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Inhibition of lipid oxidation in long-term frozen stored chicken meat by dietary oregano essential oil and α-tocopheryl acetate supplementation
Food Research International 36 (2003) 207–213 www.elsevier.com/locate/foodres
Inhibition of lipid oxidation in long-term frozen stored chicken meat by dietary oregano essential oil and a-tocopheryl acetate supplementation N.A. Botsogloua,*, D.J. Fletourisb, P. Florou-Paneria, E. Christakia, A.B. Spaisa a Laboratory of Nutrition, School of Veterinary Medicine, Aristotle University, 54006 Thessaloniki, Greece Laboratory of Milk Hygiene and Technology, School of Veterinary Medicine, Aristotle University, 54006 Thessaloniki, Greece
b
Received 18 October 2001; accepted 28 February 2002
Abstract The antioxidative effect of dietary oregano essential oil and -tocopheryl acetate supplementation on susceptibility of chicken breast and thigh muscle meat to lipid oxidation during frozen storage at 20 C for 9 months was examined. Day-old chickens (n=80) were randomly divided into four groups, and fed a basal diet containing 30 mg -tocopheryl acetate kg 1 feed as control, or basal diet plus 200 mg -tocopheryl acetate kg 1 feed, or basal diet plus 50 or 100 mg oregano essential oil kg 1 for 38 days prior to slaughter. Lipid oxidation was assessed by monitoring malondialdehyde (MDA) formation with third-order derivative spectrophotometry, after zero and 7 days of refrigerated storage at 4 C following 1, 3, 6 and 9 months of frozen storage. Results clearly demonstrated that all dietary treatments had a major impact on the oxidative stability of broiler meat. Dietary oregano essential oil supplementation at the level of 100 mg kg1 feed was significantly (P40.05) more effective in reducing lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and control, but less effective (P40.05) compared with -tocopheryl acetate supplementation. Thigh muscle was found to be more susceptible to oxidation compared to breast muscle, although the former contained -tocopherol at markedly higher levels. Mean -tocopherol levels in muscle samples decreased during the frozen storage, the decrease being sharper between 1–3 months and 3–6 months of frozen storage for breast and thigh muscle samples, respectively. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Oregano essential oil; -Tocopherol; Lipid oxidation; Frozen chicken meat; Dietary supplementation
1. Introduction Herbs and spices have been used for many centuries to improve the sensory characteristics and to extend the shelf-life of foods (Shahidi, Janitha, & Wanasundara, 1992). As a result, considerable research has been carried out on the assessment of the antioxidant activity of many herbs, spices and their extracts when added in a variety of foods and food model systems. Particular emphasis was given to herbs of the Labiatae family, particularly rosemary, sage and oregano, which have been reported to possess substantial antioxidant activity (Economou, Oreopoulou, & Thomopoulos, 1991; Man & Jaswir, 2000; Yanishlieva & Marinova, 1995).
* Corresponding author. Fax: +30-31-999967. E-mail address: [email protected] (N.A. Botsoglou).
Oregano, a characteristic spice of the Mediterranean cuisine obtained by drying leaves and flowers of Origanum vulgare subsp. hirtum plants, is well known for its antioxidative activity (Economou et al., 1991). Carvacrol and thymol, the two major phenols that constitute about 78–82% of the essential oil, are principally responsible for this activity (Adam, Sivropoulou, Kokkini, Lanaras, & Arsenakis, 1998; Yanishlieva, Marinova, Gordon, & Raneva, 1999). A high amount of carvacrol (79.58%) is frequently observed in several Greek wild populations of this taxon (Sivropoulou, Papanikolaou, Nikolaou, Kokkini, Lanaras, & Arsenakis, 1996) but, in some cases, thymol, instead of carvacrol, appears as the major component of the Greek oregano essential oil (Adam et al., 1998; Kokkini, 1994; Vokou, Kokkini, & Bessiere, 1993). In addition, other minor constituents such as -terpinene and p-cymene, two monoterpene hydrocarbons that constitute about 5
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and 7% of the total oil, respectively, also contribute to this activity but their contribution is uncertain, as it is the effect of all the constituents working together. The dry leaves and flowers of oregano, its extracts with organic solvents, and the essential oil obtained by a steam-distillation process, have all been reported to inhibit lipid oxidation when added in various food systems (Abdalla & Roozen, 2001; Bishov, Masuoka, & Kapsalis, 1977), lard (Economou et al., 1991; Lagouri, Blekas, Tsimidou, Kokkini, & Boskou, 1993; Milos, Mastelic, & Jerkovic, 2000; Vekiari, Oreopoulou, Tzia, & Thomopoulos, 1993), and mackerel oil (Tsimidou, Papavergou, & Boskou 1995), respectively. However, all of these results were obtained from in vitro experiments and do not relate to dietary supplementation. Previous studies have shown that dietary supplementation with extracts of rosemary and sage (Lopez-Bote, Gray, Gomaa, & Flegal, 1998), tea catechins (Tang, Kerry, Sheehan, Buckley, & Morrissey, 2001), or -tocopheryl acetate (Jensen, Lauridsen, & Bertelsen, 1998; Jensen, Skibsted, Jakobsen, & Bertelsen, 1995; O’Neill, Galvin, Morrissey, & Buckley, 1998; Sheehy, Morrissey, & Fynn, 1993) could improve the oxidative stability of raw and precooked broiler meat products during refrigerated or long-tern frozen storage. Dietary supplementation may be a simple and convenient strategy to introduce a natural antioxidant into meat. Special processing is not required by this strategy, whereas the dietary supplementation allows uniform incorporation of the antioxidant into phospholipid membranes where it can effectively inhibit the oxidative reactions at their localized sites (De Winne & Dirinck, 1996; Gray & Pearson, 1987; Lauridsen, Buckley, & Morrissey, 1997; Morrissey, Buckley, Sheehy, & Monahan, 1994). The present study was designed to evaluate the effect of dietary oregano essential oil and -tocopheryl acetate supplementation on susceptibility of raw breast and thigh chicken muscle to lipid oxidation during long-term frozen storage.
2. Materials and methods 2.1. Chemicals Butylated hydroxytoluene, 2-thiobarbituric acid (TBA), -tocopherol reference standard, and 1,1,3,3-tetraethoxypropane, the precursor of malondialdehyde (MDA), were obtained from Sigma Chemical Co. (St. Louis, MO). Trichloroacetic acid, petroleum ether, pyrocatechol, hexane, methanol, acetonitrile, hydrochloric acid, and sodium hydroxide from Merck (Darmstadt, Germany). All chemicals used were of analytical-grade. -Tocopheryl acetate used for feed supplementation was obtained from Roche Products Ltd. (Hertfordshire, UK), while oregano essential oil from Meriden Animal
Health Ltd. (UK). Oregano essential oil was in form of a powder commercially known as Orego-Stim (Ecopharm Hellas, SA, Kilkis, Greece) that contained 5% oregano essential oil and 95% natural feed grade inert carrier. The major components of the essential oil were carvacrol (79.6%), p-cymene (8.7%), thymol (2.5%), and g-terpinene (2.1%). 2.2. Animals and diets Day-old Cobb 500 (n=80) broiler chickens purchased from a local commercial hatchery were randomly assigned into four groups of 20 birds each. Chickens within the control group were given a commercial diet containing a basal level of 30 mg -tocopheryl acetate kg 1 (Table 1). The diets given to the remaining three groups of chickens were also based on this basal diet, but were further supplemented with 50 mg or 100 mg oregano essential oil or 200 mg -tocopheryl acetate kg 1 feed. 2.3. Sampling procedure and lipid oxidation experiment Following feeding for 38 days, broilers were slaughtered under commercial conditions. Individual carcasses from six birds per group were trimmed for breast (pectoralis major) and thigh (gastrocnemius interna) muscles by removing skin, bones and connective tissue. Following trimming, all breast and thigh samples were individually sliced, vacuum packaged, and stored at 20 C for 1, 3, 6 and 9 months. Six breast and thigh muscle subsamples from each dietary treatment, at the time of Table 1 Composition of basal diet (g kg 1) Corn, grains Herring meal Soybean meal Soybean oil dl-Methionine Avatec (15% in lasalocid) Flavomycin Limestone pulverized Dicalcium phosphate Sodium chloride, iodized Sodium bicarbonate Binder Natuphos (phytase) Vitamin premixa Trace-mineral premixb a
464.1 13.0 425.3 60.6 2.5 0.5 0.3 11.5 14.1 2.9 2.5 1.4 0.1 1.2 0.5
Supplying per kg feed: 4.82 mg all-trans retinol acetate, 62.5 mg cholecalciferol, 30 -tocopheryl acetate, 3 mg menadione sodium bisulphite, 1 mg thiamine hydrochloride, 5 mg riboflavin, 3 mg pyridoxine hydrochloride, 0.02 mg cyanocobalamine, 30 mg niacin, 10 mg pantothenic acid, 0.8 mg folic acid, 0.05 mg biotin, 10 mg ascorbic acid, and 480 mg choline chloride. b Supplying per kg feed: 100 mg Zn, 120 mg Mn, 20 mg Fe, 15 mg Cu, 0.2 mg Co, 1 mg I, and 0.3 mg Se.
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analysis, were thawed overnight at 4 C and submitted to malondialdehyde analysis for determination of lipid oxidation. Following overnight thawing at 4 C, other six breast and thigh muscle subsamples were wrapped in transparent oxygen permeable PVC wrap (6000–8000 cm3/m2/24 h at STP), placed in a refrigerated cabinet at 4 C, and oxidative changes were monitored after 0, 3, 6 and 9 days of refrigerated storage.
column (150.46 cm) contained Nucleosil C18, 5 mm, whereas the mobile phase was 100% methanol and delivered in the system at a flow rate of 1.5 ml/min. Monitoring of the column effluents was performed at 292 nm. Detector signals were quantified using peak heights and a standard calibration curve.
2.4. Determination of lipid oxidation
All data were subjected to analysis of variance (ANOVA) The statistical significance of the differences between mean malondialdehyde values were analysed by repeated measures and Tukey tests in the general linear model of SPSS statistical package (SPSS 10.05, SPSS Ltd., Woking, Surrey, UK).
Lipid oxidation was assessed on the basis of the concentration of malondialdehyde in the examined samples according to a derivative spectrophotometric method previously developed by some of the authors (Botsoglou, Fletouris, Papageorgiou, Vassilopoulos, Mantis, & Trakatellis, 1994). In brief, samples were thoroughly homogenized (Polytron homogenizer, PCU, Switzerland) in presence of 8 ml of 5% aqueous trichloroacetic acid and 5 ml of 0.8% butylated hydroxytoluene in hexane, and the mixture was centrifuged. A 2.5-ml aliquot from the bottom layer was mixed with 1.5 ml of 0.8% aqueous 2-thiobarbituric acid to be further incubated at 70 C for 30 min. Following incubation, the mixture was submitted to conventional spectrophotometry (Shimadzu, Model UV-160A, Tokyo, Japan) in the range of 400–650 nm. Third-order derivative spectra were produced by digital differentiation of the normal spectra using a derivative wavelength difference setting of 21 nm. The concentration of malondialdehyde (ng/g wet tissue) in analysed extracts was calculated on the basis of the height of the third-order derivative peak at 521.5 nm by referring to slope and intercept data of the computed least-squares fit of standard calibration curve prepared using 1,1,3,3-tetraethoxypropane. 2.5. Determination of -tocopherol in tissues Breast and thigh muscle samples were submitted in duplicate analysis for the determination of their -tocopherol content. Extraction of -tocopherol from the analysed tissues was carried out by a modification of the method of Botsoglou, Fletouris, Psomas, and Mantis (1998). In brief, 0.2-g sample was homogenized with 100 ml of pyrocatechol solution and 5 ml of a methanolic KOH solution, and the tube was immersed in a bath of 80 C for 15 min. Following saponification, 5 ml hexane and 1 ml water were added, the mixture was vortexmixed, centrifuged at 2000 g, and an aliquot of the upper phase was evaporated to dryness to be further reconstituted in methanol and injected into the liquid chromatograph (Gilson Medical Electronics, Villiersle-Bel, France). Liquid chromatography was carried out as described by Zapel and Csallany (1983). The chromatographic
2.6. Statistical analysis
3. Results and discussion The effect of dietary oregano essential oil supplementation on lipid oxidation of chicken breast muscle stored at 20 C for 1, 3, 6 and 9 months prior to refrigerated storage for 0 and 7 days is presented in Fig. 1 A and B, respectively. Results clearly demonstrated that the dietary treatments had a major impact on the oxidative stability of broiler meat. After 1 month of frozen storage, control meat exhibited the highest (P40.05) lipid oxidation among other groups, with an MDA content of 50.6 ng g 1 sample at day 0 of refrigerated storage (Fig. 1 A). This trend was maintained at 3, 6 and 9 months. Thus, both groups of dietary oregano essential oil and the -tocopheryl acetate group showed significantly (P < 0.05) lower lipid oxidation during the frozen storage period, with MDA values of 41.0, 32.7 and 25.5 ng g 1 sample at 3 months, 48.8, 36.8 and 25.0 ng g 1 sample at 6 months, and 70.4, 48.9 and 32.5 ng g 1 sample at 9 months, respectively, compared with the controls that were at 53.4, 60.4 and 83.2 ng MDA g 1 sample, respectively. However, dietary oregano essential oil supplementation at the level of 100 mg kg 1 feed was significantly (P < 0.05) more effective in reducing lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. The dietary administration of -tocopheryl acetate at the level of 200 mg kg 1 feed significantly (P40.05) reduced MDA values in breast samples at all time points. This is consistent with many pertinent reports (Ahn, Wolfe, & Sim, 1995; De Winne & Dirinck, 1996; Lopez-Bote et al., 1998; O’Neill et al., 1998). One study failed to show an antioxidative effect of dietary -tocopheryl acetate supplementation in frozen broilers (Jensen et al., 1995). The authors explained the lack of effect as a result of very low lipid oxidation activity together with a relatively high level of muscle -tocopherol found in control birds (Jakobsen et al., 1995).
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Fig. 1. Effect of dietary oregano essential oil supplementation at levels of 50 and 100 mg kg 1 feed as a function of frozen time, on lipid oxidation of raw chicken breast muscle compared with dietary -tocopheryl acetate supplementation at a level of 200 mg kg 1 feed and control: (A) test after initial thawing and prior to refrigerated storage; (B) test after 7 days of refrigerated storage following thawing. All data points that represent mean malondialdehyde (MDA) concentrations from six analyses are accompanied by standard deviation bars, some of which however lie within the data points.
Lipid oxidation in breast muscle at day 7 of refrigerated storage following 1, 3, 6 or 9 months of frozen storage (Fig. 1 B) was significantly (P< 0.05) higher compared with that on day 0 of refrigerated storage (Fig. 1 A). Storage at 4 C for 7 days increased considerably MDA concentrations in all samples, the values being higher in the control group and lower in the -tocopheryl acetate group (Fig. 1 B). Samples within the dietary oregano essential oil groups had a significantly (P < 0.05) lower tendency for lipid oxidation compared to the control group at all time points. Dietary supplementation with oregano essential oil at the level of 100 mg kg 1 feed was significantly (P < 0.05) more effective in reducing lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and the control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. Thigh muscle was found to be more susceptible to lipid oxidation than breast muscle. The susceptibility of chicken thigh muscle to lipid oxidation during 9 months of frozen storage is shown in Fig. 2 A. On day 0 of refrigerated storage, thigh samples frozen stored for 1 month showed a lipid oxidation pattern similar to that of the breast samples although MDA values in thigh samples from all dietary groups were always higher than those in breast samples from respective dietary treatments at all time points (Fig. 2 A). This pattern did not changed at 3, 6 or 9 months. Dietary oregano essential oil supplementation at the level of 100 mg kg 1 feed was constantly more effective (P < 0.05) in reducing lipid
oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and the control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. Lipid oxidation in thigh muscle at day 7 of refrigerated storage following 1, 3, 6 or 9 months of frozen storage (Fig. 2 B) was significantly (P < 0.05) higher compared with that on day 0 of refrigerated storage (Fig. 2 A). Thigh samples within the dietary oregano essential oil groups had a significantly (P < 0.05) lower tendency for lipid oxidation compared with samples in the control group at all time points. Dietary supplementation with oregano essential oil at the level of 100 mg kg 1 feed was significantly (P < 0.05) more effective in reducing lipid oxidation compared to the level of 50 mg oregano essential oil kg 1 feed and the control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. Comparing Fig. 1 A and 2 A with 1 B and 2 B, a distinct difference in the rate of lipid oxidation between the tested storage conditions appears. In Fig. 1 A and 2 A, where lipid oxidation refers to measurements at day 0 after 1, 3, 6 or 9 months of frozen storage, MDA values exhibited a clear lag phase by month 6, followed by a more rapid increase at longer storage times. In contrast, in Fig. 1 B and 2 B where lipid oxidation was examined at day 7 of refrigerated storage after 1, 3, 6 or 9 months of frozen storage, MDA values tended to increase rapidly by month 6, and then more gradually as storage time increased.
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Fig. 2. Effect of dietary oregano essential oil supplementation at levels of 50 and 100 mg kg 1 feed as a function of frozen time, on lipid oxidation of raw chicken thigh muscle compared with dietary -tocopheryl acetate supplementation at a level of 200 mg kg 1 feed and control: (A) test after initial thawing and prior to refrigerated storage; (B) test after 7 days of refrigerated storage following thawing. All data points that represent mean malondialdehyde (MDA) concentrations from six analyses are accompanied by standard deviation bars, some of which however lie within the data points.
This inconsistency might be attributed to the analytical method employed for measuring the extent of lipid oxidation. The basis for this measurement is the reaction between TBA and MDA during heating at acid pH to produce a chromogenic complex. MDA reactivity is influenced by several factors including its formation as an artifact of the analysis during the test itself, its occurrence in various bound forms, and the specificity of the methods used for its measurement. Hence, the term ‘‘thiobarbituric acid reactive substances’’ (TBARS) is frequently used to account for the contribution of other compounds to the TBA–MDA complex formed. MDA is a secondary lipid oxidation product formed subsequent to the hydrolysis of lipid hydroperoxides. Considering that the derivative spectrophotometric method (Botsoglou et al., 1994) applied in the present study eliminates interference from other reactive compounds since it is based on derivative spectral analysis of the red TBA–MDA complex, a lag phase would be expected to occur during early stages of frozen storage followed by a rapid increase of MDA as observed in the present study. The absence of a lag phase in Fig. 1B and 2B was probably due to earlier hydrolysis of lipid hydroperoxides during the refrigerated storage of the previously frozen samples. Using MDA content as an index of absorption of the dietary oregano essential oil constituents, this study indicates that the antioxidant compounds occurring in oregano essential oil are distributed, retained, and
remained functional in muscle. The research carried out in vivo lends also support to many reports showing that the in vitro addition of oregano to various food systems inhibits lipid oxidation (Abdalla & Roozen, 2001; Economou et al., 1991; Lagouri et al., 1993; Tsimidou et al., 1995; Vekiari et al., 1993). Considering that the greater the amount of -tocopherol deposited the better protection the muscle should have against oxidative attack, breast and thigh muscle samples were analysed for their -tocopherol content. It was found that -tocopherol levels in muscle tissues of chickens were influenced by both the supplementation level and the type of the tissue. Thus, mean -tocopherol levels in breast and thigh muscle samples (n=6 samples) of the control group differed significantly (P < 0.05), being 2.05 and 3.50 mg kg 1 of tissue, respectively. Supplementing broiler diets with 200 mg tocopheryl acetate kg 1 feed, -tocopherol concentrations in breast and thigh muscle samples increased to 8.22 and 17.80 mg kg 1 of tissue, respectively. These values compare well with those reported by other workers (Jensen et al., 1995; Lin, Gray, Asghar, Buckley, Booren, & Flegal, 1989; Jakobsen et al., 1995). The higher concentrations of -tocopherol in thigh muscle have been attributed to its better developed vascular system and its higher lipid content compared to breast muscle (Lin et al., 1989). Although thigh muscle was found to contain higher amounts of -tocopherol than breast muscle, it tended
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Table 2 Effect of frozen storage on the mean -tocopherol concentrations in breast and thigh muscles of broilers given dietary -tocopheryl acetate Months
0 1 3 6 9
Concentrationsa of -tocopherol in breast (mg kg 1)
Concentrationsa of -tocopherol in thigh (mg kg 1)
Control group (30 mg kg 1 -tocopheryl acetate in feed)
Treated group (200 mg kg 1 a-tocopheryl acetate in feed)
Control group (30 mg kg 1 -tocopheryl acetate in feed)
Treated group (200 mg kg 1 -tocopheryl acetate in feed)
2.05a0.45 2.00a0.15 1.15b0.12 1.03b0.06 0.95b0.08
8.22a 0.05 8.10a 0.45 7.22b 0.40 7.12b 0.80 7.10b 0.65
3.50a0.60 3.58a0.30 3.10a0.20 2.20b0.12 1.80b0.10
17.80a0.72 17.74a0.90 18.10a0.94 11.92b1.10 11.74b0.80
Mean values in the same column with a letter in common do not differ significantly (P>0.05). a Mean valuestandard deviation for six analyses.
to oxidize faster than the latter tissue. The higher susceptibility of thigh meat towards oxidation has been attributed to the higher absolute content of polyunsaturated fatty acids with more than two double bonds in this muscle (Jensen, Guidera, Skovgaard, Staun, & Skibsted, 1997). Breast meat has a higher percentage of these acids in the fat, but their absolute amount in thigh meat is three times higher compared with breast muscle, as the total fat content in thigh meat is approximately five times that of breast meat (Jensen et al., 1997). Apart from the content of muscle -tocopherol and the amount of readily oxidizable substrates, the high level of pro-oxidative species originating from meat myoglobin and other iron containing proteins in thigh muscle is another factor causing lower oxidative stability in thigh muscle (Rhee & Ziprin, 1987). The rate of a-tocopherol depletion in breast and thigh muscle samples during 9 months of frozen storage is shown in Table 2. -Tocopherol depletion from thigh muscle samples occurred to a greater extent between 3 and 6 months of frozen storage, whereas for breast muscle samples between 1 and 3 months of frozen storage. There have been several possible explanations for the -tocopherol depletion during frozen storage. At 20 C, some catalysts and antioxidants may be trapped in the frozen solid phase and, therefore, the antioxidant activity of the cytosolic phase may no longer function optimally (Wen, Morrissey, Buckley, & Sheeby, 1996). Moreover, lipid free radicals are soluble in the oil fraction and are more stable at low temperatures. This increased stability allows them to diffuse to longer distances and spread the reaction (Kanner, 1994). Therefore, free radicals may escape the antioxidants in the frozen aqueous phase and diffuse into the membrane lipid system, where they initiate and promote lipid oxidation. Wen et al. (1996) stated that if this occurs, the lipid-soluble -tocopherol becomes the first line of antioxidant defence and thus may be rapidly depleted.
4. Conclusion In conclusion, the results of the present study show that dietary oregano essential oil supplementation reduced lipid oxidation significantly (P< 0.05) during frozen storage and subsequent refrigeration. A dose response increase in the antioxidative capacity of broiler tissues as a result of feeding oregano essential oil was exhibited. Dietary oregano essential oil supplementation at the level of 100 mg kg 1 feed more effectively (P < 0.05) reduced lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed, but it was less effective (P < 0.05) compared with supplementation with -tocopheryl acetate. Therefore, oregano essential oil may be considered as a good alternative to -tocopheryl acetate supplementation of feeds. Presently, supplementation with oregano essential oil will increase slightly the cost of feed relatively to -tocopheryl acetate supplementation but this can be avoided with extensive cultivation of oregano plants.
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Man, Y., & Jaswir, I. (2000). Effect of rosemary and sage extracts on frying performance of refined, bleached and deodorized palm olein during deep-fat frying. Food Chemistry, 69, 307–3012. Milos, M., Mastelic, J., & Jerkovic, I. (2000). Chemical composition and antioxidant effect of glycosidically bound volatile compounds from oregano (Origanum vulgare L. ssp. Hirtum). Food Control, 71, 79–83. Morrissey, P. A., Buckley, D. J., Sheehy, P. J. A., & Monahan, F. J. (1994). Vitamin E and meat quality. Proceedings of the Nutrition Society, 53, 289–295. O’Neill, L. M., Galvin, K., Morrissey, P. A., & Buckley, D. J. (1998). Comparison of effects of dietary olive oil, tallow and vitamin E on the quality of broiler meat and meat products. British Poultry Science, 39, 365–371. Rhee, K. S., & Ziprin, Y. A. (1987). Lipid oxidation in retail beef, pork and chicken muscles as affected by concentrations of heme pigments and nonheme iron and liposomal enzymic peroxidation activity. Journal of Food Biochemistry, 11, 1–15. Shahidi, F., Janitha, P. K., & Wanasundara, P. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32, 67–103. Sheehy, P. J. A., Morrissey, P. A., & Fynn, A. (1993). Increased storage stability of chicken muscle by dietary -tocopherol supplementation. Irish Journal of Agricultural and Food Research, 32, 67– 73. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T., & Arsenakis, M. (1996). Antimicrobial and cytotoxic activities of Origanum essential oils. Journal of Agricultural and Food Chemistry, 44, 1202–1205. Tang, S. Z., Kerry, J. P., Sheehan, D., Buckley, D. J., & Morrissey, P. A. (2001). Antioxidative effect of dietary tea catechins on lipid oxidation of long-term frozen stored chicken meat. Meat Science, 57, 331–336. Tsimidou, M., Papavergou, E., & Boskou, D. (1995). Evaluation of oregano antioxidant activity in mackerel oil. Food Research International, 28, 431–433. Vekiari, S. A., Oreopoulou, V., Tzia, C., & Thomopoulos, C. D. (1993). Oregano flavonoids as lipid antioxidants. Journal of the American Oil Chemical Society, 70, 483–487. Vokou, S., Kokkini, S., & Bessiere, J. M. (1993). Geographic variation of Greek oregano (Origanum vulgare ssp. Hirtum) essential oils. Biochemistry and Systemic Ecology, 21, 287–295. Wen, J., Morrissey, P. A., Buckley, D. J., & Sheeby, P. J. A. (1996). Oxidative stability and -tocopherol retention in turkey burgers during refrigerated and frozen storage as influenced by dietary tocopheryl acetate. British Poultry Science, 37, 787–795. Yanishlieva, V., & Marinova, M. (1995). Antioxidant activity of selected species of the family Lamiaceae grown in Bulgaria. Nahrung, 39, 458–463. Yanishlieva, N. V., Marinova, E. M., Gordon, M. H., & Raneva, V. G. (1999). Antioxidant activity and mechanism of action of thymol and carvacrol in two lipid systems. Food Chemistry, 64, 59–66. Zapel, B. J., & Csallany, A. S. (1983). Determination of a-tocopherol in tissues and plasma by high-performance liquid chromatography. Analytical Biochemistry, 130, 130–146.
Inhibition of lipid oxidation in long-term frozen stored chicken meat by dietary oregano essential oil and a-tocopheryl acetate supplementation N.A. Botsogloua,*, D.J. Fletourisb, P. Florou-Paneria, E. Christakia, A.B. Spaisa a Laboratory of Nutrition, School of Veterinary Medicine, Aristotle University, 54006 Thessaloniki, Greece Laboratory of Milk Hygiene and Technology, School of Veterinary Medicine, Aristotle University, 54006 Thessaloniki, Greece
b
Received 18 October 2001; accepted 28 February 2002
Abstract The antioxidative effect of dietary oregano essential oil and -tocopheryl acetate supplementation on susceptibility of chicken breast and thigh muscle meat to lipid oxidation during frozen storage at 20 C for 9 months was examined. Day-old chickens (n=80) were randomly divided into four groups, and fed a basal diet containing 30 mg -tocopheryl acetate kg 1 feed as control, or basal diet plus 200 mg -tocopheryl acetate kg 1 feed, or basal diet plus 50 or 100 mg oregano essential oil kg 1 for 38 days prior to slaughter. Lipid oxidation was assessed by monitoring malondialdehyde (MDA) formation with third-order derivative spectrophotometry, after zero and 7 days of refrigerated storage at 4 C following 1, 3, 6 and 9 months of frozen storage. Results clearly demonstrated that all dietary treatments had a major impact on the oxidative stability of broiler meat. Dietary oregano essential oil supplementation at the level of 100 mg kg1 feed was significantly (P40.05) more effective in reducing lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and control, but less effective (P40.05) compared with -tocopheryl acetate supplementation. Thigh muscle was found to be more susceptible to oxidation compared to breast muscle, although the former contained -tocopherol at markedly higher levels. Mean -tocopherol levels in muscle samples decreased during the frozen storage, the decrease being sharper between 1–3 months and 3–6 months of frozen storage for breast and thigh muscle samples, respectively. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Oregano essential oil; -Tocopherol; Lipid oxidation; Frozen chicken meat; Dietary supplementation
1. Introduction Herbs and spices have been used for many centuries to improve the sensory characteristics and to extend the shelf-life of foods (Shahidi, Janitha, & Wanasundara, 1992). As a result, considerable research has been carried out on the assessment of the antioxidant activity of many herbs, spices and their extracts when added in a variety of foods and food model systems. Particular emphasis was given to herbs of the Labiatae family, particularly rosemary, sage and oregano, which have been reported to possess substantial antioxidant activity (Economou, Oreopoulou, & Thomopoulos, 1991; Man & Jaswir, 2000; Yanishlieva & Marinova, 1995).
* Corresponding author. Fax: +30-31-999967. E-mail address: [email protected] (N.A. Botsoglou).
Oregano, a characteristic spice of the Mediterranean cuisine obtained by drying leaves and flowers of Origanum vulgare subsp. hirtum plants, is well known for its antioxidative activity (Economou et al., 1991). Carvacrol and thymol, the two major phenols that constitute about 78–82% of the essential oil, are principally responsible for this activity (Adam, Sivropoulou, Kokkini, Lanaras, & Arsenakis, 1998; Yanishlieva, Marinova, Gordon, & Raneva, 1999). A high amount of carvacrol (79.58%) is frequently observed in several Greek wild populations of this taxon (Sivropoulou, Papanikolaou, Nikolaou, Kokkini, Lanaras, & Arsenakis, 1996) but, in some cases, thymol, instead of carvacrol, appears as the major component of the Greek oregano essential oil (Adam et al., 1998; Kokkini, 1994; Vokou, Kokkini, & Bessiere, 1993). In addition, other minor constituents such as -terpinene and p-cymene, two monoterpene hydrocarbons that constitute about 5
0963-9969/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(02)00095-9
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and 7% of the total oil, respectively, also contribute to this activity but their contribution is uncertain, as it is the effect of all the constituents working together. The dry leaves and flowers of oregano, its extracts with organic solvents, and the essential oil obtained by a steam-distillation process, have all been reported to inhibit lipid oxidation when added in various food systems (Abdalla & Roozen, 2001; Bishov, Masuoka, & Kapsalis, 1977), lard (Economou et al., 1991; Lagouri, Blekas, Tsimidou, Kokkini, & Boskou, 1993; Milos, Mastelic, & Jerkovic, 2000; Vekiari, Oreopoulou, Tzia, & Thomopoulos, 1993), and mackerel oil (Tsimidou, Papavergou, & Boskou 1995), respectively. However, all of these results were obtained from in vitro experiments and do not relate to dietary supplementation. Previous studies have shown that dietary supplementation with extracts of rosemary and sage (Lopez-Bote, Gray, Gomaa, & Flegal, 1998), tea catechins (Tang, Kerry, Sheehan, Buckley, & Morrissey, 2001), or -tocopheryl acetate (Jensen, Lauridsen, & Bertelsen, 1998; Jensen, Skibsted, Jakobsen, & Bertelsen, 1995; O’Neill, Galvin, Morrissey, & Buckley, 1998; Sheehy, Morrissey, & Fynn, 1993) could improve the oxidative stability of raw and precooked broiler meat products during refrigerated or long-tern frozen storage. Dietary supplementation may be a simple and convenient strategy to introduce a natural antioxidant into meat. Special processing is not required by this strategy, whereas the dietary supplementation allows uniform incorporation of the antioxidant into phospholipid membranes where it can effectively inhibit the oxidative reactions at their localized sites (De Winne & Dirinck, 1996; Gray & Pearson, 1987; Lauridsen, Buckley, & Morrissey, 1997; Morrissey, Buckley, Sheehy, & Monahan, 1994). The present study was designed to evaluate the effect of dietary oregano essential oil and -tocopheryl acetate supplementation on susceptibility of raw breast and thigh chicken muscle to lipid oxidation during long-term frozen storage.
2. Materials and methods 2.1. Chemicals Butylated hydroxytoluene, 2-thiobarbituric acid (TBA), -tocopherol reference standard, and 1,1,3,3-tetraethoxypropane, the precursor of malondialdehyde (MDA), were obtained from Sigma Chemical Co. (St. Louis, MO). Trichloroacetic acid, petroleum ether, pyrocatechol, hexane, methanol, acetonitrile, hydrochloric acid, and sodium hydroxide from Merck (Darmstadt, Germany). All chemicals used were of analytical-grade. -Tocopheryl acetate used for feed supplementation was obtained from Roche Products Ltd. (Hertfordshire, UK), while oregano essential oil from Meriden Animal
Health Ltd. (UK). Oregano essential oil was in form of a powder commercially known as Orego-Stim (Ecopharm Hellas, SA, Kilkis, Greece) that contained 5% oregano essential oil and 95% natural feed grade inert carrier. The major components of the essential oil were carvacrol (79.6%), p-cymene (8.7%), thymol (2.5%), and g-terpinene (2.1%). 2.2. Animals and diets Day-old Cobb 500 (n=80) broiler chickens purchased from a local commercial hatchery were randomly assigned into four groups of 20 birds each. Chickens within the control group were given a commercial diet containing a basal level of 30 mg -tocopheryl acetate kg 1 (Table 1). The diets given to the remaining three groups of chickens were also based on this basal diet, but were further supplemented with 50 mg or 100 mg oregano essential oil or 200 mg -tocopheryl acetate kg 1 feed. 2.3. Sampling procedure and lipid oxidation experiment Following feeding for 38 days, broilers were slaughtered under commercial conditions. Individual carcasses from six birds per group were trimmed for breast (pectoralis major) and thigh (gastrocnemius interna) muscles by removing skin, bones and connective tissue. Following trimming, all breast and thigh samples were individually sliced, vacuum packaged, and stored at 20 C for 1, 3, 6 and 9 months. Six breast and thigh muscle subsamples from each dietary treatment, at the time of Table 1 Composition of basal diet (g kg 1) Corn, grains Herring meal Soybean meal Soybean oil dl-Methionine Avatec (15% in lasalocid) Flavomycin Limestone pulverized Dicalcium phosphate Sodium chloride, iodized Sodium bicarbonate Binder Natuphos (phytase) Vitamin premixa Trace-mineral premixb a
464.1 13.0 425.3 60.6 2.5 0.5 0.3 11.5 14.1 2.9 2.5 1.4 0.1 1.2 0.5
Supplying per kg feed: 4.82 mg all-trans retinol acetate, 62.5 mg cholecalciferol, 30 -tocopheryl acetate, 3 mg menadione sodium bisulphite, 1 mg thiamine hydrochloride, 5 mg riboflavin, 3 mg pyridoxine hydrochloride, 0.02 mg cyanocobalamine, 30 mg niacin, 10 mg pantothenic acid, 0.8 mg folic acid, 0.05 mg biotin, 10 mg ascorbic acid, and 480 mg choline chloride. b Supplying per kg feed: 100 mg Zn, 120 mg Mn, 20 mg Fe, 15 mg Cu, 0.2 mg Co, 1 mg I, and 0.3 mg Se.
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analysis, were thawed overnight at 4 C and submitted to malondialdehyde analysis for determination of lipid oxidation. Following overnight thawing at 4 C, other six breast and thigh muscle subsamples were wrapped in transparent oxygen permeable PVC wrap (6000–8000 cm3/m2/24 h at STP), placed in a refrigerated cabinet at 4 C, and oxidative changes were monitored after 0, 3, 6 and 9 days of refrigerated storage.
column (150.46 cm) contained Nucleosil C18, 5 mm, whereas the mobile phase was 100% methanol and delivered in the system at a flow rate of 1.5 ml/min. Monitoring of the column effluents was performed at 292 nm. Detector signals were quantified using peak heights and a standard calibration curve.
2.4. Determination of lipid oxidation
All data were subjected to analysis of variance (ANOVA) The statistical significance of the differences between mean malondialdehyde values were analysed by repeated measures and Tukey tests in the general linear model of SPSS statistical package (SPSS 10.05, SPSS Ltd., Woking, Surrey, UK).
Lipid oxidation was assessed on the basis of the concentration of malondialdehyde in the examined samples according to a derivative spectrophotometric method previously developed by some of the authors (Botsoglou, Fletouris, Papageorgiou, Vassilopoulos, Mantis, & Trakatellis, 1994). In brief, samples were thoroughly homogenized (Polytron homogenizer, PCU, Switzerland) in presence of 8 ml of 5% aqueous trichloroacetic acid and 5 ml of 0.8% butylated hydroxytoluene in hexane, and the mixture was centrifuged. A 2.5-ml aliquot from the bottom layer was mixed with 1.5 ml of 0.8% aqueous 2-thiobarbituric acid to be further incubated at 70 C for 30 min. Following incubation, the mixture was submitted to conventional spectrophotometry (Shimadzu, Model UV-160A, Tokyo, Japan) in the range of 400–650 nm. Third-order derivative spectra were produced by digital differentiation of the normal spectra using a derivative wavelength difference setting of 21 nm. The concentration of malondialdehyde (ng/g wet tissue) in analysed extracts was calculated on the basis of the height of the third-order derivative peak at 521.5 nm by referring to slope and intercept data of the computed least-squares fit of standard calibration curve prepared using 1,1,3,3-tetraethoxypropane. 2.5. Determination of -tocopherol in tissues Breast and thigh muscle samples were submitted in duplicate analysis for the determination of their -tocopherol content. Extraction of -tocopherol from the analysed tissues was carried out by a modification of the method of Botsoglou, Fletouris, Psomas, and Mantis (1998). In brief, 0.2-g sample was homogenized with 100 ml of pyrocatechol solution and 5 ml of a methanolic KOH solution, and the tube was immersed in a bath of 80 C for 15 min. Following saponification, 5 ml hexane and 1 ml water were added, the mixture was vortexmixed, centrifuged at 2000 g, and an aliquot of the upper phase was evaporated to dryness to be further reconstituted in methanol and injected into the liquid chromatograph (Gilson Medical Electronics, Villiersle-Bel, France). Liquid chromatography was carried out as described by Zapel and Csallany (1983). The chromatographic
2.6. Statistical analysis
3. Results and discussion The effect of dietary oregano essential oil supplementation on lipid oxidation of chicken breast muscle stored at 20 C for 1, 3, 6 and 9 months prior to refrigerated storage for 0 and 7 days is presented in Fig. 1 A and B, respectively. Results clearly demonstrated that the dietary treatments had a major impact on the oxidative stability of broiler meat. After 1 month of frozen storage, control meat exhibited the highest (P40.05) lipid oxidation among other groups, with an MDA content of 50.6 ng g 1 sample at day 0 of refrigerated storage (Fig. 1 A). This trend was maintained at 3, 6 and 9 months. Thus, both groups of dietary oregano essential oil and the -tocopheryl acetate group showed significantly (P < 0.05) lower lipid oxidation during the frozen storage period, with MDA values of 41.0, 32.7 and 25.5 ng g 1 sample at 3 months, 48.8, 36.8 and 25.0 ng g 1 sample at 6 months, and 70.4, 48.9 and 32.5 ng g 1 sample at 9 months, respectively, compared with the controls that were at 53.4, 60.4 and 83.2 ng MDA g 1 sample, respectively. However, dietary oregano essential oil supplementation at the level of 100 mg kg 1 feed was significantly (P < 0.05) more effective in reducing lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. The dietary administration of -tocopheryl acetate at the level of 200 mg kg 1 feed significantly (P40.05) reduced MDA values in breast samples at all time points. This is consistent with many pertinent reports (Ahn, Wolfe, & Sim, 1995; De Winne & Dirinck, 1996; Lopez-Bote et al., 1998; O’Neill et al., 1998). One study failed to show an antioxidative effect of dietary -tocopheryl acetate supplementation in frozen broilers (Jensen et al., 1995). The authors explained the lack of effect as a result of very low lipid oxidation activity together with a relatively high level of muscle -tocopherol found in control birds (Jakobsen et al., 1995).
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Fig. 1. Effect of dietary oregano essential oil supplementation at levels of 50 and 100 mg kg 1 feed as a function of frozen time, on lipid oxidation of raw chicken breast muscle compared with dietary -tocopheryl acetate supplementation at a level of 200 mg kg 1 feed and control: (A) test after initial thawing and prior to refrigerated storage; (B) test after 7 days of refrigerated storage following thawing. All data points that represent mean malondialdehyde (MDA) concentrations from six analyses are accompanied by standard deviation bars, some of which however lie within the data points.
Lipid oxidation in breast muscle at day 7 of refrigerated storage following 1, 3, 6 or 9 months of frozen storage (Fig. 1 B) was significantly (P< 0.05) higher compared with that on day 0 of refrigerated storage (Fig. 1 A). Storage at 4 C for 7 days increased considerably MDA concentrations in all samples, the values being higher in the control group and lower in the -tocopheryl acetate group (Fig. 1 B). Samples within the dietary oregano essential oil groups had a significantly (P < 0.05) lower tendency for lipid oxidation compared to the control group at all time points. Dietary supplementation with oregano essential oil at the level of 100 mg kg 1 feed was significantly (P < 0.05) more effective in reducing lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and the control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. Thigh muscle was found to be more susceptible to lipid oxidation than breast muscle. The susceptibility of chicken thigh muscle to lipid oxidation during 9 months of frozen storage is shown in Fig. 2 A. On day 0 of refrigerated storage, thigh samples frozen stored for 1 month showed a lipid oxidation pattern similar to that of the breast samples although MDA values in thigh samples from all dietary groups were always higher than those in breast samples from respective dietary treatments at all time points (Fig. 2 A). This pattern did not changed at 3, 6 or 9 months. Dietary oregano essential oil supplementation at the level of 100 mg kg 1 feed was constantly more effective (P < 0.05) in reducing lipid
oxidation compared with the level of 50 mg oregano essential oil kg 1 feed and the control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. Lipid oxidation in thigh muscle at day 7 of refrigerated storage following 1, 3, 6 or 9 months of frozen storage (Fig. 2 B) was significantly (P < 0.05) higher compared with that on day 0 of refrigerated storage (Fig. 2 A). Thigh samples within the dietary oregano essential oil groups had a significantly (P < 0.05) lower tendency for lipid oxidation compared with samples in the control group at all time points. Dietary supplementation with oregano essential oil at the level of 100 mg kg 1 feed was significantly (P < 0.05) more effective in reducing lipid oxidation compared to the level of 50 mg oregano essential oil kg 1 feed and the control, but less effective (P < 0.05) compared with -tocopheryl acetate supplementation. Comparing Fig. 1 A and 2 A with 1 B and 2 B, a distinct difference in the rate of lipid oxidation between the tested storage conditions appears. In Fig. 1 A and 2 A, where lipid oxidation refers to measurements at day 0 after 1, 3, 6 or 9 months of frozen storage, MDA values exhibited a clear lag phase by month 6, followed by a more rapid increase at longer storage times. In contrast, in Fig. 1 B and 2 B where lipid oxidation was examined at day 7 of refrigerated storage after 1, 3, 6 or 9 months of frozen storage, MDA values tended to increase rapidly by month 6, and then more gradually as storage time increased.
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Fig. 2. Effect of dietary oregano essential oil supplementation at levels of 50 and 100 mg kg 1 feed as a function of frozen time, on lipid oxidation of raw chicken thigh muscle compared with dietary -tocopheryl acetate supplementation at a level of 200 mg kg 1 feed and control: (A) test after initial thawing and prior to refrigerated storage; (B) test after 7 days of refrigerated storage following thawing. All data points that represent mean malondialdehyde (MDA) concentrations from six analyses are accompanied by standard deviation bars, some of which however lie within the data points.
This inconsistency might be attributed to the analytical method employed for measuring the extent of lipid oxidation. The basis for this measurement is the reaction between TBA and MDA during heating at acid pH to produce a chromogenic complex. MDA reactivity is influenced by several factors including its formation as an artifact of the analysis during the test itself, its occurrence in various bound forms, and the specificity of the methods used for its measurement. Hence, the term ‘‘thiobarbituric acid reactive substances’’ (TBARS) is frequently used to account for the contribution of other compounds to the TBA–MDA complex formed. MDA is a secondary lipid oxidation product formed subsequent to the hydrolysis of lipid hydroperoxides. Considering that the derivative spectrophotometric method (Botsoglou et al., 1994) applied in the present study eliminates interference from other reactive compounds since it is based on derivative spectral analysis of the red TBA–MDA complex, a lag phase would be expected to occur during early stages of frozen storage followed by a rapid increase of MDA as observed in the present study. The absence of a lag phase in Fig. 1B and 2B was probably due to earlier hydrolysis of lipid hydroperoxides during the refrigerated storage of the previously frozen samples. Using MDA content as an index of absorption of the dietary oregano essential oil constituents, this study indicates that the antioxidant compounds occurring in oregano essential oil are distributed, retained, and
remained functional in muscle. The research carried out in vivo lends also support to many reports showing that the in vitro addition of oregano to various food systems inhibits lipid oxidation (Abdalla & Roozen, 2001; Economou et al., 1991; Lagouri et al., 1993; Tsimidou et al., 1995; Vekiari et al., 1993). Considering that the greater the amount of -tocopherol deposited the better protection the muscle should have against oxidative attack, breast and thigh muscle samples were analysed for their -tocopherol content. It was found that -tocopherol levels in muscle tissues of chickens were influenced by both the supplementation level and the type of the tissue. Thus, mean -tocopherol levels in breast and thigh muscle samples (n=6 samples) of the control group differed significantly (P < 0.05), being 2.05 and 3.50 mg kg 1 of tissue, respectively. Supplementing broiler diets with 200 mg tocopheryl acetate kg 1 feed, -tocopherol concentrations in breast and thigh muscle samples increased to 8.22 and 17.80 mg kg 1 of tissue, respectively. These values compare well with those reported by other workers (Jensen et al., 1995; Lin, Gray, Asghar, Buckley, Booren, & Flegal, 1989; Jakobsen et al., 1995). The higher concentrations of -tocopherol in thigh muscle have been attributed to its better developed vascular system and its higher lipid content compared to breast muscle (Lin et al., 1989). Although thigh muscle was found to contain higher amounts of -tocopherol than breast muscle, it tended
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Table 2 Effect of frozen storage on the mean -tocopherol concentrations in breast and thigh muscles of broilers given dietary -tocopheryl acetate Months
0 1 3 6 9
Concentrationsa of -tocopherol in breast (mg kg 1)
Concentrationsa of -tocopherol in thigh (mg kg 1)
Control group (30 mg kg 1 -tocopheryl acetate in feed)
Treated group (200 mg kg 1 a-tocopheryl acetate in feed)
Control group (30 mg kg 1 -tocopheryl acetate in feed)
Treated group (200 mg kg 1 -tocopheryl acetate in feed)
2.05a0.45 2.00a0.15 1.15b0.12 1.03b0.06 0.95b0.08
8.22a 0.05 8.10a 0.45 7.22b 0.40 7.12b 0.80 7.10b 0.65
3.50a0.60 3.58a0.30 3.10a0.20 2.20b0.12 1.80b0.10
17.80a0.72 17.74a0.90 18.10a0.94 11.92b1.10 11.74b0.80
Mean values in the same column with a letter in common do not differ significantly (P>0.05). a Mean valuestandard deviation for six analyses.
to oxidize faster than the latter tissue. The higher susceptibility of thigh meat towards oxidation has been attributed to the higher absolute content of polyunsaturated fatty acids with more than two double bonds in this muscle (Jensen, Guidera, Skovgaard, Staun, & Skibsted, 1997). Breast meat has a higher percentage of these acids in the fat, but their absolute amount in thigh meat is three times higher compared with breast muscle, as the total fat content in thigh meat is approximately five times that of breast meat (Jensen et al., 1997). Apart from the content of muscle -tocopherol and the amount of readily oxidizable substrates, the high level of pro-oxidative species originating from meat myoglobin and other iron containing proteins in thigh muscle is another factor causing lower oxidative stability in thigh muscle (Rhee & Ziprin, 1987). The rate of a-tocopherol depletion in breast and thigh muscle samples during 9 months of frozen storage is shown in Table 2. -Tocopherol depletion from thigh muscle samples occurred to a greater extent between 3 and 6 months of frozen storage, whereas for breast muscle samples between 1 and 3 months of frozen storage. There have been several possible explanations for the -tocopherol depletion during frozen storage. At 20 C, some catalysts and antioxidants may be trapped in the frozen solid phase and, therefore, the antioxidant activity of the cytosolic phase may no longer function optimally (Wen, Morrissey, Buckley, & Sheeby, 1996). Moreover, lipid free radicals are soluble in the oil fraction and are more stable at low temperatures. This increased stability allows them to diffuse to longer distances and spread the reaction (Kanner, 1994). Therefore, free radicals may escape the antioxidants in the frozen aqueous phase and diffuse into the membrane lipid system, where they initiate and promote lipid oxidation. Wen et al. (1996) stated that if this occurs, the lipid-soluble -tocopherol becomes the first line of antioxidant defence and thus may be rapidly depleted.
4. Conclusion In conclusion, the results of the present study show that dietary oregano essential oil supplementation reduced lipid oxidation significantly (P< 0.05) during frozen storage and subsequent refrigeration. A dose response increase in the antioxidative capacity of broiler tissues as a result of feeding oregano essential oil was exhibited. Dietary oregano essential oil supplementation at the level of 100 mg kg 1 feed more effectively (P < 0.05) reduced lipid oxidation compared with the level of 50 mg oregano essential oil kg 1 feed, but it was less effective (P < 0.05) compared with supplementation with -tocopheryl acetate. Therefore, oregano essential oil may be considered as a good alternative to -tocopheryl acetate supplementation of feeds. Presently, supplementation with oregano essential oil will increase slightly the cost of feed relatively to -tocopheryl acetate supplementation but this can be avoided with extensive cultivation of oregano plants.
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