significantly lower (P < 0.05) for the L pasture than for the BD and IR pastures. Pasture L had significantly higher contents of B, Ca, Cu, Na and Zn compared to the other pastures, whereas pasture IR was significantly higher in Mn (P < 0.05). The Se content could not be properly compared since most values were very low, but it appeared that the L pasture was also richer in this element. Mean values for live weight at the start and at slaughter and carcass weight per pasture group are given in Table 2. Lambs on the BD pasture group gained little weight during the trial and were significantly lighter at slaughter compared to the lambs from the two other pasture groups (P < 0.001). Carcass yield was also significantly lower for this group (P < 0.001). The very low average daily gain of the BD animals is very likely due to the low protein content of the BD pasture, that did not meet the protein requirements of growing lambs (CVB, 2004). The BD pasture group also displayed higher pH values in the LTL muscle at 1 h (P < 0.05) and 2 h (P < 0.001) postmortem compared to the two other groups, that did not differ. Pasture groups did not significantly differ in their pH values at
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Table 1 Mean values for chemical composition and content of minerals and trace elements of the pastures (n = 3) Botanically diverse
Leguminosa rich
SEM1
P
Chemical composition (% of dry matter) Crude Protein 15.9b Crude fat 3.99 Crude ash 11.3 NDF 60.8a ADF 35.0 Lignin 4.68
10.6b 3.86 10.9 61.3a 36.7 5.79
25.4a 4.03 10.5 42.8b 31.7 7.64
2.23 0.504 0.648 3.60 2.63 0.938
0.009 0.965 0.714 0.018 0.443 0.159
Mineral and trace element composition B (ppm) 4.87b Ca (ppm) 4427b Cu (ppm) 8.03b Fe (ppm) 230 K (ppm) 29100a Mg (ppm) 1557b Mn (ppm) 59.8a Na (ppm) 1720b P (ppm) 3680 S (ppm) 2340 Zn (ppm) 29.7b Se (ppb)2 0 · >4.0
6.79b 5973b 8.70b 283 18400b 1277b 23.5b 666c 2917 2363 29.1b 1 · >4.0
36.8a 14767a 17.1a 377 21633ab 2763a 25.0b 3770a 4327 1703 40.9a 2 · >4.0
2.76 1102 1.21 45.2 2410 132 6.06 86.5 357 212 2.84
<0.001 0.001 0.003 0.147 0.049 <0.001 0.009 <0.001 0.082 0.177 0.045
Intensive ryegrass
a,b
Mean values with different superscripts differ significantly (P < 0.05). Standard error of mean. 2 No mean values could be calculated for the content of Selenium, since most values were below the detection limit (4 ppb). The number of samples above the detection limit are given as an indication. 1
Table 2 Mean values for live weight at slaughter, cold carcass weight, LTL muscle pH and colour L*, a*, b* values according to pasture group (n = 7)
Live weight at start (kg) Live weight at slaughter (kg) Cold carcass weight (kg) Carcass yield (%) pH 1 h postmortem pH 2 h postmortem pH 24 h postmortem L* 1 day postmortem a* 1 day postmortem b* 1 day postmortem a,b
Intensive ryegrass
Botanically diverse
Leguminosa rich
SEM
P
22.6 36.4a 15.8a 43.3a 6.51a 5.96a 5.63 40.7 11.6 12.8
23.1 24.8b 8.9b 35.7b 6.76b 6.41b 5.80 38.8 11.3 12.6
21.1 35.6a 15.8a 44.3a 6.68ab 6.07a 5.63 37.7 11.3 12.2
1.67 2.06 1.07 1.13 0.209 0.269 0.165 0.62 0.18 0.34
0.503 <0.001 <0.001 <0.001 0.056 <0.001 0.093 0.076 0.672 0.428
Mean values with different superscripts differ significantly (P < 0.05).
24 h postmortem but there was a tendency for higher values in meat from BD lambs. Whether this slower rate of muscle pH fall in the BD animals is related to their lower growth rate is difficult to say, but it could be hypothesized that the lower growth rate resulted in a more oxidative muscle fibre type and/or lower glycogen levels at slaughter, and hence caused slightly higher muscle pH values postmortem. The effect of grazing these pastures differing in botanical composition on rumen fatty acid metabolism and on the intramuscular fatty acid composition was also studied but is not reported here. In summary the total polyunsaturated fatty acid (PUFA) proportion in the LTL intramuscular fat was significantly higher in the BD animals compared to the other groups. However, the total intramuscular fatty acid content differed between groups
(BD < IR < L), resulting in similar contents of PUFA (mg/g meat) in meat from the BD and L lambs and lower levels in meat from the IR lambs. The differences in the intramuscular fatty acid composition were not due to differences in the dietary fatty acid supply, but were mainly related to rumen metabolism. In view of the fact that no differences between pasture groups were observed in lipid oxidation (see further), differences in the intramuscular fatty composition were probably of little or no importance in the present study. These data are therefore not included. 3.2. Colour, lipid and protein oxidation There were no significant differences between the groups for the colour a* value and MetMb% at any time
M.J. Petron et al. / Meat Science 75 (2007) 737–745
a
20
BD
IR
L
a* value
15 10 5 0 0
1
2
3
4
5
6
7
8
6
7
8
b
50
% metmyoglobin
Day of display
40
BD
IR
L
30 20 10 0
0
1
2
3
4
5
Day of display Fig. 1. Effect of type of pasture (L: Leguminosa rich; IR: Intensive ryegrass; BD: Botanically diverse) on changes in a* value (a) and % metmyoglobin (b) of LTL muscle samples during storage.
during chilled storage and display (Fig. 1). The mean a* value increased from day 0 to day 1 and thereafter decreased until day 8. These results agree with those from Guidera, Kerry, Buckley, Lynch, and Morrissey (1997) in lamb meat who found the a* value to decrease during chilled storage. As expected, the MetMb% increased during display. Results for lipid (TBARS values) and protein oxidation (free thiol and carbonyl content) on samples at day 4 and 8 of storage are given in Table 3. There was no significant effect of pasture type on TBARS values. Mean values were low (<0.7 lg MDA g 1 meat) for all pasture groups, even after 8 days of chilled storage, and are in agreement with findings from other authors on lamb (Macit, Aksakal, Emsen, Esenbuga, & Aksu, 2003; Macit, Aksakal, Emsen, Table 3 Mean values for TBARS, free thiol content and carbonyl content in LTL muscle according to pasture group (n = 7) Intensive ryegrass TBARS (lg MDA g Day 4 0.32 Day 8 0.68
Botanically diverse 1
Leguminosa rich
meat) 0.41 0.63
SEM
P
0.31 0.52
0.06 0.12
0.359 0.631
protein) 70.4 64.4a
2.71 1.97
0.106 <0.001
Carbonyl content (nmol DNPH incorporated mg 1 protein) Day 4 0.65a 0.85b 0.73a 0.04 a b 1.42 0.98a 0.13 Day 8 0.98
0.009 0.046
Free thiol content (nmol of free thiol mg Day 4 62.9 62.9 Day 8 61.0a 48.6b
a,b
1
Mean values with different superscripts differ significantly (P < 0.05).
741
Aksu, et al., 2003). As expected, an increase in TBARS values was observed with storage. Protein oxidation measured by the carbonyl content was significantly affected by the type of pasture (Table 3). Samples from the BD group had significantly higher mean carbonyl contents than samples from the L and IR group on both day 4 (P < 0.01) and day 8 (P < 0.05) samples. A decrease in the free thiol content of muscle with time of storage is also indicative of protein oxidation (Batifoulier et al., 2002). Sista, Erickson, and Shewfelt (2000) suggested, using a chicken muscle model system, that sulphydryls are utilized to stabilize primary oxidation products. On samples at day 4, there was no significant difference between the pasture groups for the free thiol contents, but on samples at day 8, lamb meat of pasture BD had significantly lower values (P < 0.001) compared to the other groups. There was no significant difference at any time in the free thiol and carbonyl contents between the L and IR groups. Hence, for reasons that we cannot explain, the BD pasture appeared to induce the most pronounced protein oxidation in meat postmortem, which could be associated with increased protein degradation, fragmentation or aggregation (Martinaud et al., 1997). This effect of diet on protein oxidation was somewhat unexpected since no significant effect of diet on colour and lipid oxidation was found. In previous studies, protein oxidation was related to lipid oxidation in beef (Mercier, Gatellier, & Renerre, 1995), turkey meat (Mercier et al., 1997) and fish (Srinivasan & Hultin, 1995). Others studies on beef have shown an effect of diet on lipid oxidation but no significant dietary effect on protein oxidation (Mercier, Gatellier, & Renerre, 2004). The present study is the first report to our knowledge on protein oxidation in lamb meat. Our results show a significant effect of diet on protein oxidation, as measured by both the free thiol and carbonyl contents, that was not associated with a dietary effect on lipid oxidation. Whether the effect of the BD pasture on protein oxidation observed in this study is primarily due to a different balance in the dietary supply of pro-oxidants and antioxidants, or whether it is related to the lower growth rate of the lambs in this group and possible accompanied changes in muscle protein properties, remains to be investigated. Interference of the higher pH values in the LTL measured postmortem can also not be excluded, although it is difficult to hypothesize a causal relationship. 3.3. a-Tocopherol content and antioxidant enzyme activities Oxidation of muscle components postmortem can be retarded by antioxidants provided by the diet, of which a-tocopherol is the most important (Morrissey et al., 1998; lamb: Turner, McClure, Weiss, Borton, & Foster, 2002), and by the action of endogenous antioxidant enzymes, which seem to be relatively stable during refrigerated storage (Renerre et al., 1996). The muscle levels of atocopherol found in this study (1.1–1.7 lg g 1; Table 4) were close to those previously described in LTL muscle
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Table 4 Mean values for LTL muscle a-tocopherol content and activities of glutathione peroxidase (GSH-Px), catalase (Cat) and superoxide dismutase (SOD) according to pasture group (n = 7) a-tocopherol content (lg g 1) GSH-Px (U g 1) Cat (U g 1) SOD (U g 1) a,b
Intensive ryegrass
Botanically diverse
Leguminosa rich
SEM
P
1.72 0.090a 40.8 64.4
1.24 0.082a 30.8 68.5
1.09 0.181b 30.1 72.4
0.20 0.07 9.96 8.99
0.091 0.006 0.074 0.259
Within a row, mean values with different superscripts differ significantly (P < 0.05).
of lambs (Guidera et al., 1997; Salvatori et al., 2004). Pasture groups did not significantly differ in their muscle atocopherol contents in this study, but there was a tendency for higher levels in meat from the IR lambs compared to the BD and L lambs. Antioxidant enzymes activities are presented in Table 4. Diet did not modify muscle postmortem SOD activity, while a trend for a higher Cat activity was observed for the IR group compared to the other groups. Activities of SOD and Cat help to scavenge the highly reactive superoxide and hydroxyl ions respectively, and a decrease in the SOD and Cat activities has been associated with oxidative stress in vivo (Pigeolot et al., 1990). On the other hand, some authors considered an increase in Cat activity as a feedback response mechanism to oxidative stress (Venkatraman & Pinnavaia, 1998; Renerre, Poncet, Mercier, Gatellier, & Me´tro, 1999). The background and the impact of differences in the residual activity of these enzymes in meat remains to be further elucidated. As far as we are aware no other data are available in literature on the activity of these enzymes in lamb meat postmortem. In contrast to SOD and Cat, a significant effect of pasture type was found on the GSH-Px activity, that was higher for the L group compared to the IR and BD groups (P < 0.01). Although no clear explanation is available, the finding is in line with several studies on bovine meat that have shown an effect of finishing mode on the activity of GSH-Px (Descalzo, Insani, Eyherabide, Guidi, & Pensel, 2000; Mouty et al., 2002; Gatellier, Mercier, & Renerre, 2004; Mercier et al., 2004). GSH-Px plays a central role in the antioxidant defence system. An elevation of GSHPx activity has been associated with oxidative stress (Frank & Messaro, 1980). If residual postmortem activity of GSHPx is related to its activity in vivo, this would suggest that animals on the L pasture were subject to more oxidative stress. However, this L group showed similar values for lipid oxidation to the other groups and lower protein oxidation than the BD group. The results could therefore also suggest that the increase in GSH-Px activity on the L pasture enhanced the antioxidant defence capacity. The effect of feeding regimes on muscle antioxidant enzyme activities, and to what extent this is related to lipid and especially protein oxidation postmortem, is still largely unknown. Factors that might be responsible for this dietary effect include differences in the deposition of n-3 PUFA (e.g., white clover was shown to limit biohydrogenation of n-3 PUFA by Lee, Harris, Dewhurst, Merry, &
Scollan (2003)), but other antioxidants, trace elements and especially Se could also influence the activity of GSH-Px (Huang et al., 1994; Papas, 1999). Selenium is an essential cofactor for GSH-Px (Huang et al., 1994; Papas, 1999). Supplementation of selenium to feed can increase the selenium and GSH-Px contents of tissues in farm animals (Mahan & Parret, 1996). The L pasture showed a tendency for a higher content of Se and had clearly higher values for most trace elements, except for Mn, than the other pastures. Although levels of cofactors that are required in feed to modulate the enzyme activities in vivo are not known, these differences may have contributed to the difference in GSH-Px activity. On the other hand, the activities of SOD and Cat were not affected by the type of pasture in this study and the levels of cofactors for these enzymes (Mn, Zn, Cu) also differed between pastures. As argued by Papas (1999), not only the dietary supply of enzyme cofactors is important in this respect, interaction with other minerals and plant compounds that determines, e.g., the oxidation state of these cofactors and their absorption and bioavailability should be considered.
3.4. Relationships between measures of oxidation and antioxidant parameters In the present study, the correlations between a-tocopherol content on the one hand and lipid peroxidation, protein oxidation and endogenous enzyme activities on the other hand were not significant. However, significant correlations between GSH-Px activity and free thiol content (day 8) (r = 0.445; P < 0.05), and between Cat activity and TBARS content (day 4) (r = 0.581; P < 0.01) and carbonyl content (day 4) (r = 0.606; P < 0.01) were found. As mentioned above, the relationship between nutritional background and muscle a-tocopherol content and the effect on slowing down postmortem oxidative changes has been extensively described in literature for various species, but the relationship between a-tocopherol levels and endogenous antioxidant enzyme activities is unclear. Several studies have shown that a-tocopherol did not affect endogenous antioxidant activities in pork (Cadenas, Rojas, Perez-Campo, Lopez-Torres, & Barja, 1995), turkey (Renerre et al., 1999) and beef (Walsh, Kennedy, Goodall, & Kennedy, 1993; O’Grady, Monahan, Fallon, & Allen, 2001), but a positive significant correlation between SOD and a-tocopherol content has been reported by Gatellier
M.J. Petron et al. / Meat Science 75 (2007) 737–745
et al. (2004) in beef. Our data do not support a possible relationship. The relationship between Cat activity and lipid or protein oxidation is poorly documented. Cand and Verdetti (1989) found no correlation between SOD, Cat and GSH-Px activities and the TBARS content in rat organs.
Principal component 2 (19.44%)
a
1.0 cat carbonyl d4 tba d4
carbonyl d8
.5
tba d8
a*d4 a*d8
0.0
vit e sod
t hiol d4
m mb mmb d8d4
gsh-px
-.5 thiol d8
.0 -1.0
-.5
0.0
.5
1.0
Principal component 1 (38.28%)
b
3 GROUP L IR BD
Principal component 2
2
743
However, in the present results on lamb meat, Cat activity was positively related to lipid oxidation and protein oxidation, when the latter was measured by carbonyl content. In a study in rabbits an increase in Cat activity in response to oxidative stress was insufficient to inhibit the increased free radical production, contributing to further lipid and protein oxidation (Gumieniczek, 2005). A possible explanation for the significant correlation between GSH-Px activity and protein oxidation, as measured by free thiol content, is that glutathione (GHS) is a major non-protein thiol in living organisms. It has been shown in the muscle of rats that increased activity of GSH-Px is associated with higher levels of GSH (Kumaran, Savitha, Anusuya Devi, & Panneerselvam, 2004). This finding is consistent with our results with the higher GSHPx activity corresponding to a higher free thiol content. Principal component analysis (PCA) was carried out to further elucidate relationships between the variables in this study (Fig. 2). The first and second PC explained 38% and 19% of the variation respectively. The relationships between antioxidant enzyme activities and oxidation variables as mentioned above are also apparent from this plot. It is possible to distinguish a group of variables composed of Cat activity, TBARS values and carbonyl content in the positive axis on the PC2, far from the origin and explaining an important part of the variation. GSH-Px, SOD and free thiol content are located on the negative axis on PC2. Explaining an independent cause of variation, a* value and MetMb% are located on PC1, far from the origin, and being opposite to each other. The regression factor scores show the discrimination between the pasture groups. Although few significant differences between individual variables were apparent, there is a rather good distinction between the groups based on this factor analysis. However, variability between animals within groups also appeared to be large.
1
4. Conclusions Finishing lambs for three months on pastures that differed in botanical composition (intensive ryegrass, botanically diverse or leguminosa rich) did affect protein oxidation and GSH-Px activity of muscle postmortem. However, it did not significantly affect colour and lipid oxidation and activities of SOD and Cat. More research is needed to elucidate dietary factors that affect the oxidative status and endogenous antioxidant enzyme activities of meat.
0
-1
-2 -3
-2
-1
0
1
2
Principal component 1 Fig. 2. (a) PCA loadings plot for the variables a-tocopherol content (vit e), lipid oxidation (tba), protein oxidation (carbonyl; thiol), colour oxidation (a*; metmb) and enzyme activities (cat, sod and gsh-px) of LTL muscle according to the first two factors. d4 and d8 denotes day 4 and day 8 stored samples. (b) Regression factor scores according to the pasture groups (L: Leguminosa rich; IR: Intensive ryegrass; BD: Botanically diverse).
Acknowledgements This work was financially supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), Brussels. Marı´a Jesu´s Petro´n is grateful for the financial support provided by the Secretarı´a de Estado de Educacio´n y Universidades and to the Fondo Social Europeo during the development of this
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