Dietary supplementation with plant extracts, xantophylls and synthetic antixidants: Effect on fatty acid profile and oxidative stability of frozen stored chicken breast meat

Chickens from 22 to 42 days of age were fed a basal diet supplemented with α-tocopheryl acetate (150 mg·kg-1), extracts of coneflower, thyme or sage (560 mg·kg-1), marigold xantophylls (20 mg lutein·kg-1), mix of synthetic antioxidants (48.6 mg·kg-1) or β-apo-8-carotenoic acid ethylester (40 mg·kg-1). On day 42 the chickens were slaughtered, breast muscles were excised, frozen (-20oC) and stored for 6 months. Dietary supplementation with plant extracts, synthetic antioxidants or pigments did not affect performance. Sage extract increased the level of stearic acid (C18:0), arachidonic acid (C20:4), DHA and n-3 PUFA and decreased oleic acid and α-linolenic acid (C18:3) contents and PUFA n-6/n-3 ratio in lipids of stored breast meat. Additives did not favourably affect TBA-RS values in meat. In chickens fed the control diet or supplemented with 150 mg·kg-1 α-tocopheryl acetate, stored meat contained 1.72 or 6.45 mcg·g-1 α-tocopherol, respectively. Dietary synthetic xantophyll negatively influenced sensory indices in boiled meat.

In meat containing more unsaturated fatty acids there is a risk that their profile will change during storage (Jeremiah, 1980).It seems not unlikely that flavonoids, carotenoids, essential oils and other plant substances may affect lipid accumulation in tissues and control the changes during storage of meat.The present study was designed to evaluate the effects of dry extracts of sage, thyme and coneflower, vitamin E, marigold xantophylls, β-apo-8-carotenoic acid ethylester, and a mixture of synthetic antioxidants supplemented to a diet on performance, fatty acid composition, TBA-RS, vitamin E content, and sensory properties in breast meat of chickens fed diets containing a mixture of rape seed and fish oils.

MATERIAL AND METHODS
The study was carried out on 320, 22-day-old sexed Cobb chickens (725 g average initial body weight) allocated to 8 groups in 5 replicates, each containing 4 males and 4 females, and kept in cages with wire mesh floors.The control, basal maize-soyabean diet contained, %: rape seed oil 4 and fish liquid fat 1 (Lysi h.f.Reykjavik, stabilized with ethoxyquin in amount 250 · kg -1 ).Fish fat (Tables 1 and 2) contained (in % of total fatty acids): 4.75 eicosapentaenoic acid (EPA) and 3.55 docosahexaenoic acid (DHA).Vitamin-mineral premix contained no vitamin E or antioxidants.Experimental diets were supplemented (to 1 kg) with 150 mg dl-α-tocopheryl acetate (Lutavit ® E 50, BASF) or 560 mg dry extracts from purple coneflower (Echinacea purpurea) (more than 2.5% of polyphenolic substances), thyme (Thymus vulgaris) (phenols, thymol, essential oils) or sage (Salvia officinalis) (flavonoids, essential oils), or with 20 mg of marigold (Tagetes sp.) xanthophylls (lutein).For comparative purposes synthetic additives were used: 48.6 mg antioxidant preparation (in it: butylated hydroxytoluene 28.6 mg, ethoxyquin 14.3 mg, butylated hydroxyanisol 5.72 mg) or 40 mg β-apo-8-carotenoic acid ethylester.Diets as mash were fed ad libitum from day 22 to 42.On day 42 of life 4 males and 4 females were slaughtered, right breast muscles (M.pectoralis maior and minor) without skin and outer fat were excised, packed in plastic bags and frozen at -20 o C.After 6 months the caudal part of the muscle was taken for chemical analyses and sensory testing.
Samples were extracted as described by Folch et al. (1957), saponified, converted to methyl esters (Morrison and Smith, 1964), extracted with hexane and separated.Fatty acid composition was determined with a GC Varian 3400 gas chromatograph equipped with a CP-Wax 58, 25 m × 0.53 mm, 1.0 μm colum and He as the carrier gas, 6 ml per min.Peak areas were measured with Star Chromatography Workstation software (Varian Star 4.5).
Thiobarbituric acid reactive substances (TBA-RS) content as a measure of oxidative changes in meat lipids was determined according to Salih et al. (1987) with the modifications of Pikul et al. (1989).Breast meat α-tocopherol content was determined with a Merck-Hitachi HPLC equipped with a LiChroCART 250-4 Superspher 100 RP-18, 4 μm column and FL, EX. 295 nm, Em. 350 nm detector according to Manz and Phillip (1981).Basal chemical analysis was conducted according to AOAC (1990).
Breast meat was also boiled in slightly salted water and sensory analysis was conducted by a 6-member panel.Smell and taste, juiciness and tenderness were ranked on a 4-point scale for degree of acceptability (2-unacceptable, 3-acceptable, 4-good, 5-very good).
Data were subjected to one-way analysis of variance and differences were examined by Duncan's multiple range test (Statistica, ver.5.0 PL software).

RESULTS
The average final body weight of chickens was 2400 g.Plant extracts, α-tocopheryl acetate and synthetic antioxidants or pigment added to the basal diet had no effect on body weight gain, feed intake, feed conversion, or on carcass quality indices (Tables 3 and 4).Supplementation of diets with sage extract increased stearic acid (C 18:0 ), arachidonic acid (C 20:4 ) and DHA levels and decreased oleic acid (C 18:1 ) and α-linolenic acid (C 18:3 ) levels in lipids of stored chicken breast meat in comparison with the control group (Table 5).In breast meat of chickens fed the sagesupplemented diet, the level of polyunsaturated fatty acids n-3 (PUFA n-3) was higher and the PUFA n-6/n-3 ratio in lipids was lower than in groups fed diets with synthetic antioxidants (Table 6).As compared with other groups, a numerically lower level of PUFA n-3 was found in breast meat lipids from chickens fed the control diet and diet supplemented with synthetic xantophyll or synthetic antioxidants.
Supplementation of the basal diet with vitamin E, plant extracts, marigold xanthophylls or synthetic antioxidants had no positive effect on the TBA-RS value in stored meat, and addition of marigold xanthophylls even significantly increased TBA-RS content (Table 7).The content of vitamin E in breast meat of chickens fed diets enriched with 150 mg α-tocopheryl acetate was nearly four times larger than in the control group (Table 7).Diet supplementation with synthetic xantophyll negatively influenced (P<0.05) the smell and taste of boiled breast meat (Table 7).Nevertheless, in all groups, stored breast meat was found to be suitable for consumption.

DISCUSSION
The results of the present study indicate that dietary supplementation with natural plant extracts chosen for their antioxidative properties or with synthetic antioxidants or pigments did not affect the performance of broiler chickens fed a balanced maize-soyabean diet.However, some authors have found improved performance when active plant substances were added as a substitute for antibiotic growth promotors in chickens (Horton et al., 1991, Jamroz et al., 2003).
The lipids of the basal diet containing 4% rape seed oil and 1% of fish fat, were relatively rich in oleic acid, linoleic acid, linolenic acid, EPA and DHA.This diet ensured a satisfactory level of PUFA in breast meat lipids of control chickens.Some of the investigated additives affected the fatty acid profile of stored breast meat lipids.Supplementation of the diet with sage extract increased the levels of stearic acid, arachidonic acid, DHA and PUFA n-3 and decreased the level of monounsaturated oleic acid and polyunsaturated α-linolenic acid in comparison with the control group and chickens fed synthetic xantophyll.The PUFA n-6/n-3 ratio was also lowered as a result of adding sage.These results may suggest an effect of sage extract on fat metabolism.The effect of plant extracts on fatty acid profiles was reported in fattening pigs (Hanczakowska and Świątkiewicz, 2006;Szewczyk et al., 2006).A reduction of monounsaturated fatty acids and increase of PUFA in muscle fat of pigs fed nettle extract was confirmed by a decrease of cholesterol and triacylglicerol concentrations in blood serum (Szewczyk et al., 2006).
Synthetic xantophyll or mix of synthetic antioxidants (BHT+EQ+BHA) had no effect on the fatty acid profile of fat in stored breast meat.In the case of BHA similar results were reported in muscle of pigs (Hanczakowska and Świątkiewicz, 2006).
Natural plant extracts with antioxidative proprieties or synthetic antioxidants or pigments added to the diet had no positive effect on TBA-RS values of stored breast meat compared with the control diet.In case of marigold xantophylls added to the diet, the effect in stored meat was even negative.Woodall et al. (1996) stated that dietary β-carotene and zeaxanthin supplementation of chicken diets significantly reduced in vitro susceptibility of liver to oxidation (TBA-RS) and nonsignificantly in breast muscle homogenates.Canthaxantin did not protect tissues against oxidation, whereas supplementation with α-tocopherol (100 mg·kg -1 ) had a significant antioxidative effect in muscle.
Dietary supplementation with 500 mg·kg -1 rosemary or sage extract decreased the TBA-RS content in broiler breast meat stored for 4 months or cooked (Lopez-Bote et al., 1998).The antioxidant activities of sage extracts were higher than those of oregano (Pizzale et al., 2002).In poultry, improved abdominal fat oxidative stability was observed as a result of supplementing BHT and ethoxyquin to diets (Bartov and Bornstein, 1981).
A relatively low TBA-RS content in stored breast meat was found in chickens fed a control diet that contained in 1 kg 15.9 mg of α-tocopherol originated from natural feed components.Similar TBA-RS values were found in meat when the diet contained 160.7 mg α-tocopherol, e.g., supplemented with 150 mg α-tocopheryl acetate.Both levels of vitamin E in the diet correspond to 1.7 and 6.4 mcg·g -1 α-tocopherol in stored breast meat.The results of the present study suggest that the natural vitamin E content in the rape seed oil diet ensured as good anti-oxidative protection of meat as a four-fold higher vitamin E level.However, in our earlier experiment (Koreleski and Świątkiewicz, 2006) addition 150 or 300 mg·kg -1 α-tocopheryl acetate to a diet containing 8.1 mg·kg -1 of natural α-tocopherol, lowered the TBA-RS value in stored breast meat.A lower TBA-RS content was also noted in refrigerated turkey breast meat supplemented with 180 mg α-tocopheryl acetate (Sheldon et al., 1997).
Stored breast meat was found to be suitable for consumption, but the effect of additives on the taste and smell of meat was not the same.Dietary synthetic xantophyll negatively influenced sensory indices in boiled meat, but the worse taste and smell were not of the rancid type.Plant extracts of coneflower, thyme and sage in the diet did not affect the flavour or taste of stored boiled meat as compared with the control diet or the diet supplemented with α-tocopheryl acetate.These results are in agreement with data reported by Gardzielewska et al. (2003), who found no effect in broilers of herbal supplements (coneflower, garlic or ginger) on the sensory properties of breast meat after 4 months of frozen storage.

CONCLUSIONS
Sage extract added to the diet can beneficially affect the fatty acid profile of stored breast meat lipids in chickens.Under the conditions of meat storage in this experiment, the natural vitamin E content in the rape seed oil-containing diet provided equivalent anti-oxidative protection of meat as the diet supplemented with 150 mg α-tocopheryl acetate.

Table 3 .
Performance of chickens, 22-42 days of age

Table 4 .
Results of slaughter analysis

Table 5 .
Selected fatty acids in stored breast meat, % of total fatty acids

Table 6 .
Selected groups of fatty acids (% of fatty acids) and ratios in stored breast meat