Oxidative Stability of Dark Chicken Meat Through Frozen Storage: Inuence of Dietary Fat and -Tocopherol and Ascorbic

Acid Supplementation
A. Grau,* F. Guardiola,*,1 S. Grimpa,* A. C. Barroeta, and R. Codony*
*Nutrition and Food Science Department-CeRTA, Faculty of Pharmacy, University of Barcelona, Avinguda Joan XXIII s/n, E-08028 Barcelona, Spain; and Unitat Docent de Nutricio i Alimentacio Animal, ` Facultat de Veterinaria, UAB, E-08193 Bellaterra, Spain ABSTRACT We used factorial design to ascertain the inuence of dietary fat source (linseed, sunower and oxidized sunower oils, and beef tallow) and the dietary supplementation with -tocopheryl acetate (-TA) (225 mg/kg of feed) and ascorbic acid (AA) (110 mg/kg) on dark chicken meat oxidation (lipid hydroperoxide and TBA values and cholesterol oxidation product content). -TA greatly protected ground and vacuum-packaged raw or cooked meat from fatty acid and cholesterol oxidation after 0, 3.5, or 7 mo of storage at 20 C. In contrast, AA provided no protection, and no synergism between -TA and AA was observed. Polyunsaturated fatty acidenriched diets (those containing linseed, sunower, or oxidized sunower oils) increased meat susceptibility to oxidation. Cooking always involved more oxidation, especially in samples from linseed oil diets. The values of all the oxidative parameters showed a highly signicant negative correlation with the -tocopherol content of meat.

(Key words: ascorbic acid, chicken leg meat, dietary fat, lipid oxidation, -tocopherol) 2001 Poultry Science 80:16301642

INTRODUCTION
Meat composition can be modied by diet. Animals fed on diets rich in unsaturated fats have increased the polyunsaturated fatty acid (PUFA) content of their lipid fraction (Lin et al., 1989a; Ajuyah et al., 1993; Ahn et al., 1995; Mooney et al., 1998; Lopez-Ferrer et al., 1999). This modication is nutritionally desirable, but it increases susceptibility of meat to oxidation. Nevertheless, this drawback can be overcome by dietary supplementation with antioxidants. Among these, the protective role of tocopheryl acetate (-TA) is unquestionable (Jensen et al., 1998), whereas that of other compounds such as carotenoids or ascorbic acid (AA) remains to be established (Pardue and Thaxton, 1986; King et al., 1995; Erickson, 1998; Ruiz et al., 1999). Dietary modulation of the composition of meat may enable us to improve its oxidative stability and thus increase the nutritional value and the shelf life of meat products. The oxidative status of meat can be assessed on the basis of primary oxidation [i.e., through the measurement of lipid hydroperoxide (LHP) value] or secondary oxidation [i.e., through the measurement of malondialdehyde (MDA), or cholesterol oxidation products (COP)].
2001 Poultry Science Association, Inc. Received for publication December 15, 2000. Accepted for publication July 2, 2001. 1 To whom correspondence should be addressed: e-mail: barz@ farmacia.far.ub.es.

The ferrous oxidation-xylenol orange (FOX) method has been reported to be a simple, sensitive, precise, and specic way to measure LHP (Jiang et al., 1992; Shantha and Decker, 1994; Burat and Bozkurt, 1996; NouroozZadeh, 1998). This assay consists of the peroxide-mediated oxidation of ferrous ions in an acidic medium containing the dye xylenol orange, which binds the resulting ferric ions to produce a blue-purple complex with a maximum absorbance between 550 and 600 nm. This method has been adapted for dark chicken meat samples (Grau et al., 2000b) showing a high specicity for LHP. With regard to the measurement of MDA, the aqueous acid extraction methods [reaction of an aqueous acid extract of MDA with 2-thiobarbituric acid (TBA) to give a red complex absorbing at 530 to 537 nm] seem to be the most appropriate for routine assessment of lipid oxidation in meat and meat products (Witte et al., 1970; Pikul et al., 1989; Raharjo and Sofos, 1993). However, these methods have been criticized for their lack of sensitivity and specicity, as compounds other than MDA can also react with TBA, giving interfering complexes with absorption at the same (530 to 537 nm) or similar wave lengths (450 to 460 nm) than the MDA-TBA adduct (Ko-

Abbreviation Key: AA = ascorbic acid; BT = beef tallow; COP = cholesterol oxidation product; FOX = ferrous oxidation-xylenol orange; LHP = lipid hydroperoxide; LO = linseed oil; MDA = malondialdehyde; OSO = oxidized sunower oil; PUFA = polyunsaturated fatty acid; SO = sunower oil; -TA = -tocopheryl acetate.

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sugi et al., 1987; Guillen and Guzman, 1988; Raharjo and Sofos, 1993; Botsoglou et al., 1994). To overcome these drawbacks, Botsoglou et al. (1994) proposed an acid aqueous extraction method using third-derivative spectrophotometry, which removes the main sources of interference. Their method was further studied in order to be applied to chicken meat samples (Grau et al., 2000c). Finally, the determination of COP content in foods of animal origin has also been used as a parameter to measure lipid oxidation (Paniangvait et al., 1995; Addis et al., 1996). Another interest of this determination relies on the potentially harmful effects of these compounds (Guardiola et al., 1996; Brown and Jessup, 1999). The rst choice method for this analysis consists of lipid extraction, COP enrichment, and gas chromatography (Addis et al., 1996; Guardiola et al., 1998). A method following these steps has been adapted to dark chicken meat samples (Grau et al., 2000a). The goal of this paper was to study the inuence of the dietary fat source, -TA and AA supplementations, and cooking on the oxidative stability of dark chicken meat samples vacuum-packaged and stored at 20 C for 0, 3.5, and 7 mo. Our discussion is based on the results of several oxidative parameters (LHP and TBA values and COP content).

TABLE 1. Ingredients and composition of the basal diet Ingredients and composition Corn Corn germ meal Soybean meal, 44% protein Sorghum Sunower meal Barley Rye Beef tallow Calcium carbonate Dicalcium phosphate Sepiolite Salt Sodium bicarbonate Trace mineral-vitamin mix1 Composition2 Dry matter Crude protein Crude fat Crude ber Ash
1 2

Percentage 30.0 18.4 17.4 10.0 8.0 5.8 5.0 0.1 1.9 1.4 1.0 0.3 0.1 0.5 88.2 16.0 4.1 5.1 7.4

Did not include vitamin E. Metabolizable energy, 2,750 Kcal/kg.

randomly placed in 48 pens (ve birds per pen). Dietary treatments were randomly distributed; three pens were assigned to each treatment, and broiler chickens were fed ad libitum for 50 d.

MATERIALS AND METHODS Experimental Design

A 4 2 2 3 factorial design was planned and conducted in triplicate to study the inuence of various dietary factors (four types of fat source and two levels of -TA and AA supplementation) and storage periods (0, 3.5, and 7 mo) at 20 C on several parameters (-tocopherol content, fatty acid composition, LHP and TBA values, and COP content) in raw and cooked dark chicken meat.

Sample Preparation
Broiler chickens (57 d old) were leg-ringed and slaughtered according to commercial procedures at the Avicola Maria slaughterhouse (Begur, Spain). Carcasses were ` transported to the Cuits processing plant (Cassa de la Selva, Spain) and stored for 3 d at 4 C in a walk-in cooler. Legs with skin from each pen were divided into two groups. One group of legs was hand deboned, ground, vacuum-packaged in 13.5 16-cm polyamide/polyethylene bags and stored for 0, 3.5, or 7 mo at 20 C. The other group of ve legs was hand deboned. Two legs of this group were vacuum-packaged in 20 40-cm polyamide/ polyethylene bags and cooked at 80 C for 35 min in a pressure cooker. Thereafter, these two cooked legs were ground, vacuum-packaged in 13.5 16-cm polyamide/ polyethylene bags, and stored at 20 C for 0, 3.5, or 7 mo. The remaining three legs were vacuum-packaged in 20 40-cm polyamide/polyethylene bags, cooked at 80 C for 35 min in a pressure cooker, and stored for 13 mo at 20 C until sensory analysis and TBA value determination (Figure 1). Permeability to oxygen of all bags used was 50 cm3 bar 24 h (Deutsches Institut fur Normung; 53,380, 23 C). Results from sensory analysis have been discussed in a previous work (Bou et al., 2001).

Dietary Treatments and Animals

Sixteen isocaloric dietary treatments were prepared from a basal diet (Table 1) by the following combination of the dietary factors studied (4 2 2): fat source2 [beef tallow (BT), sunower oil (SO), oxidized sunower oil (OSO), and linseed oil (LO); 6% fat supplementation in all cases]; -TA (0 and 225 mg/kg of feed); and AA (0 and 110 mg/kg of feed).3 Sunower oil [unrened; specic absorbances at 232 (K232) and 270 nm (K270) were 2.87 and 0.25, respectively] was oxidized by heating in a fryer for 12 h at 160 C and then leaving the oil in the fryer at room temperature for 6 d (K232 = 4.40, K270 = 0.83). Specic absorbances were measured as described by Guardiola et al. (1995). Two hundred forty female broiler chicks (Ross, 1 d old) were fed a control diet for 6 d. One-week-old birds were

Analysis of Samples
Lipid hydroperoxide values were assessed according to the FOX method described by Grau et al. (2000b). Reaction mixtures containing 75 L of sample extract were incubated for 4 h.

` Dietary fats supplied by Caila i Pares S.A., E-08040 Barcelona, Spain. -TA (Rovimix E-50 Adsorbate) and AA (Rovimix C) were from Hoffmann-La Roche, Ltd., CH-4070 Basel, Switzerland.
3

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FIGURE 1. Experimental design. This experimental design was conducted in triplicate (80 3 female broiler chicks were randomly assigned to 16 3 treatments). AA = ascorbic acid supplementation in mg/kg of feed; BT = beef tallow; COP = cholesterol oxidation product; LHP = lipid hydroperoxide; LO = linseed oil; OSO = oxidized sunower oil; SO = sunower oil; -TA = -tocopheryl acetate supplementation in mg/kg of feed. One leg of each bird in each group of legs.

EFFECT OF DIET ON CHICKEN MEAT OXIDATION

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Thiobarbituric acid values were determined by an acid aqueous extraction method with third-derivative spectrophotometry, as described by Grau et al. (2000c). Analyses for COP and cholesterol content were performed as described by Grau et al. (2000a). Fatty acid composition and -tocopherol content were determined as in Grau et al. (2000b). For all analytical methods, reagents and standards used were previously detailed by Grau et al. (2000a,b,c). Statistics. To determine whether the factors studied (fat source, -TA and AA supplementations, cooking, and storage time) had any signicant effect on the responses, 12 multifactor ANOVA were performed as follows: (1 to 3) raw samples at 0, 3.5, and 7 mo, respectively (n = 48); (4) all raw samples (0 + 3.5 + 7 mo, n = 144); (5 to 8) the same for cooked samples; (9) raw + cooked samples at 0 mo (n = 96); (10 and 11) the same at 3.5 and 7 mo, respectively, and (12) all samples (raw + cooked at 0 + 3.5 + 7 mo, n = 288). In all cases, interactions higher than order two were ignored and P 0.05 were considered to be signicant. When dietary fat source or storage time showed a signicant effect, the Scheffe test for a posteriori contrasts ( = 0.05) was applied to determine statistical differences between least-squares means. Pearson correlation coefcients were used to examine possible linear correlations between responses (LHP values, TBA values, COP content, and -tocopherol content).

RESULTS AND DISCUSSION Inuence of Dietary Factors

The dietary fat signicantly inuenced the values of the oxidation parameters in both raw and cooked dark chicken meat stored for 0, 3.5, or 7 mo at 20 C, except for LHP values in raw samples stored for 0 or 3.5 mo (Table 2). At any storage time, raw and cooked samples from chickens fed BT or LO diets gave the lowest and the highest TBA values, respectively. Other authors also reported higher TBA values for chicken leg meat from unsaturated fat sources (Lin et al., 1989a; Ajuyah et al., 1993; Ahn et al., 1995; Maraschiello et al., 1999; Ruiz et al., 1999), and, as in our case, it is a consequence of the higher PUFA content in meats from unsaturated diets (Table 3). When the fat source signicantly inuenced LHP values, samples from diets containing unsaturated fat sources also gave the highest values. In contrast, the highest COP content for raw and cooked samples was observed in SO- and BT-fed groups, respectively. The cholesterol content of samples cannot account for this effect of dietary fat on COP content, as there were no signicant differences between the cholesterol content of meat from different fat sources (Table 3). Li et al. (1996) also failed to nd any differences in cholesterol content in freeze-dried chicken breast meat samples from animals fed diets supplemented with sh, ax, palm, or sunower oils. Feeding OSO did not signicantly increase the oxidation of dark chicken meat (Table 2). However, other stud-

ies carried out with chickens fed on diets containing similar amounts of OSO reported signicantly higher TBA values as a result of feeding oxidized oil (Lin et al., 1989b; Galvin et al., 1993, 1997). In addition, Galvin et al. (1993) and Sheehy et al. (1993, 1994) showed that ferrous-ascorbate-induced lipid oxidation in chicken tissue homogenates (measured by TBA values after incubation at 37 C) was much higher for the OSO-fed group than for the SOfed group. This nding is consistent with the nding that in cooked samples TBA values tended to be higher for OSO groups, although it was not signicant (Table 2). However, as argued by Monahan et al. (1992), lack of a clear prooxidant effect of dietary OSO could be attributed to an insufcient degree of oxidation of the heated SO. We do not accept the dose of the oxidized oil as a cause of this absence of signicant effect, because the amount of oil used by Lin et al. (1989b), Sheehy et al. (1993, 1994), and Galvin et al. (1993, 1997) ranged from 40 to 80 g/kg of feed and that used in our study was 60 g/kg. Specic absorbances at 232 and 270 nm were, respectively, 2.87 and 0.25 for the SO and 4.40 and 0.83 for the OSO, which indicates that conjugated dienes in OSO were only approximately 1.5-fold greater than in SO. Thus, PUFA peroxide content in our OSO is much lower than in OSO used by other authors (Lin et al., 1989b; Galvin et al., 1993, 1997; Sheehy et al., 1994). Moreover, these studies reported that supplying oxidized oils decreased plasma and muscle -tocopherol levels. This result was attributed to the destruction of the endogenous -tocopherol of SO during heating, the destruction of -tocopherol in the gastrointestinal tract by free radicals from the oxidized oil, and the absorption of LHP, resulting in a higher rate of oxidation and higher consumption of -tocopherol in vivo. Despite that earlier studies indicated that LHP were not absorbed in the intestinal tract (Andrews et al., 1960; Bunyan et al., 1968), it has been recently shown that, in humans, LHP are absorbed and incorporated into chylomicrons to some extent (Naruszewicz et al., 1987; Staprans et al., 1994). In addition, studies conducted in rats showed that levels of LHP in serum very-low-density lipoprotein plus low-density lipoprotein fraction correlated with LHP in the diet (Staprans et al., 1993). Thus, chylomicrons could deliver LHP to the liver, where they could be secreted in very-low-density lipoprotein and therefore to reach peripheral tissues. However, in the present study, although the -tocopherol content of meat samples from OSO diets was slightly lower than that from SO, this difference was not signicant (Table 2). This nding is possibly due to the low LHP level of the OSO. Furthermore, Galvin et al. (1997) reported that, in cooked ground breast or leg chicken meat stored for different periods at 4 or 20 C, samples from OSO diets always resulted in higher TBA values than those from SO. In contrast, in our study, no signicant differences were detected between OSO- and SO-fed groups during the 7 mo of frozen storage. -TA supplementation resulted in a signicant protection against oxidation for both raw and cooked samples at any storage time, which was reected by all the oxidation

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parameters (Table 2). The decrease in TBA and COP values as a consequence of dietary tocopherols has been reported by several authors and has been attributed to the accumulation of the dietary -tocopherol in muscle tissues of chickens (Lin et al., 1989a; Ajuhah et al., 1993; Ahn et al., 1995; Jensen et al., 1995; King et al., 1995; de Winne and Dirinick, 1996; Li et al., 1996; Lauridsen et al., 1997a; ONeill et al., 1998; Marashiello et al., 1999; Ruiz et al., 1999). In fact, from our data, supplementation of broiler diets with 225 mg of -TA/kg of feed led to an overall increase of tocopherol in meat of 39.19 and 35.56 mg/kg for raw and cooked samples, respectively (Table

2). In contrast, no studies have been found concerning LHP values in chicken meat from animals fed tocopherolsupplemented diets. AA supplementation resulted in a prooxidant effect in terms of TBA values, which was only signicant for raw meat (at all storage times) (Table 2). For the other oxidation parameters, the effect of dietary supplementation with AA was not signicant in any case. In fact, whether AA prevents or promotes lipid oxidation in meat is controversial (Benedict et al., 1975; Bartov, 1977; Pardue and Thaxton, 1986; King et al., 1995; Lauridsen et al., 1997b). Low concentrations of AA in meat lead to a prooxidant

TABLE 2. Least-squares grand means (global means) and least-squares means for responses at different times of storage as inuenced by factors1 -TA supplementation (mg/kg of feed) LO 193.0 181.0x 1.445x 6.574x 21.9 16.9 159.8 212.8x 1.407x 5.778x 21.7 17.8 172.2xy 198.1x 1.436x 5.715x 1.43x 2.45y 20.6 17.8 0 381.3x 287.1x 1.041x 5.024x 3.9y 1.7y 312.7x 363.1x 1.095x 4.465x 3.7y 1.7y 327.4x 344.3x 1.174x 4.435x 2.15x 4.20x 3.5y 1.5y 225 17.7y 23.8y 0.092y 2.914y 42.5x 34.4x 11.9y 21.6y 0.125y 2.577y 44.8x 39.2x 11.4y 19.9y 0.192y 2.502y 0.85y 2.33y 41.6x 37.9x AA supplementation (mg/kg of feed) 0 201.9 160.1 0.396y 3.854 22.9 17.6 156.9 194.7 0.478y 3.459 23.4 20.8 167.9 186.6 0.558y 3.407 1.42 3.40 22.7 19.9 110 197.2 150.8 0.736x 4.084 23.3 18.5 167.7 190.0 0.743x 3.584 24.7 20.1 170.8 177.6 0.809x 3.530 1.58 3.13 22.4 19.5

Responses2 0 mo of storage LHP valueDE TBA valueABCDE -Tocopherol contentE 3.5 mo of frozen storage LHP valueAE TBA valueABDE -Tocopherol contentA 7 mo of frozen storage LHP valueAD TBA valueABDE Total COPABE -Tocopherol contentE

Meat Raw Cookeda Rawabc Cookeda Raw Cooked Raw Cookeda Rawabc Cooked Rawa Cookeda Raw Cookeda Rawabc Cooked Rawab Cooked Raw Cooked

Global means 199.5X 155.4*,X 0.566 3.969*,X 23.2 18.1* 162.3Y 192.3*,Y 0.610 3.521*,Y 24.0 20.5* 169.4Y 182.1*,Y 0.683 3.468*,Y 1.50 3.26* 22.5 19.7*

Fat source BT 183.7 124.7z 0.155x 2.554z 25.2 16.0 141.9 147.6y 0.184y 2.347z 26.7 19.8 150.0y 135.5y 0.273y 2.306y 1.26x 3.70x 24.6 19.2 SO 205.7 162.2xy 0.404y 3.186yz 23.5 21.5 173.3 199.0x 0.509y 2.802yz 26.5 23.4 174.8xy 198.3x 0.571y 2.760y 1.90y 3.59x 24.1 21.7 OSO 215.6 153.8y 0.262y 3.562y 21.8 17.8 174.2 210.0x 0.341y 3.158y 21.3 20.8 180.5x 196.5x 0.454y 3.092y 1.42x 3.31x 20.9 20.2

a Interaction of dietary fat source -TA supplementation signicant at P 0.05. P-values obtained from multivariate ANOVA (MANOVA) (n = 48) for raw or cooked samples. A Interaction of dietary fat source -TA supplementation signicant at P 0.05. P-values obtained from MANOVA for all raw and cooked samples together (n = 96). b Interaction of dietary fat source AA supplementation signicant at P 0.05. P-values obtained from MANOVA (n = 48) for raw or cooked samples. B Interaction of dietary fat source AA supplementation signicant at P 0.05. P-values obtained from MANOVA for all raw and cooked samples together (n = 96). c Interaction of -TA AA supplementation signicant at P 0.05. P-values obtained from MANOVA (n = 48) for raw or cooked samples. D Interaction of dietary fat source cooking signicant at P 0.05. P-values obtained from MANOVA for all raw and cooked samples together (n = 96). E Interaction of -TA supplementation cooking signicant at P 0.05. P-values obtained from MANOVA for all raw and cooked samples together (n = 96). xz Means in the same row for a certain factor bearing no common superscripts are statistically different (P 0.05; P-values obtained from MANOVA, n = 48, for raw or cooked samples). Superscripts obtained by means of the Scheffe test ( = 0.05). XY Global means for the same response and the same kind of meat (raw or cooked) bearing no common superscripts are statistically different (P 0.05; P-values obtained from MANOVA n = 144, for raw or cooked samples). Superscripts obtained by means of the Scheffe test ( = 0.05) indicate the inuence of the storage time on global means. 1 AA = ascorbic acid; COP = cholesterol oxidation products; LHP = lipid hydroperoxide; TA = tocopheryl acetate. Determined COP were: cholestanetriol, cholesterol-5,6-epoxide; cholesterol-5,6-epoxide, 7-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol. 2 LHP values expressed as mg cumene hydroperoxide/kg of sample, TBA values as milligram of malondialdehyde per kilogram of sample, and -tocopherol and COP content (mg/kg of sample). *Denotes a statistically signicant difference between global means for raw and cooked samples (P 0.05). P-values obtained from MANOVA for all raw and cooked samples together (n = 96).

EFFECT OF DIET ON CHICKEN MEAT OXIDATION

TABLE 3. Fatty acid composition, expressed as compensated area normalization (%), and cholesterol content (mg/100 g of sample) of raw and recently cooked dark meat from chickens fed diets containing different fat sources Raw meat BT Total SFA Total MUFA C18:2 n-6 C18:3 n-6 C20:2 n-6 C20:3 n-6 C20:4 n-6 C22:4 n-6 C22:5 n-6 Total n-6 PUFA C18:3 n-3 C18:4 n-3 C20:4 n-3 C20:5 n-3 C22:5 n-3 C22:6 n-3 Total n-3 PUFA Total PUFA Cholesterol
1 2 2 1

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Cooked meat LO 20.38 33.39 20.73 0.06 0.12 0.13 0.27 0.04 0.01 21.36 23.44 0.24 0.12 0.47 0.47 0.16 24.90 46.26 97.65 BT 34.53 47.68 15.82 0.12 0.13 0.14 0.44 0.12 0.03 16.80 0.89 0.02 0.01 0.02 0.07 0.04 1.05 17.85 ND4 SO 22.97 33.63 40.67 0.33 0.25 0.27 0.87 0.26 0.08 42.74 0.64 0.02 0.02 0.01 0.03 0.02 0.74 43.47 ND OSO 22.43 35.22 38.40 0.29 0.22 0.24 0.77 0.21 0.06 40.19 1.89 0.04 0.02 0.03 0.08 0.04 2.10 42.29 ND LO 20.30 32.70 20.87 0.05 0.12 0.15 0.26 0.03 0.01 21.50 24.04 0.24 0.13 0.47 0.48 0.17 25.53 47.03 ND

SO 22.62 34.96 39.69 0.32 0.23 0.25 0.92 0.27 0.08 41.75 0.63 0.02 TR3 0.01 0.04 0.02 0.72 42.47 98.93

OSO 22.31 35.25 38.51 0.26 0.22 0.26 0.89 0.23 0.06 40.43 1.83 0.04 0.02 0.03 0.09 0.04 2.05 42.48 96.09

34.41 47.29 16.09 0.16 0.13 0.15 0.56 0.14 0.04 17.26 0.88 0.03 0.01 0.02 0.08 0.05 1.07 18.34 99.61

Dietary fat source (BT = beef tallow; SO = sunower oil; OSO = oxidized sunower oil; LO = linseed oil). SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids. 3 TR = traces. 4 ND = not determined.

effect by reducing free transition metals such as Fe(III) or Cu(II) to lower valence states [Fe(II) and Cu(I)], at which the catalyst is more active in decomposing LHP to free radicals. In contrast, at high concentrations, AA shows an antioxidant effect because its ability to scavenge oxygen and lipid free radicals predominates (Decker and Xu, 1998; Frankel, 1998). This observation has been made when AA is an endogenous meat component and when it has been added postmortem. In the latter case, up to 200 to 300 mg of AA/kg of meat results in a prooxidant activity, whereas at higher concentrations, AA acts as an antioxidant (Roozen, 1987; Mielche and Bertelsen, 1994; Decker and Xu, 1998; Frankel, 1998). In addition, ascorbate may also release iron from ferritin, membrane lipids, and insoluble protein promoting lipid oxidation (Decker and Welch, 1990; Decker et al., 1993). Furthermore, the observation that AA showed a signicant activity only in raw samples could be attributed to the low concentrations of AA in meat as a result of the small quantities of AA usually found in chicken muscle tissues proceeding from self-synthesis (40 to 65 mg/kg, as reviewed by Pardue and Thaxton, 1986) and of the low supplementation applied in this study. Moreover, in our study, broilers were slaughtered after 18 h of feed withdrawal, and plasma AA levels decrease rapidly after the removal of dietary supplementation (Pardue and Thaxton, 1986). Furthermore, this low amount of AA present in raw meat may be easily degraded during cooking, leading to the lack of prooxidant effect observed in cooked samples. The degradation of AA during cooking can be due to the fact that AA is readily oxidized by heat in the presence of metal ions. This process occurs even when only residual oxygen is present in the food package (Greg-

ory, 1996). In addition, in systems with low levels of oxygen, such as vacuum-packaged samples, anaerobic degradation of AA can take place, and trace-metal catalysis of this reaction has been demonstrated (Gregory, 1996).

Interactions Between Dietary Factors

In raw samples, interaction between dietary fat source and AA supplementation was signicant for COP content (only determination at 7 mo was performed) and for TBA values at any storage time. AA resulted in a prooxidant effect when an unsaturated fat (LO, SO, or OSO), especially LO, was fed (Figure 2). This effect may be caused by the higher PUFA content in meats from these diets (Table 3), which rendered them more labile to prooxidant agents. In contrast, AA tended to prevent oxidation in meat from saturated diets (supplemented with BT). In raw samples, interaction between dietary fat source and -TA supplementation was signicant for all oxidative parameters except for LHP values at 0 and 3.5 mo of storage, although a similar trend was observed. -TA supplementation canceled the inuence of the dietary fat, resulting in no signicant differences between samples from animals fed -TA-supplemented diets from the different fat sources. In contrast, when this supplement was not added, samples from unsaturated diets gave higher oxidation values (Figure 3), which means that basal level of this compound in feed is insufcient to protect enriched-PUFA meats from oxidation. Furthermore, -tocopherol content in muscle from -TA-supplemented diets was higher for BT and SO than for LO or OSO groups (only signicant in samples stored for 3.5 mo, although a similar trend was observed at the other time

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points) (Figure 3), but oxidation of these groups was not different, which indicates that feed supplementation with 225 mg of -TA/kg was more than enough to protect raw meat with a high PUFA content against oxidation. Cooked samples showed behavior similar to raw samples (Figure 4), although this interaction was not signicant for TBA and COP values at 3.5 or 7 mo (Table 2). Despite that meat from LO diets showed the greatest decrease in TBA values due to -TA supplementation, oxidation values were not as low as those from the other fat sources. In addition, at 0 mo, -tocopherol in raw samples from LO diets was similar to that of samples from BT and OSO groups. Similarly, Ahn et al. (1995) reported that mixed tocopherols (200 mg/kg of feed) did not reduce TBA values of vacuum-packaged cooked chicken meat from a diet containing full-fat ax seeds to those of samples from a diet containing corn-soybean meal. In contrast, -TA supplementation reduced LHP values to the same level, regardless of the dietary fat source (Figure 4). Interaction between -TA and AA was only signicant for TBA values from raw samples (at any storage time).

In the absence of -TA, AA resulted in a prooxidant activity, which was canceled by -TA supplementation (Figure 5). It is noteworthy that, for both raw and cooked samples, and for all the storage periods and parameters studied, no differences were found between samples from -TA- and from -TA-plus-AA-supplemented groups. Accordingly, AA supplementation had no inuence on -tocopherol content of meat (Table 2), which was also observed by Lauridsen et al. (1997b) in breast and leg meat from chickens fed different doses of AA (0, 210, 420 or 840 mg/kg of feed).

Inuence of Cooking
Cooked samples showed signicantly higher TBA and COP values (Table 2). Cooking-related oxidation has been reported in the literature for different cooking methods (Love and Pearson, 1974; Pikul et al., 1984; King et al., 1995; Paniangvait et al., 1995; Kesava Rao et al., 1996; Kowale et al., 1996; Galvin et al., 1997; Maraschiello et al., 1998) and has been attributed to various factors. Between them, protein denaturation, which can lead to the loss of antioxidant enzyme activity (e.g., inactivation of catalase and glutathione peroxidase) or the release of iron from metalloproteins (mainly myoglobin); disruption of cell membranes, which brings PUFA and cholesterol into contact with prooxidants; transformation of myoglobin from an antioxidant to a prooxidant species; and thermal decomposition of hydroperoxides to prooxidant species, such as alkoxyl and hydroxyl radicals (Rhee, 1988; Jadhav et al., 1996; Decker and Xu, 1998; Frankel, 1998). Also the loss of endogenous -tocopherol content observed in cooked samples compared with the raw ones (Table 2) may be related to the higher oxidation in cooked samples.

Interaction Between Cooking and Dietary Factors

Interaction between cooking and dietary fat source was always signicant, except for LHP values at 3.5 mo of storage. After cooking (0 mo of storage), the highest difference in TBA values between cooked and raw samples corresponded to meat from LO diets, and the lowest to that from BT diets (Table 2). This higher susceptibility to cooking related oxidation in chicken leg meat from unsaturated diets has also been observed by other authors (Ruiz et al., 1999) and has been attributed to the higher PUFA content of these samples (Table 3). Rhee et al. (1996) compared the lipid oxidation potential of beef, chicken and pork, and concluded that the amount of PUFA was the main determinant of interspecies differences in lipid oxidation rate in cooked meats. Furthermore, Pikul et al. (1984) stated that the initial concentration of TBA-reactive substances in raw chicken meat is a key factor that determines the nal content of those compounds in cooked meat. In addition, cooking had a greater inuence on TBA values of samples from OSO than SO diets, which was also reported by Galvin et al. (1997) for breast and leg meat.

FIGURE 2. TBA values and total COP content of raw dark chicken meat samples stored at 20 C for 7 mo as affected by dietary ascorbic acid (AA) (110 mg/kg) and fat supplementation (6%). BT = beef tallow; COP = cholesterol oxidation products; LO = linseed oil; MDA = malondialdehyde; OSO = oxidized sunower oil; SO = sunower oil.

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FIGURE 3. Lipid hydroperoxide (LHP) and TBA values and total cholesterol oxidation products (COP) and -tocopherol content of raw dark chicken meat samples stored at 20 C for 7 mo as affected by dietary -tocopherol acetate (-TA) (225 mg/kg) and fat supplementation (6%). BT = beef tallow; CHP = cumene hydroperoxide; LO = linseed oil; MDA = malondialdehyde; OSO = oxidized sunower oil; SO = sunower oil.

On the other hand, LHP values from samples at 0 mo of storage were signicantly lower in cooked than in raw meat. This was evident for all fat sources, although the lower decrease, which was not signicant, corresponded to meat samples from LO diets (Table 2). This result could be because, during cooking, destruction of LHP to secondary oxidation products predominates over the formation of new LHP molecules. This unbalance is less marked in samples from unsaturated fat sources, which would be more prone to LHP formation. In contrast, at 3.5 and 7 mo of storage, unsaturated diets resulted in cooked samples with higher LHP values than the raw ones (Table 2), because prooxidant conditions favored by cooking involved greater LHP formation during storage of samples with a higher unsaturated fatty acid prole. In addition, it must be remembered that the FOX method used to measure LHP values is, to a certain extent, an induced method (Grau et al., 2000c). In this case, FOX reaction was incubated for 4 h and, during this time, lipids from the sample undergo some oxidation that may be enhanced by the prooxidants released during cooking. While AA did not affect sample oxidation during cooking, -TA supplementation reduced it (Table 2). For all oxidative parameters (TBA, LHP, and COP), oxidation induced by cooking was signicantly lower in -TA-supplemented groups (Table 2). Oxidative stability toward cooking, given by -TA, has been also described in terms of TBA values by other authors (King et al., 1995; Galvin et al., 1997). In addition, at 0 mo of storage, meat samples from diets without -TA supplementation gave lower LHP values after cooking (Table 2), which could be attrib-

uted to the above-mentioned decomposition of LHP to secondary oxidation products during the cooking procedure (Frankel, 1998). In contrast, in samples from supplemented diets, LHP values did not change signicantly after cooking, because initial LHP values were much lower and, consequently, thermal decomposition of LHP was much less important.

Inuence of the Storage Time and Its Interaction with Other Factors
Oxidation of samples was inuenced by storage. However, the behavior of LHP and TBA values from raw or cooked samples was different (Figure 6). In raw samples, LHP values signicantly decreased during the rst 3.5 mo of storage. Besides, TBA values tended to increase during the whole period, although nonsignicantly. Such an increase in TBA values during frozen or chilled storage has also been reported for raw samples packaged in air (Pikul et al., 1984, 1989; Rhee et al., 1996). Differences in the magnitude of this increase [from 4.9 times after 6 mo in the study of Pikul et al. (1989) to 1.2 after 7 mo in the present study] could be attributed to the protective effect of vacuum-packaging (assessed in chicken leg meat by Ang and Huang, 1993). In contrast, in cooked samples, LHP signicantly increased after the rst 3.5 mo, and TBA values decreased (Figure 6). For both parameters, changes during the second period (3.5 to 7 mo) were not signicant. This decrease in TBA values was also observed by other authors (King et al., 1995; Galvin et al., 1997) in non-vacuum-

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packaged raw and cooked chicken meat samples and it may be due to the reaction of MDA with proteins or to MDA polymerization reactions (Buttkus, 1967; Whang et al., 1986; Esterbauer et al., 1991; Aubourg, 1993; Wen et al., 1996). The different behavior of LHP values of raw and cooked samples during storage could be explained by the

FIGURE 5. TBA values of raw dark chicken meat samples stored at 20 C for 0, 3.5, or 7 mo as affected by dietary ascorbic acid (AA) and -tocopheryl acetate (-TA) supplementation (110 and 225 mg/kg of feed, respectively).

prooxidant conditions induced by cooking, as previously explained (such as liberation of iron and disruption of cellular membranes), which led to a higher LHP production through the storage and during the incubation of the FOX reaction (Grau et al., 2000c). According to this, Rhee et al. (1996) found a higher content of nonheme iron in cooked chicken leg meat compared with raw. Furthermore, this nonheme iron increased during storage of the meat at 20 C from 0 to 2.5 mo, but it remained steady from 2.5 to 5 mo. This agrees with the fact that LHP values in cooked samples increased signicantly only during the rst period of storage (Table 2). In addition, changes in

FIGURE 4. Lipid hydroperoxide (LHP) and TBA values, and tocopherol content of cooked dark chicken meat samples at 0 mo of storage at 20 C as affected by dietary -tocopherol acetate (-TA) (225 mg/kg) and fat supplementation (6%). BT = beef tallow; CHP = cumene hydroperoxide; LO = linseed oil; MDA = malondialdehyde; OSO = oxidized sunower oil; SO = sunower oil.

FIGURE 6. Evolution of lipid hydroperoxide (LHP) and TBA values of raw or cooked dark chicken meat as inuenced by storage period at 20 C. CHP = cumene hydroperoxide; MDA = malondialdehyde.

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nonheme iron of raw leg meat during the 5 mo of frozen storage were not signicant (Rhee et al., 1996). In contrast, Kanner et al. (1988) described an increase in free iron in raw turkey and chicken meat (dark and white muscles) during storage at 4 C for 3 or 7 d, which was claimed to be important in lipid oxidation in chilled stored meat. Endogenous -tocopherol of raw or cooked samples did not change throughout the 7 mo of frozen storage, which justies the nding that supplemented samples were highly protected from oxidation during the whole study (Table 2). Stability of -tocopherol during frozen storage has been previously described by King et al. (1995). Several studies also reported benecial effects of dietary tocopherols on chilled or frozen storage stability of chicken, turkey, and pork raw or cooked meat (reviewed by Jensen et al., 1998). In addition, the protective role of endogenous tocopherols throughout storage seems to be additive to other preservation methods such as vacuum-packaging (Jensen et al., 1998). On the other hand, dietary fat source or AA supplementation did not inuence meat stability during storage. Other authors reported that meat samples from chickens fed diets supplemented with unsaturated fatty acids were more susceptible toward oxidation during chilled (Lin et al., 1989a; Ruiz et al., 1999) or frozen storage (Lin et al., 1989a). However, those studies were carried out in nonvacuum-packaged samples, which means an increased amount of oxygen, thus favoring oxidation (Ang and Huang, 1993). In addition, Ahn et al. (1993) reported that total lipid and fatty acid composition of turkey patties affected lipid oxidation only if oxygen was freely accessible to the patties during storage. The inuence of AA during storage has been poorly studied. Lauridsen et al. (1997b) also reported a lack of effect of different doses of AA (210 to 840 mg/kg feed) on the oxidative stability of breast or thigh raw chicken meat under chill (4 C, 10 d) or freezer storage (12 C, 9 mo).

Correlations Between Results from the Different Methods

Correlations between results are stated in Tables 4 and 5. Only TBA and COP values for cooked samples failed to show a signicant correlation. Positive correlation between COP content and TBA values has been reported in cooked samples from chicken and turkey (Park and Addis, 1987; Galvin et al., 1998), beef (Park and Addis, 1987; de Vore, 1988), and pork (Monahan et al., 1992). However, in those works, samples had been trimmed of extramuscular fat, cooked after grinding, non-vacuumpackaged, and stored at 4 C (0 to 12 d). In contrast, our samples containing meat plus skin were ground after cooking, vacuum-packaged, and stored at 20 C for 7 mo. These differences may account for the disagreement in the results. Moreover, for both raw and cooked samples, there was always a highly signicant negative correlation between oxidative parameters and -tocopherol content. Negative correlation between TBA values and plasma or muscular -tocopherol has also been reported for chickens fed diets containing fresh or heated SO with no -TA supplementation (Sheehy et al., 1994), and SO or lard supplemented with 200 mg/kg of -TA (Marashiello et al., 1999). In addition, several studies showed that high levels of tissue or plasma TBA or COP values always correspond to low levels of endogenous -tocopherol (Lin et al., 1989a; Sheehy et al., 1993; Galvin et al., 1997; Ruiz et al., 1999). To sum up, despite that TBA and COP values are not correlated in cooked samples, all oxidative parameters are inversely correlated with -tocopherol content, which allows the conclusion that -tocopherol is a key factor in avoiding the formation of fatty acid and cholesterol oxidation products. Furthermore, the negative correlation between TBA values and -tocopherol content was more pronounced

TABLE 4. Correlations between oxidative parameters and -tocopherol content in raw dark chicken meat samples Storage time Oxidation values1 TBA-LHP TBA-COP LHP-COP TBA--tocopherol LHP--tocopherol COP--tocopherol
1 2

0 mo (n = 48) 0.42042 (0.0029) 3 0.4863 (0.0005) 0.9477 (<0.00005)

3.5 mo (n = 48) 0.5149 (0.0002) 0.5331 (0.0001) 0.9306 (<0.00005)

7 mo (n = 48) 0.5928 (<0.00005) 0.6466 (0.0001) 0.8320 (<0.00005) 0.5842 (<0.00005) 0.9329 (<0.00005) 0.7994 (<0.00005)

Whole period (n = 144) 0.4931 (<0.00005) 0.5304 (<0.00005) 0.9287 (< 0.00005)

COP = cholesterol oxidation products; LHP = lipid hydroperoxide values; TBA = 2-thiobarbituric acid values. Pearsons correlation coefcient. P-values are stated in parentheses. 3 COP values were only determined in samples stored for 7 mo.

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TABLE 5. Correlations between oxidative parameters and -tocopherol content in cooked dark chicken meat samples Storage time Oxidation values1 TBA-LHP TBA-COP LHP-COP TBA--tocopherol LHP--tocopherol COP--tocopherol
1 2

0 mo (n = 48) 0.62122 (<0.00005) 3 0.5236 (0.0001) 0.9056 (<0.00005)

3.5 mo (n = 48) 0.6204 (<0.00005) 0.5845 (<0.00005) 0.9368 (<0.00005)

7 mo (n = 48) 0.6250 (<0.00005) NS 0.7048 (<0.00005) 0.5812 (<0.00005) 0.9191 (<0.00005) 0.7260 (<0.00005)

Whole period (n = 144) 0.5953 (<0.00005) 0.5610 (<0.00005) 0.9094 (<0.00005)

COP = cholesterol oxidation products; LHP = lipid hydroperoxide values; TBA = 2-thiobarbituric acid values. Pearsons correlation coefcient. P-values are stated in parentheses. 3 COP values were only determined in samples stored for 7 mo.

after 3.5 or 7 mo of storage than at 0 mo, and in cooked than in raw samples (Tables 4 and 5), which indicates that the protective effect of the antioxidant is more relevant when these processing operations are involved.

REFERENCES
Addis, P. B., P. W. Park, F. Guardiola, and R. Codony, 1996. Analysis and health effects of cholesterol oxides. Pages 199 240 in: Food Lipids and Health. R. E. McDonald and D. B. Min, ed. Marcel Dekker, New York, NY. Ahn, D. U., A. Ajuyah, F. H. Wolfe, and J. S. Sim, 1993. Oxygen availability affects in prooxidant catalyzed lipid oxidation of cooked turkey patties. J. Food Sci. 58:278282. Ahn, D. U., F. H. Wolfe, and J. S. Sim, 1995. Dietary -linolenic acid and mixed tocopherols, and packaging inuences on lipid stability in broiler chicken breast and leg muscle. J. Food Sci. 60:10131018. Ajuyah, A. O., D. U. Ahn, R. T. Hardin, and J. S. Sim, 1993. Dietary antioxidants and storage affect chemical characteristics of -3 fatty acid enriched broiler chicken meats. J. Food Sci. 58:4346, 61. Andrews, J. S., W. H. Grifth, J. F. Mead, and R. A. Stein, 1960. Toxicity of air-induced soybean oil. J. Nutr. 70:199210. Ang, C. Y. W., and Y. W. Huang, 1993. Internal temperature and packaging system affect stability of cooked chicken leg patties during refrigerated storage. J. Food Sci. 58:265269, 277. Aubourg, S. P., 1993. Review: Interaction of malondialdehyde with biological moleculesNew trends about reactivity and signicance. Int. J. Food Sci. Technol. 28:323335. Bartov, I., 1977. Lack of effect of dietary ascorbic acid on stability of carcass fat and meat of broilers. Br. Poult. Sci. 18:553555. Benedict, R. C., F. D. Strange, and C. E. Swift, 1975. Effect of lipid antioxidants on the stability of meat during storage. J. Agric. Food Chem. 23:167173. Botsoglou, N. A., D. J. Fletouris, G. E. Papageorgiou, V. N. Vassilopoulos, A. J Mantis, and A. G. Trakatellis, 1994. Rapid, sensitive, and specic thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples. J. Agric. Food Sci. 42:19311937. Bou, R., F. Guardiola, A. Grau, S. Grimpa, A. Manich, A. Barroeta, and R. Codony, 2001. Inuence of dietary fat source, -tocopherol, and ascorbic acid supplementation on sensory quality of dark chicken meat. Poultry Sci. 80:800807. Brown, A. J., and W. Jessup, 1999. Oxysterols and atherosclerosis. Atherosclerosis 142:128. Bunyan, J., J. Green, E. A. Murrell, A. T. Dipolck, and M. A. Cawhorne, 1968. On the postulated peroxidation of unsaturated lipids in the tissues of vitamin E-decient rats. Br. J. Nutr. 22:97110.

Conclusions
Supplementation of broilers diets with -TA (225 mg/ kg of feed) resulted in effective protection of meat from fatty acid and cholesterol oxidation, regardless of the dietary fat source (BT, SO, OSO, or LO). On the contrary, AA supplementation (110 mg/kg) was clearly useless and showed no synergism with -TA. Combining vacuumpackaging and dietary -TA supplementation seems a feasible approach to greatly reduce fatty acid and cholesterol oxidation of raw or cooked chicken meat during frozen storage (7 mo at 20 C). In addition, the results demonstrate that, in meats, the oxidation values obtained by means of different analytical methods greatly depend on several factors, such as processing and storage conditions, unsaturation degree of the sample, and other intrinsic features (i.e., endogenous natural antioxidants or free iron content). Consequently, it is very difcult to compare oxidation values from different studies.

ACKNOWLEDGMENTS
A. Jordan, C. Ferrer, D. Alonso, and M. Erra (all from University of Barcelona) helped in laboratory work, and S. Lopez-Ferrer (UAB) in breeding animals. HofffmanLa Roche Ltd. provided -TA and AA. Cuits provided slaughter and processing facilities. This work was supported in part by research grants from the Comissio Interdepartamental de Recerca i Inno ` vacio Tecnologica (CIRIT), the Comision Interministerial de Ciencia y Tecnologa (CICYT), and the Instituto Danone.

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