Effect of Dietary Grape Pomace and Vitamin E on Growth Performance, Nutrient Digestibility, and Susceptibility to Meat Lipid Oxidation in Chickens

Effect of Dietary Grape Pomace and Vitamin E on Growth Performance, Nutrient Digestibility, and Susceptibility to Meat Lipid Oxidation in Chickens I. Gon˜i,* A. Brenes,†1 C. Centeno,† A. Viveros,‡ F. Saura-Calixto,† A. Rebole´,‡ I. Arija,‡ and R. Estevez† *Departamento de Nutricio´n I, Facultad de Farmacia, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain; †Departamento de Metabolismo y Nutricio´n, Instituto del Frı´o, CSIC, Avda. Ramiro de Maeztu s/n, Ciudad Universitaria, Madrid 28040, Spain; and ‡Departamento de Produccio´n Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain thigh meats was lower in the birds fed the supplemented vitamin E diet than the control diet after 1, 4, and 7 d of refrigerated storage. Results showed a linear reduction of lipid oxidation in breast and thigh meats at 4 and 7 d with increasing content of GP in the diet. Oxidative stability in breast and thigh meats at 1, 4, and 7 d of storage was equivalent or less effective in GP diets compared with the vitamin E diet. A linear increase was observed in liver α-tocopherol concentration with increasing content of GP in the diet, but it was inferior to the supplemented vitamin E diet. In conclusion, the results showed that a dietary inclusion rate up to 30 g/kg of GP did not impair chickens growth performance and protein and amino acids digestibilities and increased antioxidant activity in diet and excreta. Grape pomace and vitamin E diets reduced the lipid oxidation of meat during refrigerated storage and increased liver α-tocopherol concentration, although these effects were greater, in some cases, by adding vitamin E to the diet.

Key words: grape pomace, chick, lipid oxidation 2007 Poultry Science 86:508–516

Alonso et al., 2002; Torres et al., 2002). Grape skins and seeds are rich source of flavonoids, including monomeric phenolic compounds such as (+)-catechins, (-)-epicatechin, and (-)-epicatechin-3-O-gallate and dimeric, trimeric, and tetrameric procyanidins. Studies have shown flavonoids have the capacity to act as powerful antioxidants by scavenging free radicals and terminating oxidative reactions (Gonzalez-Parama´s et al., 2004). Flavanols and flavanol oligomers and polymers (proanthocyanidins) have been proven to possess powerful antioxidant properties (Yilmaz and Toledo, 2004). The application of GP compounds in food technology has also demonstrated a potent edible oil antioxidant capacity and an inhibitor of the oxidation of fish lipids, frozen fish muscle, and cooked, cold stored turkey meat (Wanasundara and Shahidi, 1994; Pazos et al., 2005; Mielnick et al., 2006). In experiments with rats, the inclusion of grape flavonoids causes a diminution of tissue lipid peroxidation in kidney, liver, and lung (Preuss et al., 2001; Rodrigo et al., 2005) and

INTRODUCTION Grape pomace (GP) is the residue left after juice extraction by pressing grapes in the wine industry. In Spain alone, over 250 million kilograms of this by-product (constituted by seeds, skin, and stem) are used every year either as animal feed (with low nutritional value) or for ethanol production by fermentation and distillation (lowlevel benefit). This material is under-exploited, and most of it is generally disposed in open areas, leading to serious environmental problems (Botella et al., 2005). Recent investigations have stressed the importance of this by-product from wine processing as plant material particularly rich in a wide range of polyphenols (Bonilla et al., 1999;

©2007 Poultry Science Association Inc. Received June 27, 2006. Accepted October 26, 2006. 1 Corresponding author: [email protected]

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ABSTRACT Grape pomace (GP) is a source of polyphenols with powerful antioxidant capacity. An experiment was conducted to investigate the effect of inclusion of GP at levels of 5, 15, and 30 g/kg and α-tocopheryl acetate (200 mg/kg) in a corn-soybean basal diet on growth performance, protein and amino acid digestibilities; antioxidant activity of diet, serum and excreta, lipid oxidation of breast and thigh meats during refrigerated storage, and liver vitamin E concentration. Growth performance and protein and amino acid digestibilities were not affected among the different treatments. Total intake and digestibility of extractable polyphenols in the birds fed the GP diet were increased compared with birds fed supplemented and unsupplemented vitamin E diets. Antioxidant activity in vitamin E and GP diets and excreta exhibited higher scavenging free radical capacity than the control diet using 3-ethylbenzthiazoline-6-sulfonic acid and ferric reducing antioxidant power methods. Lipid oxidation (malondialdehyde concentration) in breast and

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MATERIALS AND METHODS Test Product Red GP (peels and seeds; Vitis vinifera var. Cencibel) was obtained from a winery (Vinı´cola de Castilla S.A., Manzanares, Ciudad Real, Spain). Proximate composition of GP is shown in Table 1. Grape pomace was used as a source of dietary fiber and polyphenols in the chicken diets. The α-tocopheryl acetate used in the diets was donated by DSM Nutritional Products Iberia S.A., Alcala´ de Henares, Madrid, Spain.

Table 1. Proximate composition of grape pomace Item

DM (g/kg)

Protein Soluble sugars Fat Fiber Ash Extractable polyphenols Nonextractable polyphenols

137.9 20.7 102.6 325.0 24.1 48.6 160.4

± ± ± ± ± ± ±

1.2 0.3 6.4 8.5 0.3 2.7 7.4

Data are the mean of 4 determinations ± SD.

1

Birds and Diets A total of 120, one-day-old male broiler chicks (Cobb strain) were obtained from a commercial hatchery. The birds were housed in electrically heated stainless steel starter battery brooders in an environmentally controlled room with 23 h of constant overhead fluorescent lighting during 3 wk. Diets in mash form and water were provided for ad libitum consumption. Chicks were allocated to 20 cages, each cage containing 6 chicks, to receive 5 dietary treatments with 4 replicates of each treatment. Celite (Celite Corp., Lompoc, CA), a source of acid insoluble ash (AIA), was added at 10 g/kg to all diets as an indigestible marker. All diets were formulated to meet or exceed the minimum NRC (1994) requirements for broiler chickens. At the end of the experimental period, birds were weighed, and feed consumption was recorded for feed efficiency computation. All housing and handling were approved by the University Complutense of Madrid Animal Care and Ethics Committee in compliance with the Ministry of Agriculture, Fishery and Food for the Care and Use of Animals for Scientific Purposes. Ingredients and nutrient composition of diets are shown in Table 2. Experimental diets were as follows: 1) control cornsoybean diet + 30 g/kg of cellulose (CS), 2) CS + vitamin E (200 mg/kg of α-tocopheryl acetate), 3) CS + 5 g/kg of GP, 4) CS + 15 g/kg of GP, 5) CS + 30 g/kg of GP. The cellulose was substituted by GP in the experimental diets.

Collection of Samples and Measurements At 21 d of age, 8 birds were randomly selected from each treatment (2 per replicate). Serum was prepared from blood obtained by cardiac puncture for subsequent determination of antioxidant activity. The blood samples were allowed to clot in polypropylene tubes for 2 h at room temperature at 1,500 × g for 10 min, and the supernatant was removed. All samples were stored at −20°C until assayed. After killing the chicks by cervical dislocation, liver was removed, cleaned from adhering tissue, and frozen at −20°C. The ileum was quickly dissected out and the content expressed by gentle manipulation into a plastic container and stored at −20°C. Digesta were pooled from 2 birds of each replicate within the same treatment. Ileal contents were freeze-dried and ground (1-mm screen) and subsequently analyzed for CP, amino acids, and celite. Clean stainless steel collection trays were also

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considerable antioxidant activity within the large intestine and feces derived from excreted extractable polyphenols (EP) and nonextractable polyphenols (Gon˜i and Serrano, 2005). Poultry meat is relatively rich in polyunsaturated fatty acids and is, therefore, readily susceptible to oxidative deterioration (Kanner, 1994). Increasing the unsaturation degree of the muscle membrane by dietary manipulation increases the susceptibility of chicken meat to oxidative deterioration during storage (Enberg et al., 1996), and as a consequence, flavor and nutritional value are decreased. Synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole have long been used to control lipid oxidation in stored meat and meat products, but concern over their use (Imaida et al., 1983; Okada et al., 1990), has created a need and prompted research for alternative antioxidants, particularly from natural sources. The oxidative stability of poultry meat depends largely on the contained α-tocopherol present in cell membrane phospholipids, which in turn is dependent on the level of α-tocopheryl acetate added to the diet (Wen et al., 1997). Dietary supplementation with this antioxidant has been shown to increase vitamin E in muscle tissues, improving the oxidative stability of meat during storage (Carreras et al., 2004). Apart from α-tocopherol, research has shown that dietary supplementation of essential oils from rosemary, sage, and oregano to broilers could improve the oxidative stability of chicken and turkey meat during refrigerated or long-term storage (LopezBote et al., 1998; Botsoglou et al., 2002; Botsoglou et al., 2003a). Evidence is also available on the antioxidative effect of added tea catechins on susceptibility of chicken meat to lipid oxidation (Tang et al., 2000, 2001). Wine by-products represent sources of antioxidants that have been relatively unexploited to date, but they are subjects of increased industrial interest. No evidence is available on the potential antioxidant properties of GP when added in poultry diets. The objective of this study was to evaluate the effect of dietary GP and vitamin E on broiler chicken performance, nutrient digestibility, antioxidant activity of diet, serum, and excreta. The susceptibility to oxidation of breast and thigh refrigerated meats and liver α-tocopherol concentration was also determined.

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Table 2. Ingredients and nutrient composition of experimental diets (g/kg as fed) Item

Control + vitamin E

Control + 5 GP1

Control + 15 GP

Control + 30 GP

430.7 404.4 85.0 30.0 — 19.3 10.5 3.0 5.0 2.1 10.0

430.7 404.4 85.0 30.0 — 19.3 10.5 3.0 5.0 2.1 10.0

432.6 402.6 85.0 25.0 5.0 19.3 10.4 3.0 5.0 2.1 10.0

436.4 398.9 85.0 15.0 15.0 19.3 10.3 3.0 5.0 2.1 10.0

442.2 393.4 85.0 — 30.0 19.3 10.0 3.0 5.0 2.1 10.0

219.0 2.6 95.3

228.0 2.8 250.5

221.0 NA4 83.7

227.3 NA 74.2

223.0 4.1 86.8

3,101 9.0 10.0 4.5

3,101 9.0 10.0 4.5

3,105 9.0 10.0 4.5

3,111 9.0 10.0 4.5

3,122 9.0 10.0 4.5

1

GP = grape pomace. Vitamin and mineral mix supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol, 1,000 IU; vitamin E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 12.5 ␮g; riboflavin, 5.5 mg; Ca panthotenate, 11mg; niacin, 53.3 mg; choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; delquin, 125 mg; DL-Met, 500 mg; amprol, 1 g; Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.18 mg; NaCl, 2,500 mg. 3 Celite Corp, Lompoc, CA. 4 NA = not analyzed. 5 Calculated value (FEDNA, 2003). 2

placed under each cage, and excreta from the birds were collected for 48 h. A subsample of excreta was collected in polyethylene bags and freeze-dried for subsequent determination of celite, extractable polyphenols, and antioxidant activity. Eight birds (2 per replicate with the live weight closest to the particular replicate average) per treatment were slaughtered, and carcasses were immediately trimmed for breast and thigh meat. These tissues were individually sliced and sampled for lipid oxidation studies. Tissue samples, breast excluding skin and thigh with skin, were wrapped in transparent O2-permeable polyvinyl chloride film (13,500 cm3/m2 per d), frozen, and stored at −20°C until required. After thawed, the raw meat samples were placed in a nonilluminated refrigerated cabinet at 4°C, and the progress of lipid oxidation was determined after 1, 4, and 7 d of storage.

Chemical Analysis Dry matter (930.15), CP (976.05), crude fiber (978.10), and ash (942.05) were analyzed according to the methods of the Association of Official Analytical Chemists (1995). Crude fat was determined by extraction in petroleum ether following acidification with a 4 N HCl solution (Wiseman et al., 1992). Analysis of soluble sugars was carried out by using anthrone and thiourea as a reagent following the conditions described by Southgate (1976). The AIA contents of diet, ileal digesta, and excreta were measured after ashing the samples and treating the ash with boiling 4 M HCl (Siriwan et al., 1993). Amino acids

in the diets and the ileal contents were analyzed (994.12) following AOAC (1995) procedures and separated using a Beckman model 6300 autoanalyzer (Beckman Coulter, Monheim, Germany). Determination of the amino acid (AA) Trp was not possible under the conditions of analysis used. The extent of lipid oxidation was determined by measuring the TBA-reacting substances at 1, 4, and 7 d of storage and was expressed as micrograms of malondialdehyde (MDA) per gram of muscle using the procedure described by Salih et al. (1987). Ten grams of ground meat was homogenized with 35 mL of 3.86% perchloric acid in an Ultra-Turrax (IKA Works Inc., Wilmington, NC) at 21,280 × g for 1 min. Butylated hydroxyanisole was added before homogenization at a level of 125 ␮g/mg of fat. The blended sample was filtered through Whatman number 2V filter (Whatman International Ltd., Maidstone, UK) into 50-mL Erlenmeyer flasks. Five milliliters of the filtrate was mixed with 5 mL of 0.02 M TBA in distilled water in capped test tubes. Tubes were incubated at room temperature in the dark for 15 to 17 h or heated in boiling water for 30 min. The absorbance was determined at 531 nm against a blank containing 5 mL of distilled water and 5 mL of 0.02 M TBA solution. Polyphenols were extracted in diet and excreta by shaking at room temperature with methanol water (50:50 vol/ vol, 50 mL/g sample during 60 min, at room temperature and with constant shaking). After centrifugation (15 min, 3,000 × g) supernatants were combined and used to measure the antioxidant capacity by the 2, 2-azinobis (3-ethilenzotiazolin)-6-sulfonate (ABTS; Fluka Chemicals, Madrid, Spain) method.

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Ingredients Corn (8.1% CP) Soybean (48% CP) Sunflower oil Cellulose GP (13.2% CP) Dicalcium phosphate Calcium carbonate Salt Vitamin-mineral premix2 DL-Met Celite3 Analyzed composition CP Total polyphenols Vitamin E (mg/kg) Calculated composition AME5(kcal/kg) Met + Cys Ca Available P

Control

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GRAPE POMACE IN CHICKEN DIETS

Table 3. Performance of broiler chicks (0 to 21 d) fed diets containing grape pomace1 (GP) and vitamin E

Treatments Control Control + vitamin E Control + 5 GP Control + 15 GP Control + 30 GP Pooled SEM Statistical significance2 Control vs. vitamin E GP vs. no GP Vitamin E vs. GP Type of response3 Linear Quadratic

Weight gain (g)

Feed consumption (g)

Feed efficiency (g:g)

656 649 655 651 673 39

856 839 862 855 869 47

1.31 1.29 1.32 1.31 1.29 0.03

NS NS NS

NS NS NS

NS NS NS

NS NS

NS NS

NS NS

1

Data are means of 4 pens of 6 chicks. Probability value of contrast; NS = P > 0.05. 3 Due to percentage GP in diet. 2

concentration in feed/AIA concentration in ileal digesta or excreta) × (CP and AA concentration in ileal digesta and EP concentration in excreta/CP and AA concentration in feed)]. Data were subjected to ANOVA using the GLM procedures of SAS (SAS Institute, 2001), and single df linear contrasts were used to separate treatments. Linear and quadratic effects were also analyzed. Significant differences among treatment means were determined at P < 0.05 by Duncan’s multiple-range test.

RESULTS Growth Performance The addition of increasing concentration of GP in the chicken diets did not impair growth performance (BW, feed consumption, and feed efficiency) compared with those birds fed the unsupplemented and supplemented vitamin E diets (Table 3).

Apparent Digestibilities of Protein and Amino Acids The inclusion of graded concentrations of GP did not affect the apparent ileal digestibility of CP and essential and nonessential amino acids. Statistical analysis also demonstrated a reduction of Arg (P < 0.05), Leu (P < 0.05), Phe (P < 0.01), Glu (P < 0.01), Pro (P < 0.01), Tyr (P < 0.01), His (P < 0.05), and Cys (P < 0.01) digestibilities in birds fed GP diets compared with those fed the vitamin E diet. Likewise, a quadratic effect (P < 0.05) was observed in Lys, Thr, Glu, and Ser with increasing dietary GP (Table 4).

Calculations and Statistical Analysis

Antioxidant Activity in Diets, Excreta, and Serum

Apparent ileal CP, AA, and EP digestibilities were calculated using the following formula: 100% ( [100% × (AIA

Total intake and digestibility of EP in the birds fed the GP diet were significantly increased (P < 0.05) up to 1.6

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Extractable polyphenols were determined in methanol, acetone, and water extracts obtained from GP, diet, and excreta by the Folin-Ciocalteau procedure (Montreau, 1972) using gallic acid as a standard. Residues from the extract were treated with 5-mL/L of HCl-butanol during 3 h at 100°C (Reed et al., 1982). Nonextracted polyphenols were calculated from the absorbance at 550 nm of the anthocyanidin solutions. Condensed tannins from Mediterranean carob pod (Ceratonia siliqua L.) supplied by Nestle´ S.A. (Vevey, Switzerland) were treated under the same conditions to obtain standard curves. The ABTS assay was determined in extracted samples (GP, diet, and excreta) and serum. The antioxidant activity was estimated following the procedure described by Re et al. (1999) with some modifications. The ABTS radical cation (ABTS+ⴢ) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS+ⴢ solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 658 nm. After addition of 100 ␮L of extracted samples or Trolox standard (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid; Sigma-Aldrich Co, St Louis, MO) to 3.9 mL of diluted ABTS+ⴢ solution, absorbance readings were taken every 20 s using a Beckman DU-640 (Beckman Instruments Inc, Fullerton, CA). The reaction was monitored for 6 min. The percentage inhibition of absorbance vs. time was plotted, and the area below the curve (0 to 6 min.) was calculated. Methanolic solutions of known Trolox concentrations were used for calibration of the measurement of EP antioxidant activity. The ABTS determination on serum was similar to the method previously indicated, but 10 ␮L of serum was added to 3 mL of ABTS+ solution and an aqueous solution of Trolox was used for calibration of the measurement of antioxidant activity. The ferric antioxidant power (FRAP) of the samples was estimated according to the procedure previously described (Benzie and Strain, 1996; Pulido et al., 2000). Briefly, FRAP reagent was mixed with distilled water and either the sample or appropriate reagent blank. Readings at 30 min were selected for calculation of FRAP values. Reduction power activities were as micromolars of Trolox equivalents per gram of DM. α-Tocopherol content of diets and liver was determined by the method of Butriss and Diplok (1984), which includes saponification with saturated KOH in the presence of pyrogallol; for liver, 1 mL of 25% liver homogenate was used. The α-tocopherol was then extracted with hexane and measured by normal-phase HPLC using a Hypersil Si 100 (5 ␮m) column and a mobile phase of hexaneisopropanol (98:2 vol/vol) and detected by fluorescence using a HPLC system (Hewlett-Packard 1100, Agilent Technologies GmbH, Waldbronn, Germany).

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NS 0.05

NS NS 0.01 NS NS 0.01

NS NS NS 0.05

NS NS

NS NS

NS NS

The extent of lipid oxidation, as measured by MDA formation, in breast and thigh meats was significantly lower (P < 0.05) in the supplemented vitamin E diet, in a range of 25 to 58%, than the control group after 1, 4, and 7 d of refrigerated storage. The inclusion of GP in the diets significantly reduced MDA values in breast samples after 4 (P < 0.05, up to 33%) and 7 d (P < 0.001, up to 47%) of refrigerated storage and in thigh samples (P < 0.001, up to 30%) at 7 d compared with samples obtained from birds fed the control diet. Malondialdehyde values of thigh meat samples from birds fed GP diets at 1, 4, and 7 d were significantly increased (67, 53, and 32%, respectively) compared with samples obtained from birds fed the vitamin E diet. A linear response was observed in breast and thigh meats (P < 0.01) at 4 and 7 d, respectively, with increasing content of GP in the diet. Likewise, a quadratic response was also observed in breast meat (P < 0.001) at 7 d of refrigerated storage (Table 6).

NS NS NS 0.05

NS NS

NS NS NS NS NS NS NS NS 0.05

NS NS 0.01

NS NS NS

NS NS NS

NS NS 0.05

NS NS NS

NS NS NS

NS NS 0.01 NS NS NS NS NS 0.01

NS NS NS

2

Data are means of 8 chicks for each treatment. Probability value of contrast; NS = P > 0.05. 3 Due to percentage GP in diet.

NS NS NS NS NS NS NS NS

NS NS NS NS NS 0.05 NS NS NS

1

70.08 73.69 71.07 69.24 67.96 3.47 88.60 89.96 88.65 88.71 88.09 1.40 84.00 86.55 86.77 85.65 84.57 1.72 85.02b 87.59a 86.49ab 84.31b 84.18b 1.67 82.35b 84.68b 89.41a 82.10b 81.30b 2.65 90.23 91.30 87.68 89.62 89.63 1.57 86.85 88.74 88.47 87.46 87.28 1.83 91.66 93.19 93.18 92.64 92.79 1.47 88.49 90.47 91.15 89.11 88.09 2.01 91.51 93.44 92.75 92.49 91.66 1.17 85.03 85.38 85.26 84.73 84.50 1.41

Control Control + vitamin E Control + 5 GP Control + 15 GP Control + 30 GP Pooled SEM Statistical significance2 Control vs. vitamin E GP vs. no GP Vitamin E vs. GP Type of response3 Linear Quadratic

87.83 89.14 88.85 88.06 87.10 1.73

88.41 90.33 89.84 88.78 88.84 1.40

81.84 83.65 82.44 81.81 80.73 2.28

81.34 83.36 83.82 82.25 81.26 2.33

86.12 87.50 87.26 86.00 84.99 2.18

% 87.82 89.61 87.95 87.01 84.75 2.12

84.97 86.30 86.32 85.81 84.48 1.74

Pro Gly Glu Asp Ala His Val Thr Phe Met Lys Leu CP

Ile

MDA Concentration

Liver α-Tocopherol

Treatments

Arg

and 2.3 times, respectively, compared with birds fed the supplemented and unsupplemented vitamin E diets (Table 5). Antioxidant activity in vitamin E and GP diets exhibited significantly higher scavenging free radical capacity than control diets using ABTS (3.4 and 6.6 times, respectively) and FRAP (1.4 and 1.5 times, respectively) methods. Similarly, the birds fed vitamin E and GP diets exhibited significantly higher scavenging free radical capacity in excreta than those fed control diets using ABTS (1.2 and 1.2 times, respectively) and FRAP (1.2 and 1.2 times, respectively) methods. The dietary treatment did not affect the antioxidant activity measured on serum (Table 6). Animals fed diets containing GP or vitamin E showed more elevated values than the control group, but the difference was not significant.

The inclusion of vitamin E in the diet increased P < 0.001) liver α-tocopherol concentration (47%) compared with the control diet. Liver α-tocopherol concentration was increased (P < 0.01; up to 33%) in birds fed GP diets compared with those fed control diet. Likewise, the inclusion of GP in the diets reduced (P < 0.001) liver α-tocopherol concentration (20%) compared with the vitamin E diet. A linear response (P < 0.01) was observed in liver α-tocopherol concentration at 21 d of age with increasing content of GP in the diet (Table 6).

DISCUSSION The performance of chicks for each experimental group indicated that GP exerted no growth-promoting effect when administered up to 30 g/kg. There are few references in the literature in relation to the use of grape byproducts in chicken feed. Hughes et al. (2005) and Lau

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Table 4. Apparent ileal digestibility (%) of protein and essential and nonessential amino acids of broiler chicks (0 to 21 d) fed grape pomace1 (GP) and vitamin E

Ser

Tyr

Cys

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Table 5. Total intake, digestibility, and antioxidant activity of extractable polyphenols in diets, excreta, and serum of broiler chicks fed diets containing grape pomace (GP) and vitamin E Antioxidant activity (␮mol of Trolox equivalent/g) Treatment Control Control + vitamin E Control + 30 GP Pooled SEM

Total intake1 (g)

Digestibility (%)

2.23b 2.35b 3.57a 0.11

14.3b 16.0b 32.8a 5.96

1

2

Diet (ABTS method)

Diet2 (FRAP method)

Excreta1 (ABTS method)

Excreta1 (FRAP method)

Serum3 (ABTS method)

1.87 6.29 12.26 0.33

8.63 11.96 12.95 0.69

63.2b 75.1 75.8a 7.54

75.7b 88.6a 89.9a 6.89

370 390 391 33.51

Means in columns with no common superscript differ significantly (P < 0.05). Data are means of 4 pens of 6 chicks each. 2 Data are the mean of 3 determinations. ABTS = 2, 2-azinobis (3-ethilenzotiazolin)-6-sulfonate; FRAP = ferric antioxidant power. 3 Data are means of 8 chicks. a,b 1

In the current experiment, apparent ileal digestibility of protein and essential and nonessential amino acids were not affected. This lack of effect could be attributed to the low content of polyphenols in the experimental diets to cause detrimental effect. Polyphenols bind to proteins due to the interaction of their reactive hydroxyl groups with the carbonyl group of protein. As a result of this complexation, protein and AA digestibility were reduced by the inclusion of sorghum and faba bean polyphenols in chicken and pig diets (Rostagno et al., 1973; Jansman et al., 1989; Ortiz et al., 1993). Inhibition of different digestive enzymes by tannins has also been reported (Reddy and Pierson, 1985). There are many references in the literature to the composition and antioxidant properties of grape polyphenols (Gonzalez-Parama´s et al., 2004; Yilmaz and Toledo, 2004), but there have been very few studies on the digestibility and intestinal degradation of polyphenols and other major grape constituents. Available data on the absorption and metabolism of polyphenols suggest negligible bioavailability of polymeric proanthocyanidins (De`prez et

Table 6. Effect of refrigerated storage on lipid oxidation of breast and thigh meats and liver α-tocopherol content of broiler chicks fed diets containing grape pomace1 (GP) and vitamin E Malondialdehyde concentration (mg/kg of meat) d1 Treatments Control Control + vitamin E Control + 5 GP Control + 15 GP Control + 30 GP Pooled SEM Statistical significance2 Control vs. vitamin E GP vs. no GP Vitamin E vs. GP Type of response3 Linear Quadratic

Breast

α-Tocopherol (␮g/g of tissue)

d4 Thigh a

Breast a

d7 Thigh a

Breast a

1

Thigh a

Liver

a

0.13 0.09b 0.10ab 0.13a 0.11ab 0.029

0.13 0.09b 0.14a 0.15a 0.14a 0.034

0.30 0.24b 0.29a 0.21b 0.20b 0.043

0.30 0.19c 0.29a 0.27ab 0.23b 0.038

0.47 0.32c 0.38b 0.25dc 0.27d 0.031

0.43 0.28d 0.37b 0.35bc 0.30cd 0.433

18.19c 26.66a 21.39bc 23.56ab 24.25ab 3.03

0.05 NS NS

0.05 NS 0.001

0.05 0.05 NS

0.05 NS 0.001

0.05 0.001 NS

0.001 0.001 0.001

0.001 0.01 0.001

NS NS

NS NS

0.001 NS

0.01 NS

0.001 0.001

0.01 NS

0.01 NS

Means in columns with no common superscript differ significantly (P < 0.05). Data are means of 7 chicks for each treatment. 2 Probability value of contrast; NS = P > 0.05. 3 Due to percentage GP in diet. a–d

d 21

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and King (2003) reported growth depression in chickens fed diets containing grape seed extract. The poor response obtained from the former studies could be justified, because grape seed extract was a pure form containing 90.2% of total phenolics, expressed as a gallic acid equivalent by the Folin method, and incorporated in the diet at 30 g/kg. In the current experiment, grape seed pomace contained 4.86% of total polyphenols by the Folin method. Thus, the total EP in the diet containing the highest proportion of GP were 0.41%. Similarly, the concentration of condensed tannins present in the higher concentration of GP diet could be relatively low to produce a growth depression effect. The effect of polyphenols has also been studied in chickens using ingredients like sorghum and faba bean. In general, relatively high dietary concentrations of polyphenols by the addition of these ingredients reduced performance in chickens as well as other livestock (Gualtieri and Rapaccini, 1990; Jansman et al., 1989; Nyachotti et al., 1997). Polyphenolic compounds are also known for their ability to interact with different molecules such as proteins.

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2006). The antioxidant activity caused by the presence of these compounds could have additional effects, sparing other antioxidants and protecting molecules from oxidative damage during digestion and preserving the intestinal epithelium from potential oxidative damage caused by dietary factors or bacterial metabolism (Scalbert and Williamson, 2000; Gon˜i and Serrano, 2005). Using the MDA content and the digestibility of EP as an index of absorption of the dietary grape seed constituents indicates that the antioxidant compounds occurring in this wine by-product could be distributed, retained, and remained functional in muscle and liver. This research carried out in chickens lends support to observations previously reported by Tang et al. (2000) on the significant effect of tea catechin in chicken meat quality and in the comparable effectiveness of this dietary polyphenol as compared with dietary α-tocopheryl acetate. Similarly, this study confirms in vitro observations that the addition of wine polyphenols to various food systems (fish lipids, frozen fish, and turkey meat) inhibits lipid oxidation (Lau and King, 2003; Pazos et al., 2005; Mielnik et al., 2006) and those that provide an enhancement of the antioxidant defense potential in kidney and liver of rats by flavonolrich red wine (Fremont et al., 2000; Rodrigo et al., 2002, 2005). In the current experiment, the liver concentration of vitamin E was similar to those reported by Villaverde et al. (2004) in chickens using a high polyunsaturated fatty acid diet. A reduction in α-tocopherol deposition in chicks fed unsaturated diets was also reported by Surai and Sparks (2000) and Sijben et al. (2002). The increase in liver vitamin E content by the inclusion of GP could be due to the sparing effect of GP on vitamin E in the intestine. Less vitamin E would be destroyed through oxidation, resulting in greater amounts of absorbed vitamin E. It has been reported that polyphenols [quercetin, (-)-epicatechin and (+)-catechin], owing to their 1-electron reduction potentials, may spare vitamin E to delay lipid oxidation and to regenerate tocopherol in rat and human models (Frank, 2005). Hence, addition of feedstuffs rich in these bioactive compounds may prove beneficial for the enhancement of vitamin E status and for the reduction in lipid oxidation of the tissues. Results in this study also confirm that dietary GP and vitamin E can delay lipid oxidation in breast and thigh chicken meats and reduce the potential risk induced by lipid oxidation products. Similar results have been reported using tea catechins by Tang et al. (2000, 2001) and vitamin E in chickens by Maraschiello et al. (1999) and De Winne and Dirinck (1996). Dietary GP supplementation at the level of 15 and 30 g/kg was effective in delaying lipid oxidation compared with 200 mg/kg α-tocopheryl acetate diet in breast (4 and 7 d of storage) and thigh meat (7 d of storage). The greater efficiency in preventing lipid peroxidation in breast meat compared with thigh meat by dietary GP could be justified by the greater susceptibility of thigh meat to oxidation attributed to the higher absolute content of polyunsaturated fatty acids and the large amount of prooxidative agents originating from myoglobin and other Fe-containing proteins in thigh mus-

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al., 2000). In the current experiment, the digestibility of EP was 32.8%; this means that a higher amount of EP was available in the intestinal tract of the animal-fed diet containing GP that could be partially absorbed in the small intestine. However, the antioxidant capacity of serum from GP-fed birds did not show significant differences compared with the other dietary groups. The digestibility of nonextracted polyphenols present in the diets has also been determined (Brenes et al., unpublished data), but negative digestibility values were obtained. Possible interferences between polyphenols with a high degree of polymerization and polyphenols associated with high molecular weight compounds and other compounds like amino acids and sugars, together with limitations in the extraction techniques, make the determination difficult. The increase in the antioxidant activity of EP in the excreta (20%) in the highest grape pomace-fed birds compared with those fed the control diet suggests that part of polyphenols are degraded by intestinal microflora. Gon˜i et al. (2005) reported that intestinal bacteria showed a high capacity to degrade EP in rats. De`prez et al. (2000) and Ward et al. (2004) also reported that major polyphenolic constituents of GP (polymeric proanthocyanidins) were degraded by human colonic microflora into smaller compounds including phenolic acids that could be ab¨ zkan et al. (2004), Dolara et al. sorbed and metabolized. O (2005), and Papadopoulou et al. (2005) also demonstrated that phenolic grape extracts and red wine polyphenols influenced intestinal microflora, decreasing the number of Propionibacteria, Bacteroides, and Clostridia and increasing Lactobacilli and Bifidobacteria numbers. Similarly, dietary fiber associated with polyphenols in GP could play an additional mechanism to stimulate the intestinal fermentation and to influence the production of particular microbial metabolites. Such findings on the effectiveness of polyphenolic compounds may be beneficial as alternatives to the dietary antimicrobial growth promoters, which are currently banned within the European Union. Future studies must be done to determine the nature and extent of specific components of polyphenols with potential antimicrobial properties. Nutritional interest in polyphenolic compounds has increased greatly in light of their antioxidant capacity (Scalbert and Williamson, 2000). The relative contribution of EP to the total antioxidant activity in excreta, obtained by the FRAP and ABTS methods, depends on the diet. The influence of different factors on the effectiveness of antioxidants in complex heterogeneous foods and biological systems cannot be evaluated using only 1 assay. The 2 systems chosen to evaluate the antioxidant activity (FRAP and ABTS) measure the total reduction power and the free radical-scavenging activity. In the current experiment, the vitamin E and the GP diets exhibited the highest antioxidant activity in comparison with the control diet using both methods. The antioxidant compounds present in grape have already been identified as phenolic acids (benzoic and hydroxycinnamic acids), stilbene derivatives, flavan-3-ols (catechin and epicatechin), flavonols (quercetin and and myricetin), and anthocyanidins (Caillet et al.,

GRAPE POMACE IN CHICKEN DIETS

ACKNOWLEDGMENTS We thank the Ministerio de Educacio´n y Ciencia for financial support of this investigation (project AGL200610312/GAN).

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cle tissues compared with breast muscle tissues (Wen et al., 1997; Higgins et al., 1998; Botsoglou et al., 2003b). In conclusion, the results presented in this study showed that increasing the concentration of GP up to 30 g/kg did not impair performance and protein and AA digestibilities. An increase in the antioxidative activity of broiler diet, excreta, and meat as a result of the dietary administration of GP and vitamin E was also reported. The better oxidative stability of meat samples receiving the diet supplemented with the higher concentration of GP was accompanied by an increase in the concentration of vitamin E in the liver. Therefore, GP could be considered as a good alternative to α-tocopheryl acetate supplementation of feeds and to improve vitamin E status. Antioxidant constituents (mainly flavonoids), present in GP, might be considered responsible for this mechanism and on the sparing effect of vitamin E in liver. Currently, work is in progress with the aim to assess the effects of dietary concentrations of polyphenols by the addition of GP or their isolated bioactive ingredient on performance, on sparing effect of vitamin E, and to study the susceptibility to lipid oxidation of breast and thigh tissues of broiler chickens during a 42-d growth period.

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