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Research Note Effects of thymol and carvacrol feed supplementation on lipid oxidation in broiler meat A. Luna,* M. C. Lábaque,* J. A. Zygadlo,* and R. H. Marin*†1 *Instituto de Ciencia y Tecnología de los Alimentos, and †Cátedra de Química Biológica, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, 5016, Argentina ABSTRACT Poultry meat is particularly prone to oxidative deterioration due to its high concentration of polyunsaturated fatty acids. The present study evaluates the effects of thymol and its isomer carvacrol on lipid oxidation when supplemented to the feed. Supplementation with the antioxidant butylated hydroxytoluene was used as a positive control. Thus, broiler chickens were assigned to 1 of 4 treatments: vehicle (control), 150 mg/kg of butylated hydroxytoluene (positive control), 150 mg/kg of thymol, or 150 mg/kg of carvacrol. Breast and thigh samples were taken at 0, 5, and 10 d of 4°C storage. Lipid oxidation was deter-

mined by the analysis of 2-TBA reactive substances (TBARS). Sample storage for 5 to 10 d significantly increased the levels of TBARS. Feed supplementation did not significantly affect breast sample oxidation. However, after 5 and 10 d of storage, increasingly higher values of TBARS were detected in thigh samples of the control group in comparison to the 3 supplemented groups. Interestingly, the same lower values of TBARS were detected between those feed-supplemented groups. Therefore, the application of the natural antioxidants thymol or carvacrol could be useful to improve poultry meat quality.

Key words: broiler meat, thymol, carvacrol, lipid oxidation, feed supplementation 2010 Poultry Science 89:366–370 doi:10.3382/ps.2009-00130


oxidative stability of tissue after feeding poultry with antioxidant compounds added into the diet (Botsoglou et al., 2002; Lee et al., 2004; Govaris et al., 2005), that could consequently enhance the commercial value of the products obtained. Feed supplementation may be a simple and convenient strategy to introduce lipidsoluble antioxidants into the phospholipid membrane tissues, where they can effectively inhibit the oxidative reactions at their localized sites (Luaridsen et al., 1997). Synthetic antioxidants such as butylated hydroxytoluene (BHT) or butylated hydroxyanisole have been widely used as antioxidants (Chastain et al., 1982). However, there is a trend to search for compounds that may allow a shift from synthetic to natural antioxidants (Sheehy et al., 1995; Yanishlieva, 2001; Botsoglou et al., 2002). This trend is justified because a carcinogenic potential from the use of synthetic antioxidants has been suspected (Chen et al., 1992). Oregano is an aromatic plant with a wide distribution throughout the Mediterranean area (Sivropoulou et al., 1996). This plant contains molecules that have intrinsic bioactivities on animal physiology and metabolism and possesses intense antimicrobial (Dorman and Deans, 2000; Ultee et al., 2002), antifungal (Daouk et al., 1995), and antioxidant properties (Yanishlieva et al., 1999; Cervato et al., 2000). In pigs, it was demonstrated that it reduces wastes and odor emissions

Consumer concern on the quality of meat and meat products has greatly increased during the past decades (Min and Ahn, 2005). In fact, quality and healthfulness were reported to be 2 of the most important factors influencing the choice of the consumer for foods (Lennernas et al., 1997). One factor that affects the quality of meat is the oxidation of lipids. In general, lipid oxidation has long been recognized as a major deterioration process affecting both the sensory and nutritional quality of foods (Sheehy et al., 1995; Yanishlieva et al., 1999). The rate and extent of lipid oxidation in muscle tissue appears to be dependent on the degree of muscle tissue damages during preslaughtering events such as stress and physical damages and postslaughtering events such as early postmortem conditions, pH, and carcass temperature (Morrisey et al., 1998). Poultry meat is particularly prone to oxidative deterioration due to its high concentration of polyunsaturated fatty acids (PUFA; Igene and Pearson, 1979). There are many studies showing an improvement in the ©2010 Poultry Science Association Inc. Received March 11, 2009. Accepted July 29, 2009. 1 Corresponding author: [email protected]

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in intensive farming (Varel, 2002). Oregano activity is mainly attributed to its main components carvacrol and thymol, substances that modify the bacterial cell membrane permeability (Lambert et al., 2001) and react with lipid and hydroxyl radicals converting them into stable products (Yanishlieva, 2001). Although oregano or its essential oils have already been used with the intention of improving the quantity and quality of the products of an animal, the results obtained in poultry are still controversial (Young et al., 2003; Botsoglou et al., 2004, 2005; Giannenas et al., 2005; Simitzis et al., 2008). Given that thymol and carvacrol are molecules that have intrinsic bioactivities on animal physiology and metabolism (Reiner et al., 2009), these 2 compounds could have antioxidant activity on chicken meat when supplemented in the feed. Thus, the objective of the present study was to evaluate the effects of thymol, and its isomer carvacrol, on lipid oxidation when supplemented in the feed. Supplementation with the antioxidant BHT was used as a positive control.

MATERIALS AND METHODS Twenty-four 1-d-old broiler chickens (Cobb 500) were purchased from a local hatchery (INDACOR S.A., Córdoba, Argentina). On arrival, they were wing-banded for individual identification and randomly allocated in 12 pens (102 cm × 70 cm × 80 cm, length × width × height; 2 birds per pen). Water and feed were supplied ad libitum throughout the experiment. A corn-soybeanbased broiler starter ration [23% CP, 3,190 kcal of ME/ kg, 6% crude fat (50% triglycerides, 28% mono- and diglycerides, 9% nonpolar lipids and 13% polar lipids)] was provided from 1 d to 3 wk of age followed by grower ration [18% CP, 3,190 kcal of ME/kg, 5.5% crude fat (52% triglycerides, 23% mono- and diglycerides, 10% nonpolar lipids, and 12% polar lipids)] thereafter. A 14L:10D cycle was applied. Birds from each pen were assigned to 1 of 4 treatments that differed in the supplement added to the feed until slaughter age (42 d): vehicle (control; CON), 150 mg/kg of BHT (BHT), 150 mg/kg of thymol (THY), and 150 mg/kg of carvacrol (CAR). A 0.5% ethanolic solution of those supplements was pulverized weekly to fresh commercial feed mash. At 42 d of age, broilers were slaughtered by cervical dislocation. Carcasses were immediately processed, individually placed in sterile polypropylene bags, and stored in a nonluminated refrigerated cabinet at 4°C until sampling. Samples of breast (pectoral major) and thigh (biceps femoris) were taken from each carcass at 0, 5, and 10 d of storage at 4°C (refrigerated storage). Samples were individually packaged and stored at −20°C in oxygen-impermeable vacuum bags until further analysis of lipid oxidation. All chemicals used in this study were reagent-grade commercial products. Solution A consisted of 5% trichloroacetic acid (99%, Anedra, Buenos Aires, Argentina) in deionized water with 0.08% propyl galate (purum Downloaded from by guest on 23 January 2018

98%, Fluka, Zwijndrecht, the Netherlands) 0.1% in ethanol (96%, Porta, Córdoba, Argentina). Solution B consisted of 0.17% TBA (purum 98%, Fluka, Germany) in deionized water with 10% dimethylsulfoxide (99.9%, Sintorgan, Buenos Aires, Argentina). The compounds used as supplements were THY (E. Merck, Darmstadt, Germany), CAR [purum 97% (gas chromatography), Fluka, St. Louis, MO]), and BHT (Fluka AG, Buchs SG, Switzerland). Lipid oxidation of meat samples were determined by the analysis of 2-TBA reactive substances (TBARS) according to Nielsen et al. (1997), slightly modified. 2-Thiobarbituric acid was expressed as nanograms of malondialdehyde (MDA) per gram of wet tissue. Briefly, 2 g of meat sample (breast or thigh) was thoroughly homogenized in the presence of 2 mL of solution A (see chemical description above) per gram of meat and vortexed. Ten minutes later, 2 mL of solution B (see chemical description above) was added into the tube and the mixture was vortexed again. After a further 10 min, the mixture was incubated in a 90 ± 2°C water bath for an hour and then cooled for 30 min in an ice bath. Finally, the sample was centrifugued at 2,200 × g for 20 min. The absorbance of the resulting upper layer was read at 532 and 700 nm. To reduce the turbidity measurements, the concentration of TBARS in the extracts analyzed was calculated as the difference between the absorbances obtained at 532 and 700 nm. Tetraethoxypropane was used as a standard. Data were evaluated with repeated measures ANOVA, with time of storage (3 levels) as the within-subject variable and feed supplementation effect (4 levels) as the between-subject variable. Fisher’s least significant difference tests were used for post hoc comparisons of means. The homogeneity of the variances was tested. A probability level of less than or equal to 0.05 was considered to represent significant differences.

RESULTS The effect of refrigerated storage on the lipid oxidation of chicken breast samples was analyzed in birds that had been fed feed supplemented with THY, CAR, or BHT (Figure 1). The MDA content in breast samples was not found to be significantly influenced by feed supplementation (F4,24 = 0.85; P = 0.68). However, as expected, storage for 5 or 10 d significantly increased the levels of MDA in those samples (F2,48 = 0.51; P < 0.001). The susceptibility of broiler thigh to lipid oxidation as a function of storage time and feed supplementation is illustrated in Figure 2. As expected, storage during 5 to 10 d increased the levels of MDA (F2,48 = 15.11; P < 0.001). Interestingly, the extent of lipid oxidation was also influenced by feed supplementation (F4,24 = 5.06; P < 0.001). At 0 d of storage (nonstored samples), no differences were detected in the levels of MDA registered in thigh samples from different feed-supplemented groups. However, after 5 d and, particularly, 10 d of


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Figure 1. Effect of refrigerated storage on the lipid oxidation of chicken breast as a function of feed supplementation with vehicle (CON), 150 mg/kg of thymol (THY), 150 mg/kg of carvacrol (CAR), or 150 mg/kg of butylated hydroxytoluene (BHT). Data points represent mean malondialdehyde (MDA) contents and their SD.

refrigerated storage, increasingly higher values of MDA were detected in broiler thigh of the CON groups in comparison to the supplemented groups. Specifically, at 5 d of storage, the CON group showed significantly higher (P < 0.03) values of MDA than the THY and CAR groups. Likewise, after 10 d of storage, values registered in thigh samples from the CON group were significantly higher (P < 0.001) than those found in the other 3 (THY, CAR, and BHT) feed-supplemented groups. No differences were detected in MDA values obtained between the 3 feed-supplemented groups neither after 5 nor after 10 d of storage (Figure 2).

of PUFA in thigh compared with breast tissue (Jensen et al., 1997). This contention is supported by the fact that thigh meat has a higher percentage of PUFA in comparison to breast meat. In thigh meat, the absolute amount of PUFA is 3 times higher, and its total fat content is approximately 5 times higher than that of breast meat (Jensen et al., 1997). The content, composition, and quality of dietary fat in feed, and the tendency of animal species to store fatty acids into membrane phospholipids, can affect the fatty acid composition of membranes and their susceptibility to lipid oxidation (Ahn et al., 1993; Song and Miyazawa, 2001). Here, the feed supplementation with THY, CAR, and BHT in broiler diets retards lipid oxidation (as MDA formation) in thigh meat when refrigerated. This provides indirect evidence that these antioxidants could be absorbed and enter in the systemic circulatory system after ingestion. However, because these 2 oregano oil constituents at trace levels in tissues have not been studied, their bioavailability cannot be directly demonstrated yet. Thymol and CAR seem to have similar effectiveness, meaning, according to the definition of Yanishlieva et al. (1999), that the possibility of blocking the radical chain process by interaction with peroxide radicals is similar in both compounds. However, they probably differ in the mechanism of action on broiler meat deterioration because their molecular asymmetries differ. Yanishlieva et al. (1999) also proposed that during the oxidation of lipids at ambient temperature, THY is a more effective and more active antioxidant than CAR, and both compounds differ in the mechanism of their inhibiting action, which depends on the character of the lipid medium. These authors also suggest that under those experimental conditions, THY is a better antioxidant than CAR, due to

DISCUSSION Refrigerated storage in both breast and thigh muscles increased lipid oxidation (MDA formation) compared with fresh tissues, in accordance with previous studies in poultry and mammal species (Nielsen et al., 1997; Botsoglou et al., 2003). The susceptibility of meat to lipid oxidation depends on the animal species, muscle type, and anatomical location (Rhee and Ziprin, 1987; Rhee et al., 1996). Herein, feed supplementation with THY, CAR, and BHT was found to delay lipid oxidation in thigh, but not in breast muscle, after its storage. Similarly, when chickens were fed with oregano essential oil (Botsoglou et al., 2002), thigh muscle seemed to be more susceptible to oxidation compared with breast muscle samples. Also, Salih et al. (1989) reported that turkey thigh meat was more susceptible to oxidation than turkey breast meat. Considering that MDA is produced as a result of unsaturated fatty acid oxidation (Fernández et al., 1997; Ulu, 2004), differences in the effect of the same antioxidant compounds on the muscle type could be explained by higher absolute content

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Figure 2. Effect of refrigerated storage on lipid oxidation of chicken thigh as a function of feed supplementation with vehicle (CON), 150 mg/kg of thymol (THY), 150 mg/kg of carvacrol (CAR), or 150 mg/ kg of butylated hydroxytoluene (BHT). Data points represent mean malondialdehyde (MDA) contents and their SD.



the fact that the former has greater steric hindrance of phenolic group than the later one. Future research experiments could be designed to elucidate whether these 2 isomers differ in their mechanism of action. Also, it would be interesting to optimize the condition in which each compound would have the greater antioxidant effect on broiler meat or other meat products. The antioxidant activity of both THY and CAR feed supplementation have similar effectiveness to retard lipid oxidation than supplementation with BHT. Indeed, both natural compounds seem to show antioxidant activity earlier than BHT because at 5 d of storage, thigh samples obtained from THY- and CAR (but not BHT)-supplemented groups had shown significant differences compared with the CON samples. Nevertheless, TBARS levels registered in samples derived from the BHT-supplemented group also showed significant differences compared with the CON group when 10 d of storage was reached. Our results allow us to suggest that supplementation with the natural antioxidants THY or CAR could be applied in the future to improve poultry meat quality. Lee et al. (2004) mentioned that accumulation of essential oil in the body is unlikely due to their fast metabolic conversion and excretion, but if chickens are continuously fed diets containing essential oil constituents, those compounds may be deposited in various tissues. Botsoglou et al. (2002) showed that essential oils can be deposited in a dose-dependent fashion, and thus can be consumed by humans. Consequently, whether this consumption with poultry meat will evoke negative effects needs to be assessed. It should however be emphasized that the compounds THY and CAR are given generally recognized as safe status by the Food and Drug Administration (Furia and Bellanca, 1975), implying that their use is safe. In conclusion, the feed supplementation with THY or CAR has similar effectiveness to retard lipid oxidation than the supplementation with BHT and could be considered useful natural supplements to be applied in the poultry industry to improve meat quality.

ACKNOWLEDGMENTS This research was supported by grants from Fondo Nacional de Ciencia y Tecnología (FONCYT) and Secretaría de Ciencia y Tecnológia (SECyT), Universidad Nacional de Cordóba (UNC), Argentina. MCL, JAZ, and RHM are career members of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. AL holds research fellowships from the latter institution and is a graduate student of the Doctorado en Ciencias Biológicas, Facultad de Ciencias Exactas, Físicas y Naturales (FCEFyN), UNC. We thank very much Eng. Felix Serrano (INDACOR S.A.) and DISE S.A. for their support in the developing of this study.

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Research Note Effects of thymol and carvacrol feed supplementation on lipid oxidation in broiler meat A. Luna,* M. C. Lábaque,* J. A. Zygadlo,* and R...

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