9"! 0.. {.11 I! 1 .1... "IIII.1.‘I>‘ .lnl' . :53. .i-.V viil’ v ‘p' .1: A... w (wilifiixuo .12! I)». ' .w”:uc - Q i ‘ a? “man 32.3.1!» .. €013.14! .0 !, A ' fizz-x . . 17A)... 3 < .1513)?! . .91.}: :‘flillflitl at" . [5.- pl .1!£¢\.Il !. In... (at! 01.1 cl .7... In)": '35. .orah o .80. 0!} ’3’: .5! Tassels Date lllllllll llllllllllllllllllllllllllllllllllllliilill r .‘I This is to certify that the thesis entitled STABILIZATION OF TURKEY LIPIDS THROUGH DIETARY SUPPLEMENTATION WITH VITAMIN E presented by Kuang-Hua Hsieh has been accepted towards fulfillment of the requirements for Master of Science degree in Food Science y// 63% Major professor(/ 8’—-4¢~02 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution L LIIRARY Hiohlzan State University J PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. r DATE DUE DATE DUE DATE DUE : 1*_ Vi“. e v.5. Him a 2133i 1 1 i226 ii 5.x) fi=j MSU Is An Affirmative Action/Equal Opportunity Institution ammo-m IIIBILIIIEIOI'OI EUR!!! LIPIDS IIIOUII 0131.3! SUPPLIIIIIIIIOIHIIII‘VIIIIII’I BY IDAHO-EUR 881!“ A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1992 ABSTRACT STABILIZATION OF TURKEY LIPIDS THROUGH DIETARY SUPPLEMENTATION WITH VITAMIN E BY KUANG-HUA HSIEH The study was designed to examine the influence of >three levels of dietary vitamin E (12.5, 50 and 100 Ifi/kg feed) on the deposition of a-tocopherol in turkey muscle, and on selected quality characteristics of raw turkey meat (oxidative stability of lipids, color, drip loss) during refrigerated and frozen storage. Muscle and membranal lipids from turkeys fed the higher levels of vitamin E (50 and 100 IU/kg feed) had significantly (p < 0.05) higher a- tocopherol concentrations and greater oxidative stability than those from turkeys fed the control diet. The beneficial effect of dietary vitamin E on color stability "and drip loss was also observed during storage. The . stability of cholesterol in cooked turkey meat during refrigerated storage was improved by dietary vitamin E. Cholesterol oxidation was significantly (p < 0.05) influenced by supplementation at the 100 IU vitamin E/kg feed level. Lipid oxidation and cholesterol oxide product formation were positively correlated in cooked dark and light turkey meat (r-0.79 and r-0.55). The author expresses sincere gratitude to her academic advisor, Dr. J. Ian Gray, for his guidance and consistent encouragement throughout my graduate studies. Appreciation is extended to my committee, Dr. Alden Booren, Dr. James F. Price and Dr. Cal J. Flegal, for their technical assistance and critical review of this manuscript. Further_acknowledgements are also due to Dr. A. Asghar and Dr. E. Comma for their technical assistance. special thanks are to ur. Napolitano Angelo, Poultry Science Research and Teaching Center, Michigan State University, for his assistance during turkey feeding periods. The author recognizes laboratory associates Dr. Frank J. Monahan, Shu-Hui Chan and Cheng-Hsiung Chen for their assistance throughout this research program. Finally, I would like to thank to express my heartfelt appreciation to my parents and family for all of the love, 'encouragement and support during this graduate study. iii Page LIST OT TABLES ......................................... 1X LIST 0! EIGURES ........................................ Xi INTRODUCTION ........................................... 1 REVIEN 0? LITERATURE ................................... 3 Lipid Oxidation in Meat Systems ................... 3 Mechanism of lipid oxidation .................. 3 Initiation and catalysis of lipid oxidation ... 4 Composition of poultry lipids . . . . . . . . . . . . . . . . . 11 Lipid oxidation products ...................... 14 Cholesterol Oxidation in Meat Systems ............. 16 Cholesterol distribution in meat .............. 16 Mechanism of cholesterol oxidation ............ 18 Cholesterol oxidation products in foods . . . . . . . 20 Stabilization of Lipids with Antioxidants ......... 23 Synthetic phenolic antioxidants ............... 23 Naturally occurring antioxidants .............. 24 Alpha-Tocopherol in Heat Systems .................. 25 Alpha-tOCOpherOI distribution ooooooooooooooooo 26 The biochemical function of alpha-tocopherol .. 28 Effect of Dietary Vitamin E Supplementation on the Quality Characteristics of Meat .................. 34 Effect of Dietary Vitamin E Supplementation on Growth Performance Of Mina]. ......OOOOOOOOOCOOO000...... 37 EEPERINENTAL Cholesterol Oxide Standards ....................... 40 Reagents .......................................... 40 Turkey Feeding Regimen ............................ 41 Sample Preparation ................................ 41 iv Measurement of Lipid Oxidation in Muscle Tissue ... Isola tion and Quantitation of Alpha-Tocopherol in Blood Plasma and Muscle Tissue ................... ( ( ( ( a) Plasma ..................................... b) Muscle tissue ............................. c) Microsomal fraction ....................... d) HPLC quantitation of alpha-tacopherol ..... calor Ana1Y818 ......OOOOOOOOOOOOOO00......00...... Determination Of Drip was ......OOOOOOOOOOOOOOOOOO Determination of Cholesterol Oxides in Cooked Turkey Meat Cholesterol Determination ......................... Oxida tive Stability of the Microsomal Lipids of Turkey ”usele ......OOOOOOOOOOOOCOOO0.0.0.0000...O Stati stical Analysis .............................. usnramnlswsslm ......OOOOIOOOOOOO0......00...... Effect of Dietary Vitamin E Supplementation on Gram Performance ......OOOOOOOOO......OOOOOOO0.0 Effect of Vitamin E Supplementation on Alpha- Tocopherol Concentration in Plasma and Muscle Tiss Effec ue ............OOOOOOOOOOOOOOO0.0.0....0....O. t of Vitamin E Supplementation on the Quality of Raw Meat During Refrigerated and Frozen Storage Lipid stability ......OOOOOOOOIOO......OOOOOOOO Drip 1°38 ......OOOOOOOOOOOCOO00......0.0.0.... calor ......OOOOOOOOOOOO000.......0...00.0.0... Effect of Vitamin E Supplementation on Lipid Stability and Cholesterol Oxidation in Cooked Meat Lipid Stability ......OOOOOOOOOOOOOOOOOOCOOS... Cholesterol oxidation ......................... Relationship between lipid and cholesterol Effec Lipi OXidation ........0.........OOOOOOOOOOOOOO.... t of Vitamin E Supplementation on Microsomal dStability ......OOOOOOIOOOOOOOOOOO00.0.00... Concentration of a-tocopherol in the microsomal fractions of turkey meat ..................... Metmyoglobin/hydrogen peroxide-catalyzed lipid peroxidation .....OOOOOOOOOOOOO0.000.000.0000. Bm‘mmcnusro‘s ......OOOOOOOOOOOO0.0.0.0.000... APPENDICES 44 45 45 46 47 48 48 49 49 51 52 53 54 54 56 61 61 66 7O 77 77 79 84 9O 95 98 9. 10. Concentrations of a-tocopherol (pg/g meat) in dark and light meat from turkeys fed diets containing three levels of vitamin E ............... The effect of vitamin E supplementation on TEARS values (mg malonaldehyde/kg sample) of raw dark turkey meat stored at 4°C for 8 days .... The effect of vitamin E supplementation on TEARS values (mg malonaldehyde/kg sample) of raw light turkey meat stored at 4°C for 8 days . . . . . Effect of different levels of dietary vitamin E on TEARS values (mg malonaldehyde/kg sample) dark meat stored at -20°C for 6 months . . . of raw Effect of different levels of dietary vitamin E on TEARS values (mg malonaldehyde/kg sample) light meat stored at -20°C for 6 months . . of raw Effect of vitamin E supplementation on the development of cholesterol oxidation products (expressed as a percentage of the initial cholesterol concentration) in cooked ground dark turkey meat during storage at 4°C .. Effect of vitamin E supplementation on the deVelopment of cholesterol oxidation products (expressed as a percentage of the initial cholesterol concentration) in cooked ground light turkey meat.during storage at 4°C . Analysis of variance for TEARS values of raw dark turkey'meat (refrigerated study) ........ Analysis of variance for TEARS values of raw light turkey meat (refrigerated study) ...... Analysis of variance for ”a" values of color of raw dark turkey meat (refrigerated study) 11.Ana1ysis of variance for ' a” values of color of raw light turkey meat (refrigerated study) 12.Ana1ysis of variance for ”L" values of color of raw dark turkey meat (refrigerated study) 13.Analysis of variance for "L" values of color of raw light turkey meat (refrigerated study) 14. Analysis of variance for "b" values of color of raw dark turkey meat (refrigerated study) vi 98 99 100 101 102 103 104 105 105 106 106 107 107 108 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.' 27. 28. 29. 30. 31. Analysis of variance for ”b" values of color of raw light turkey meat (refrigerated study) ........ Analysis of variance for total cholesterol oxidation product concentrations in cooked dark turkey meat (cooked study) Analysis of variance for total cholesterol oxidation product concentrations in cooked light turkey'meat (cooked study) Analysis of variance for TEARS values of cooked dark turkey meat (cooked study) ................... Analysis of variance for TEARS values of cooked light turkey'meat (cooked study) .................. Analysis of turkey meat Analysis of turkey’meat Analysis of turkey'meat Analysis of turkey'meat Analysis of turkey'meat Analysis of turkey'meat Analysis of turkey'meat Analysis of variance for drip loss of raw dark (refrigerated study) .................. variance for drip loss of raw light (refrigerated study) .................. variance for drip loss of raw dark (3 months frozen study) ............... variance for drip loss of raw light (3 months frozen study) ............... variance for drip loss of raw dark (6 months frOZBn StUGY) oeeooooooooeooo variance for drip loss of raw light (6 months frozen study) ............... variance for TEARS values of raw dark (frozen study) variance for TEARS values of raw light turkey meat (frozen study) .................. Analysis of variance for "a” value of color of raw dark turkey meat (frozen study) ............... Analysis of variance for "a” values of color of raw light turkey meat (frozen study) .............. Analysis of variance for "L” value of color of raw dark turkey meat (frozen study) ............... Analysis of variance for "L" values of color of raw light turkey meat (frozen study) .............. vii 108 109 109 110 110 111 111 112 112 113 113 114 114 115 115 116 116 32. 33. 34. 35. 36. 37. Analysis of variance for "b" value of color of raw dark turkey meat (frozen study) . . . . . . . . . . . . . . . Analysis of variance for ”b” values of color of raw light turkey meat (frozen study) .............. Analysis of variance for a-tocopherol contents in plasma samples of turkey during feeding period . . . . . Analysis of variance for body weights of turkey during feeding period Analysis of variance for a-tocopherol contents in raw dark meat of turkeys fed three dietary levels OfVitaminE 00............OOOOOOOOOOOO0.0.0.000... Analysis of variance for a-tocopherol contents in raw light meat of turkeys fed three dietary levels of vitamin E Rune” .0.........OOOOOOOOOOO......OOOOOOOOOOOOOOOOO viii 117 117 118 118 119 119 120 LIST OI TABLES Table Page 1. Proposed initiators of lipid oxidation ............ 7 2. Autoxidation and antioxidant reactions involving VitaminE O.............OOOOOOOOOOOOOOOO0.0.0.0.... 29 3. Composition of turkey rations used in the feeding Study 00............OOOOOOOOOOOOOOOO0.00.0.0....... 42 4. Effects of dietary vitamin E level on the feed consumption, feed conversion and body weights of turkeys ....OOO.......OOOOOOOOOOOO0.0.0.0000......O 55 5. Alpha-tacopherol content (pg/ml plasma) in plasma samples of turkeys fed three dietary levels of VitaminE ......OOOOOOOOOOOOOOOOOO......OOOOOOOOOOO 57 6. Concentration of a-tocopherol (pg/g tissue) in the light and dark meat of turkeys fed three dietary levels Of VitaminE O.C...OO..OO0.0000000.00000.0'... 59 7. The effect of vitamin E supplementation on TEARS values of raw dark and light meat during storage at 4°C O....O.......OOOOOOOOOOOOOOOOOO0.00.00.00.00 62 8. The effect of vitamin E supplementation on TEARS values of raw dark and light meat during storage at -20.C ........OOOOOOOOOOOOOOOOOOO....0.0.0.0.... 64 9. Percentage drip loss from meat from turkeys fed vitamin E-supplemented diets when stored under fluorescent light at 4°C .......................... 67 10. Percentage drip loss from turkey meat which had been frozen for 3 and 6 months, and subsequently stored under fluorescent light at 4°C .................... 68 11. Changes in the color characteristics of dark turkey meat stored at 4°C for8days .. 71 12. Changes in the color characteristics of light turkey meat stored at 4°C for8days 72 ix 13. 14. 15. 16. 17. 18. Changes in the color characteristics of dark turkey meat stored at -20°C for 3 and 6 months . . . . . . . . . . . Changes in the color characteristics of light turkey meat stored at -20°C for 3 and 6 months . . . . . . . . . . . The effect of dietary vitamin E supplementation on TEARS values of cooked turkey dark and light meat during storage at 4°C ....O.......OOOOOOOIOOOOOOOOO Effect of dietary vitamin E supplementation on the concentration of individual cholesterol oxidation products (ug/g meat) in cooked ground dark and light turkey meat during refrigerated storage ........... Effect of vitamin E supplementation on the development of total cholesterol oxidation products in cooked dark and light turkey meat during storage at 4°C 0......00.00....0..........OOOOOOOOOOOOOO... Concentration of a-tocopherol in the microsomes of dark and light meat from turkeys fed control, and Vitamin E-supplemented diets eeooooooooooooooooeooo 73 74 78 81 82 89 Figure 1. 2. 3. 4. lantern-u Proposed scheme of lipid peroxidation ............. Scheme for cholesterol.oxidation .................. Glutathione peroxidase system ..................... Protection of membranal lipids by a-tocopherol .... Flow diagram of the feeding, slaughtering, and analytical protocols .............................. Relationship between TEARS and total COP formation in cooked ground dark turkey meat stored foruptoadays at 4°C 0.0.0.0000........OOOOOOOOO Relationship between TEARS and total COP formation in cooked ground light turkey meat stored for up to 3 days at 4°C ........................... Metmyoglobin/hydrogen peroxide-catalyzed lipid peroxidation in the microsomal membranes from dark meat of turkeys fed a control and vitamin E- supplemented (50 and 100 IU/kg feed) diets ........ Metmyoglobin/hydrogen peroxide-catalyzed lipid 'peroxidation in the microsomal membranes from light meat of turkeys fed control vitamin E-supplemented (so and 100 IU/kg feed) diets ......OOOOOOOOOOOOOOOO xi Page 19 32 33 43 ‘ 85 86 91 92 Oxidative deterioration of meat lipids can directly affect many quality characteristics of meat products including color, flavor, texture, nutritive value and even safety (Pearson et al., 1983). The lipids associated with the subcellular organelles are especially susceptible to oxidation by virtue of their high content of phospholipids containing relatively large amounts of polyunsaturated fatty acids (Pearson et al., 1977). Recent studies have shown the advantages of vitamin E supplementation at supranutritional levels in the diets of broilers (Asghar et al., 1989b, 1990; Lin et al., 1989b) and pigs (Buckley et al., 1989; Monahan et al., 1992b) in terms of increasing the oxidative stability of lipids in the subcellular membranes in muscle meat systems. In addition, dietary supplementation of vitamin E for the subsequent benefit of increased lipid stability in animal food products has been extensively reported. Furthermore, vitamin E might help to exploit the full growth potential of meat animals and yield better quality meat (Ashgar et al., 1991a). More recently, interest in the possible toxicological effects of lipid oxidation products, particularly 2 cholesterol oxidation products (COPS), has increased (Addis and Park, 1989; Kubow, 1990). Animal studies provide some compelling evidence implicating dietary cholesterol oxides as initiators of atherosclerotic lesions in blood vessels (Taylor et al., 1979; Imai, 1980; Peng et al., 1985, 1987). In a recent study, Engeseth (1990) reported that the environment in which cholesterol exists in muscle tissues may play an important role in determining its susceptibility to oxidation, and that the oxidation of nearby unsaturated lipids can result in the formation of free-radicals which may attack cholesterol. Therefore, the stabilization of membrane lipids by dietary vitamin E might enhance the stability of cholesterol in meat products. The overall objective of this study was to evaluate the effects of three levels of dietary vitamin E on the growth performance of male turkeys, and on the quality of turkey meat during refrigerated and frozen storage. Specific objectives of the study included the evaluation of the effects of dietary vitamin E on lipid oxidation, including cholesterol, in cooked ground turkey meat, and on the peroxidative stability of membranal lipids. WW Lipid oxidation is one of the major deteriorative processes responsible for the loss in quality of frozen meat or precooked meat products during storage (Pearson et al., 1977, 1983; Tichivangana and Morrissey, 1985; Gray and Pearson, 1987). Oxidative deterioration of meat lipids causes undesirable changes in the color, flavor, texture, nutritive value, and safety of meat products (Pearson et al., 1983). Lipid oxidation occurs in all stored foods to some extent and there is some concern about the toxicity of oxidation products (Pearson, 1981). With increasing emphasis on the role of diet in human diseases such as coronary heart disease, cancer, and stroke (Senate Select Committee, 1977a,b), questions have been raised relative to the role of lipids and their breakdown products in the etiology of these diseases. W The overall mechanism of lipid oxidation consists of three distinct phases: initiation, propagation and termination (Gunstone and Morris, 1983). 4 Initiation: RH ------ > R. + H. Propagation: R. + O ------ > ROO. ROO.+ ------ > ROOH + R. Termination: R. + R. ------ > RR R. + R00. ------ > ROOR ROO.+ R00. ------ > ROOR The reaction is initiated when a labile hydrogen is abstracted from a site on the unsaturated lipid (RH), with the production of a lipid alkyl radical (R.). In the propagation step, the reaction of the alkyl radical with oxygen which yields a peroxyl radical (ROO.) is followed by the abstraction of another hydrogen. A hydroperoxide (ROOH) and another free alkyl radical (R.) capable of perpetuating the chain reaction, are formed. In the termination step, the chain reaction can be stopped by the interaction of free radicals which produces non-initiating and non-propagating products. The important lipids involved in oxidation are the unsaturated fatty acid moieties, oleic, linoleic, and linolenic. The rate of oxidation of these fatty acids increases somewhat geometrically with the degree of unsaturation (Labuza, 1971). WWW Lipid oxidation in meat systems may be initiated and/or catalyzed by nonenzymic and/or enzymic reactions (Asghar et al., 1988; Rhee, 1988). In nonenzymic catalysis, the meat pigment myoglobin has 5 been implicated by many studies as playing an important role in the catalysis of lipid oxidation in uncooked red meat (Greene, 1969; Govindarajan et al., 1977; Verma et al., 1984; Rhee et al., 1986, 1987). Prior to 1970, myoglobin was viewed as the major catalyst of lipid oxidation in meat (Tappel, 1952, 1953; Younathan and Watts, 1959). However, Liu (1970a,b) determined the effects of pH and additives on linoleate oxidation catalyzed by metmyoglobin (Meth), Fe”; EDTA (1:1) chelate, and raw beef homogenates, and concluded that the catalytic activity of raw beef homogenates was due to both heme iron and nonheme iron. Later, Sato and Hegarty (1971), Love and Pearson (1974), and Igene et al.(1979) proposed that nonheme iron plays a major role in accelerating lipid oxidation in cooked meat and that heme iron does not catalyze the oxidative process. Tichivangana and Merrissey (1985) reported that purified metmyoglobin (Meth), when added to water-extracted beef or lamb muscle fibers containing the indigenous lipids, exerted virtually no catalytic effect on lipid oxidation. Rhee et al. (1987) determined the effect of Meth plus hydrogen peroxide (Meth-Mzoz) on lipid oxidation in a model meat system where ' Meth-IIZO2 was added to water-extracted beef muscle residue, rather than to a subcellular membrane fraction isolated from beef muscle. They demonstrated that the lipid-oxidizing activity of Meth-HZOZ in the raw system may be due primarily to the activated Meth and secondarily to the nonheme iron released from Meth by H202. 6 Recently, Johns et al. (1989) reported that ferric heme pigments are more powerful catalysts of lipid oxidation than inorganic iron compounds in raw muscle residue systems. They suggested that the apparently contradictory results obtained in previous studies with muscle model systems may be due to differences in sample storage time over which oxidation was monitored, and to the levels of prooxidants incorporated into the model systems. Menahan et al. (1992a) also determined that myoglobin and hemoglobin were more effective prooxidants than FeSO‘ in raw and cooked muscle systems. It is now generally accepted that these catalytic species decompose preformed hydroperoxides in cooked meat and are thus catalysts of the propagation stage of the oxidative process (Asghar et al., 1988). However, they do not account for the initiation of the oxidation process. A number of initiators of lipid oxidation in raw meat and other biological systems have been suggested and are summarized in Table 1. The extremely reactive hydroxyl (EO') radical has received much attention as one such initiator and is formed in biological tissues either by the Haber-Weiss reaction or by the.superoxide-driven Fenton reaction. The hydroxyl radical has the ability to abstract hydrogen from unsaturated lipids. These reactions occur by the following mechanisms: Haber-Weiss reaction: 02" + Fe?" ------ > 02 + Fez" 202" + 211* ------ > 02 + 11202 Fenton reaction: Fe” + 11202 ------ > Fe3+ + on' + no- Many of the proponents of the hydroxyl radical theory hold the Fenton-type reaction to be responsible for hydroxyl radical formation in biological systems (Fong et al., 1976; Gutteridge, 1984; Wilson, 1984). Table 1. Proposed initiators of lipid oxidation Proposed initiators Singlet oxygen Superoxide radical Hydroperoxyl radical Hydroxyl radical Crypto-hydroxyl radical Perferryl radical Ferryl radical Oxygen-bridged di-iron Porphyrin cation radical From Asghar et al. (1988) The initiation of lipid oxidation in biological membranes by perferryl and ferryl radicals has been extensively studied by Aust and his associates (Svingen et al., 1979; Tien and Aust, 1982). They reported that lipid oxidation is initiated by NADPH-cytochrome P450 reductase and by xanthine oxidase in liver microsomes (Figure 1). The 8 cytochrome P-450 reductase catalyzes the transfer of an electron from NADPH to ferric ion to generate ferrous ion, which can form a complex with dioxygen to produce perferryl or ferryl radicals. They assumed that these radicals have the ability to abstract hydrogen from polyunsaturated lipids to initiate the oxidation process. In these systems, chelation of iron by adenosine diphosphate (ADP) or a similar nucleotide or pyrophosphate was needed to enhance the solubility of iron in aqueous solution (Aust and Svingen, 1982). NADPH Xaithhe mum-cum» mun. Rosanna (hmhmo MI!” e- um LH AlP-Fe’3 02 EDTA-F62 Elam-Fir3 02 L- LOOH>é Lo,i.00 cnmso Ll-l m LH .9450 J Endlfiodunscflkt [INTJIXNH1: Lipid Peroxidatlon Figure 1. Proposed scheme of lipid peroxidation (Tien and Aust, 1982) 9 A direct involvement of heme protein (Meth and methe- moglobin) in lipid oxidation in muscle tissues has been suggested by Kanner and Karel (1985) and Harel and Kanner (1985) . They reported that the interaction of H202 with Meth generated activated Meth capable of initiating lipid oxidation. The active Meth complex is called the porphyrin cation radical (P.‘- Fe“ - O ). Initiation of lipid oxidation by the porphyrin cation radical proceeds via the two electron-reduction of the catalyst (Manner and Harel, 1985): p.’-Fe‘*-0+RH --------- >p-re‘*=o+R-+H* R~+oz --------- >300- Roo-+Rn --------- >ROOH+R' P-Fe‘*-O+ROOH --------- >P-Fe3*+ROO'+OH' ROO'+RH --------- >ROOH+R° Later, Manner et al. (1988a,b,c) reported that lipid peroxidation in muscle foods is initiated by "free" iron ions and hydrogen peroxide. This reaction is cycled by superoxide anion (Haber-Weiss) or by reducing compounds, e.g., ascorbic acid (redox cycling)(Ranner et al., 1987). Furthermore, Kanner et al. (1991) found the cytosolic extract of beef muscle can inhibit the activity of H202- activated myoglobin. Membrane lipid peroxidation initiated by an iron-redox cycle (ascorbic acid-ferric ions and NADPH enzymatic reaction) was inhibited partially by the high molecular weight fraction of the cytosolic extract, but 10 accelerated by the low molecular weight fraction. Enzymatic oxidation of muscle microsomes has been reported in numerous studies (Hochstein and Ernster, 1963; Lin and Multin, 1976; Rhee et al., 1984; German and Kinsella, 1985; McDonald and Hultin, 1987). Lipoxygenase catalyzes the hydroperoxidation of polyunsaturated fatty acids interrupted by a cis-methylene group (Tappel, 1963). Lipoxygenase has also been reported to initiate microsomal lipid oxidation in fish skin (German and Kinsella, 1985). Grossman et al. (1988) reported that lipoxygenase may be responsible for some of the oxidative changes occurring in fatty acids on frozen storage of chicken meat. Hultin and his colleagues (Lin and Hultin, 1976; Player and Hultin, 1977; McDonald et al, 1979; Hultin, 1980; Slabyj and Hultin, 1982) demonstrated the presence of enzymic systems in chicken and fish skeletal muscles that were capable of catalyzing the oxidation of microsomal lipids in the presence of cofactors, NADPH, ADP and ferric ions. Recent studies also revealed the presence of similar oxidation systems associated with beef and pork muscle microsomes (Rhee et al., 1984, 1985, 1987). The enzymic lipid peroxidation in skeletal muscle microsomes is dependent on NADH or NADPH - the relative effectiveness of the two differing among species - and requires ADP and Fea'or Fe” for maximum activity. Current research data indicate that enzymes or enzyme systems and possibly activated metmyoglobin are responsible 11 for initiation of lipid oxidation in raw muscle tissues and that metal catalysts (e.g., nonheme iron in red meat) also promote lipid oxidation in raw tissues, apparently through their role in the propagation of free radicals. WWW Lipids in meat are commonly classified as depot or adipose lipids and as intramuscular or tissue lipids (Watts, 1962; Love and Pearson, 1971). The depot fats are largely present as subcutaneous deposits and are appreciably present in the thoracic and abdominal cavities and between the muscles as intermuscular deposits. Depot fat tissue consists mainly of triacylglycerols which are composed of straight-chain, even-numbered carbon fatty acids, typically containing 16 or 18 carbon atoms (Dugan, 1971). The predominant fatty acids of depot fat of poultry are saturated or contain only one or two double bonds (Pearson et al., 1977). Tissue lipids are composed of deposits of triacylglycerols in fat cells, commonly called marbling in red meats, and membrane-bound lipids consisting chiefly of the phospholipids and lipoproteins (Love and Pearson, 1971). The phospholipids are susceptible to oxidation (Euege and Aust, 1978) and are believed to be the site of initiation of the oxidative process (Asghar et al., 1988) because of their high levels of polyunsaturated fatty acids (Marion and Woodroof,1966; Love and Pearson, 1971; Salmon and O'Neil, 12 1973; Igene et al., 1980; Melton, 1983). Although the phospholipid content of meat is relatively small, it still is very important in determining meat quality. The differences in fatty acid composition of the triacylglycerol and phospholipid fractions increase ongoing successively from poultry to pigs through ruminants, in which the polyunsaturated fatty acids are confined largely to the phospholipid fraction (Shorland, 1962). The main components of phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SP), and other minor components. Dawson et al. (1990) reported the fatty acids from the neutral lipid fraction contributed 94% of the total lipid fatty acids of chicken meat and the fatty acids from phosphatidylcholine and lysophosphatidylethanolamine fractions contributed 38% of the total phospholipid fatty acids. Within the phospholipid fraction, the lysophosphatidylethanolamine fraction has the greatest concentration of polyunsaturated fatty acids (70%) and is believed to be the most important phospholipid component contributing to development of warmed-over flavor (WOF). Pikul and Kummerow (1991) also indicated that the PC, PE, PI, and PS fractions accounted for 86-97% of the total PL, and that PC and PE were present in greater amounts than the other phospholipid fractions in chicken liver, heart, plasma, and egg yolk. The structural and metabolic functions of phospholipids 13 indicate that they are actively involved in oxidation during processing and storage. Igene et al. (1980) demonstrated that during frozen storage of model meat systems, the polyenoic fatty acids of PC and PE are not stable and undergo oxidation. The role of phospholipids in the formation of thiobarbituric acid-reactive substances (TEARS) has been extensively studied by Pikul et al. (1984), while Pikul and Kummerow (1991) reported that the concentration of TEARS and the percentage of polyenoic fatty acid, especially arachidonic acid, were highest in PI followed by PE, PS, PC and SP. The composition of tissue lipids is relatively constant (Koch et al., 1968). There is still considerable variation in the phospholipid content among species (Rancher et al., 1944; Waku and Uda, 1971; Tahin et al., 1981), from location to location within the carcass (Dugan, 1971), and as a consequence of the activity of muscle (Eloor, 1943; Acosta et al., 1966). The lipid content and composition of different poultry tissues vary considerably and influence the oxidative potential (Dawson and Gartner, 1983). The lipid content of turkey breast muscle is approximately half of that in thigh muscle (Wilson, 1974). Wilson et al. (1976) concluded that the red oxidative muscles have more phospholipid than white glycolytic muscles taken from the same species. However, the proportion of phospholipids in the lipids of white poultry meat is greater than that in dark meat (wangen et al., 1971; Pikul et al.,1984). On a 14 weight-percentage basis, the white and dark meat of chicken contain about the same amount of phospholipid (Katz et al., 1966). L121§_931§AIIQD_RIQQEELE Lipid oxidation in muscle foods leads to the formation of short-chain aldehydes, ketones, and fatty acids, as well as to the development of some polymers (Frankel, 1962; Keeney, 1962; Lundberg, 1962), all of which are believed to contribute to the undesirable flavor in meat, poultry and fish (Olcott, 1962; Watts, 1962; Love and Pearson, 1974). Hydroperoxides which do not contribute to off-flavor are the primary products of the reaction of oxygen with unsaturated lipids. The hydroperoxides are unstable and can undergo carbon-carbon cleavage on either side of the alkoxy radical. The alkoxy radicals also react with other radicals. These reactions account for many of the aliphatic aldehydes, alcohols, ketones, and hydrocarbons (Frankel, 1984) which are secondary products of lipid oxidation and which are responsible for the oxidized flavor in meat (Lillard, 1987; Love, 1987). Aldehydes, such as hexanal and heptanal, are major contributors to the loss of desirable flavor in meats because of their high rate of formation during lipid autoxidation and their low flavor thresholds (Ullrich and Grosch, 1987). Hexanal is the major autoxidation product of linoleic acid (Eadings, 1970; Ullrich and Grosch, 1987). 15 Alcohols contribute measurably less to the undesirable flavor quality of meat than the aldehydes due to the erelatively higher flavor thresholds (Drumm and Spanier, 1991). Pentanol and 1-octen-3-ol are the major alcohols formed during the storage of beef patties (Ullrich and Grosch, 1987). Drumm and Spanier (1991) found that the major ketones developed during the refrigerated storage of cooked beef were 2-heptanone and 2,3-octanedione. Hydrocarbons, on the other hand, have high flavor thresholds and make minimal contribution to desirable or undesirable flavors (Min et al., 1979; Frankel, 1985). Further oxidation may occur in the original peroxides or in the unsaturated aldehydes, which then undergo further degradation to form epoxides, cyclic peroxides and bicyclic endoperoxides (Enser, 1987). The interaction between lipids and protein/amino acids is contributed to by both lipid hydroperoxides and their secondary products of decomposition. Hydroperoxide radicals are very reactive with sulfur and amino functional groups of amino acids, whereas aldehydes and epoxides react with thiols from cysteine (Gardner, 1979). Cysteine and methionine side chains are especially susceptible to these oxidative, free radical reactions (Gardner, 1983; Lillard, 1987; Ladikos and Lougovois, 1990). Non-enzymic browning (Maillard) reactions also give rise to loss in nutritional evalue as well as to organoleptic changes. Carbonyls, such as aldehydes and dialdehydes, form Schiff bases with amines 16 and contribute less to the rancid odor; however, high molecular weight brown materials known as melanoidins, resulting from polymerization of Schiff bases, are unstable and produce new volatiles which affect flavor characteristics, especially during cooking and processing (Frankel, 1984). The Maillard reaction produces acyclic sulfur compounds, aromatic and non-aromatic heterocyclic compounds, and lactones which contribute to off-flavors (Chang and Peterson, 1977; Golovnja and Rothe, 1980; Shibamoto, 1980; Bailey, 1983; Shahidi et al.,1986). Protein solubility, emulsification and water-binding capacity associated with texture and rheological properties, also appear to be affected by the interaction between lipid oxidation products and proteins (Hall, 1987). MW Cholesterol, a member of the family of animal sterols, is the most abundant sterol in animal tissue, and occurs predominantly either in the free form or esterified to any one of a number of long chain fatty acids (Kritchevsky and Tapper, 1961; Smith et al., 1983). W In an earlier report, Stromer et al. (1966) reported that the Longissimus beef muscle contained 36-46 mg cholesterol [1009 raw tissue, while the subcutaneous adipose tissue contained 113-150 mg cholesterol/1009 lipid. Rhee et 17 al. (1982a) determined the cholesterol concentration in raw beef intermuscular fat (108 mg/lOOg raw tissue) and subcutaneous fat (114 mg/lOOg raw tissue) to be much higher than that of raw beef Longissimus muscle (62 mg/100g raw tissue). Most researchers agree that adipose tissue has a much higher concentration of cholesterol (mg per 1009 tissue, wet weight) than muscle tissue. Furthermore, researchers have sought to clarify the distribution of cholesterol between the muscle and the intramuscular fat by studying cholesterol distribution at the subcellular level. Hoelscher et al. (1988) found that the membrane contained 60-80% of the total cholesterol in muscle, while the cytosolic storage component contained 20-40%. In the adipose tissue, the membrane contained 8-12%, while the cytosolic storage component contained 88-92% of the total cholesterol. These investigations also showed that most of cholesterol in muscle tissue was contributed by the membrane fraction, while cholesterol in the adipose tissue, was contained primarily in the cytosolic storage fraction. They also demonstrated that increasing adipose tissue did not increase the total cholesterol content but changed the cholesterol distribution in muscle. Sweeten et al. (1990) determined the cholesterol content and subcellular distribution within intramuscular adipose tissue (marbling) and reported an average cholesterol concentration of 118 mg per 100 g intramuscular adipose tissue, with 54% in the cytoplasm and 18 46% in the membrane. Msshanism_2f_chelssterol_9xidatign Cholesterol, being an unsaturated lipid, is susceptible to oxidation (Smith, 1981, 1987; Maerker, 1987), and cholesterol oxidation products (COPS) have been detected in food products of animal origin (Missler et al., 1985; Park and Addis, 1985a, 1987; Nourooz-Zadeh and Appelqvist, 1989; Pie .t al., 1991). Cholesterol may be oxidized in vivo chemically, enzymically by specific enzymes, and perhaps by lipid peroxidation products (Smith et al., 1982). Initial stable cholesterol autoxidation products are the epimeric cholesterol 7-hydroperoxides, with the quasiequatorial 7E- hydroperoxide predominating (Teng et al., 1973a; Smith et al., 1973b), formed by reaction of ground-state molecular oxygen (302) with a C-7 cholesteryl radical (Figure 2). Formal reduction of the 7-hydroperoxides yields the corresponding 7-alcohols, cholest-5-ene-3E,7a-diol (7-a- hydroxycholesterol) and cholest-5-ene-3B,78-diol (7-8- hydroxycholesterol). Formal dehydration yields the 7- ketone, 3a-hydroxycholest-5-en-7-one (7-ketocholesterol) (van Lier and Smith, 1970a; Smith et al., 1973a; Tang et al., 1973b). Dehydration of the 7-ketone gives cholesta- 3,5-dien-7-one. The formation of epimeric cholesterol 5,6- epoxides during the autoxidation of cholesterol is facilitated by direct attack of an unoxidized cholesterol -19 8.... £1... .3 .3382“... .3 .5 .../.2. .5- en :8... ...- ... 24.3.85 ..8. in 5.2:? a. 55390 ...a. .anaa. :o.»oo.xo .oueumo.oao sou cacao» .~ ouau.s waxing»: . 1.28.: 1.33.8.8 - 1- . a 08.. - asses-3.8.0 . a a 33¢.88»...3.8.¢n 1.23.2.0 -... ... V/\/WWD 8.9.... £753.85; 5992672334808 0... _ ......W :24 (#90. \Y (mm? fees... ....iifmmwe... 2%.... ...... ....“ 8-31.3353 O o. 20 molecule by either 70- or 8-hydroperoxy cholesterol to form cholestan-S,6a-epoxy-38-ol and cholestan-S,68-epoxy-3E-ol, respectively. Hydration of either 5,6-epoxides gives Sa- cholestane-3B,5,68-triol (Smith and Kulig, 1975). Other autoxidation reactions occur, including attack on the side chain to form hydroperoxides, chief among which is cholesterol 25-hydroperoxide from which cholest-S-ene-3E, 25-diol (ZS-hydroxycholesterol) is derived (Van Lier and Smith, 1970a,b; Smith et al., 1973a; Teng et al., 1973b; Smith and Rulig, 1975). W Recently, the possible oxidation of cholesterol in foods has caused some concern due to undesirable biological implications of COPS. These products may interrupt sterol metabolism, and also produce cytotoxic, angiotoxic, mutagenic and even carcinogenic responses (Kandutsch et al., 1978; Taylor et al., 1979; Smith, 1981; Addis et al., 1983; Addis, 1986). Many studies have demonstrated the presence of COPS in animal foodstuffs such as egg products (Missler et al., 1985; Morgan and Armstrong, 1987; Nourooz-Zadeh and Appelqvist, 1987), heated tallow (Eascoul et al.,1986; Park and Addis, 1986), dairy products (Nourooz-Zadeh and Appelqvist, 1988; Sander et al., 1989), and meat products (Higley et al., 1986; Park and Addis, 1987; De Vore, 1988; Engeseth, 1990; Pie et al., 1991; Zubillaga and Maerker, 1991). 21 Eight common COPS have been identified: 25-hydroxy- cholesterol, cholestane-triol, a- and E-epoxide, 7a- and 7B- hydroxycholesterol, 7-ketocholesterol and cholesta-3,5-dien- 7-one, which is actually an artifact derived from 7-keto- cholesterol during isolation (Peng et al., 1978, 1979; Smith, 1980, 1981; Tsai, 1984; Missler et al., 1985; Park and Addis, 1985a,b, 1987; Nourooz-Zadeh and Appelqvist, 1987). Egg products have been the most extensively studied as they have high cholesterol contents. Missler et al. (1985) analyzed a scrambled egg mix which had been stored 5 years and identified 7 COPS. Nourooz-Zadeh and Appelqvist (1987) did not detect COPS in fresh egg yolk, but a dehydrated egg mix stored for 12 months contained a- and B-epoxide, 7a- and 7E-hydroxycholesterol and 7-ketocholesterol. After 8 years of storage, a dehydrated egg yolk sample contained the above COPS plus 25-hydroxycholesterol and cholestane-triol. Finocchiaro et al. (1984) reported COPS in 4 of 8 brands of grated cheeses but not in fresh ungrated cheeses. No COPS were found in freshly bleached butter oils but after storage for 90 days at 15°C, the 7-hydroxycholesterols were identified. Park and Addis (1987) reported finding similar cholesterol oxides in various meats, with 7-ketocholesterol being most predominant. Cholestane-triol, a very atherogenic cholesterol oxide, was also detected for the first time in a meat sample. Sander et al. (1989) reported 22 7-ketocholesterol and the two epoxides in dehydrated chicken, beef and turkey, but the method which involved solvent extraction (Folch et al., 1957) and overnight cold saponification was somewhat cumbersome. Zubillaga and Maerker (1991) demonstrated that the concentrations of 7- keto-cholesterol and a- and 8-epoxide in raw veal, beef, pork and chicken muscle tissues are less than 1 mg/kg, while 7-ketocholesterol constituted more than 50% of the cholesterol oxidation products. Engeseth (1990) reported 7- ketocholesterol, 7-8-hydroxycholesterol and the B-epoxide as the predominant cholesterol oxides in veal and beef samples. The study of Sander et al. (1989) indicated that the concentrations of COPS in fresh products were generally lower than those in processed products. Foods containing the greatest concentrations of COPS were dehydrated egg and meat products. Generally, the greater the cholesterol content and the more severe the conditions of processing, the greater the resultant COP content. Park and Addis (1987) and Monahan et al. (1992b) noted greater cholesterol oxide development with increasing degrees of rancidity (as determined by TEARS value) in meat. Pie et al. (1991) also reported that cooking and frozen storage lead to cholesterol oxidation in meat products. The percentage of oxysterols to cholesterol varied on average from 0.5 to 0.8%. Engeseth (1990) also showed that dietary vitamin E supplementation decreased cholesterol oxidation in veal because the membranal lipids would be stabilized by a-tocopherol, thus 23 protecting cholesterol from oxidation. Monahan et al. (1992b) demonstrated that after two days of refrigerated storage, cholesterol oxides represented 2.7% of the initial cholesterol content of cooked pork from pigs fed a basal level of vitamin E (10 mg/kg diet) and 1.6% of the initial cholesterol content of cooked pork from pigs fed a supplemented diet (200 mg/kg diet). WW Phenolic antioxidants are compounds capable of slowing the rate of oxidation in fat or fat-containing foods. Their action is due to donation of hydrogen or an electron which reacts with free radicals to form inert products, thus terminating the chain reaction mechanisms of lipid oxidation (Dziezak, 1986). The stability of lipids can be improved by antioxidants, such as synthetic phenolic antioxidants (Chastain et al., 1982) and natural antioxidants (Pratt, 1972; Rhee et al., 1983; Rhee, 1987). W Synthetic phenolic antioxidants include butylated hydroxyanisole (EHA), butylated hydroxytoluene (EHT), propyl gallate (PG) and tertiarybutyl hydroquinone (TEHQ). A number of studies have indicated that these antioxidants can effectively inhibit lipid oxidation in meat (Greene, 1969, Greene et al., 1971; Chastain et al., 1982). However, there are two major limitations to the use of synthetic 24 antioxidants in meats. First, they are approved for use in only a limited number of meat products and each new synthetic antioxidant must be subjected to a lengthy and costly process of evaluation for proof of safety. Second, consumers are increasingly concerned about the use of chemical additives in foods (Rhee, 1987). W A promising approach to retarding lipid oxidation in meat products is through the use of naturally occurring antioxidants. Compounds possessing antioxidative activity have been found in vegetable extracts (Pratt, 1965), citrus products (Park et al., 1983), oilseed products (Whittern et al., 1984), oat flour (Daniels and Martin, 1968), rosemary extracts (Houlihan and Ho, 1985) and retorted meat extracts (Einerson and Reineccius, 1978). The predominant antioxidant compounds include flavonoids, phenolic acids, tocopherols, and phenolic terpenes (Rhee, 1987). Flavones and flavonoids have molecular structures which make them useful as antioxidants. Their antioxidant activity, which is related to the ketone in the 4 position of the pyrone ring and the 2,3-olefin group, has been investigated by several workers (Simpson and Uri, 1959; Metha and Seshadri, 1959; Crawford et al., 1961). However, the flavones (e.g., quercetin) have structural characteristics associated with mutagenic and perhaps carcinogenic activity (McGregor and Lard, 1978). Oleoresin rosemary (OR) is one of the 25 important spices which has antioxidant properties and contains a number of compounds such as rosmanol, carnosol, rosmaridiphenol, and rosmariquinone which impart antioxidant activity similar to or greater than that of EHT (Houlihan and Ho, 1985). Lai (1989) found that sodium tripolyphosphate (STPP)/OR and STPP/TEHQ both effectively retarded the oxidative degradation of polyunsaturated fatty acids in chicken nuggets during frozen storage. Stoick et al. (1991) also demonstrated that OR was an effective antioxidant which, when combined with sodium tripolyphosphate, produced an additive protective effect in restructured beef steak during frozen storage. Development of such products as antioxidants is desirable because they are derived from naturally occurring substances. Recently, St. Angelo et al. (1990) reported that lipid oxidation in ground beef patties was retarded by the addition of metal chelators, free radical scavengers, rosemary and sodium .alginate. iA1EhI:I9£22h9£21_1l_l!l!_fi!l&lll Alpha-tocopherol is the most prominent natural antioxidant in meat (Dugan, 1976; Watts, 1962). It is a powerful antioxidant and functions as a lipid-soluble free radical scavenger (Linder, 1985). It has been extensively' studied, with its antioxidant properties being widely used in meat products. 26 Whittier: At least eight compounds with vitamin E activity ((a-, 8-, 7-, J-tocopherol and a-, 8-, y-, 6-tocotrienol) have been isolated from plant sources. All have a 6-chromonal ring structure and a side chain. The tocols have a side chain, and the trienols have a similar structure, with double bonds at the 3', 7', and 11' positions of the side chain. Eoth tocols and trienols occur as a variety of isomers that differ by the number and location of methyl groups on the chromanol ring. Alpha(a)-tocopherol is the most active form of vitamin E, and in foods of animal origin it accounts for almost all the vitamin E activity (Eauernfeind, 1980). -Many studies on the distribution of tocopherols in animal tissue have been made for several species. The uptake of tocopherol by tissues varies directly with the logarithm of the tocopherol intake (Eieri, 1972; Gallo- Torres, 1980). This is illustrated by data on the tocopherol content of lung in rats and in the muscle of chickens (Machlin, 1991). This relationship represents a departure from what occurs with most other vitamins, which usually have distinct deposition thresholds in tissues other than the liver and may provide an explanation for the pharmacological effects of vitamin E. Tissues vary considerably in their vitamin E levels with no consistent relationship to lipid parameters. Asghar et al. (1989b) reported tissue a-tocopherol concentration in swine in the 27 following order: liver >heart >lung >kidney. Monahan et al. (1989) found the same trend in tissue samples of pigs supplemented with 200 international units (IU) vitamin E/kg feed. However, Engeseth (1990) reported the concentrations of a-tocopherol for the tissues of veal calves decreased in the following order: liver >adipose tissue >kidney >lung >heart. With supplementation, the same order of tocopherol concentration was maintained, but adipose tissue had the highest concentration of a-tocopherol because it is one of the major storage deposits of vitamin E. In addition, the rate of depletion of tocopherol in adipose tissue is slower than in other tissues (Machlin, 1980). Tissue distribution studies demonstrated significant increases in a-tocopherol of various tissues; however, the percentage increase of each tissue varied from study to study. The overall deposition of a-tocopherol in the muscle tissues was studied by Lin et al. (1989a). They reported that approximately 0.88% of the total a-tocopherol consumed by the broilers was deposited in the tissues, with 0.56% in the dark meat and 0.32% in the light meat. The different rates of deposition apparently result from physiological variations in the vascular network of dark and white muscle tissues (Sheldon, 1984). Dark meat from broilers has a more highly developed vascular system and a higher lipid content than white meat. Therefore, a-tocopherol is deposited to a greater degree in dark meat than in white meat. In membranes, the earliest reliable estimate of the 28 subcellular distribution of a-tocopherol was made by Krishnamurthy and Eieri (1963) who found that 52-62% of the total tocopherol in the homogenate of rat liver was in the mitochondria, 21-23% was in the microsomal fraction, 6-9% was in the nuclei and 15-19% was in the soluble fraction. Mellors and Barnes (1966), on the other hand, reported values of 14%, 12%, 44%, 31% for the nuclei, mitochondria, microsome and supernatant fraction, respectively. These results are clearly different from those of previous workers. Recent studies reported higher a-tocopherol concentrations in microsomes than in mitochondria. (Asghar et al., 1989b; Monahan et al., 1989; Engeseth, 1990). WWW It has been proposed that vitamin E plays a role in the regulation of heme biosynthesis (Nair, 1972), acts as a modulator in immune response (Eendich et al., 1986) and in gene regulation (Patnaik and Nair, 1977), and protects the selenium-containing proteins (Caygill et al., 1971). The primary function of the tocopherols is as antioxidants in hydrophobic environments. All of the four tocopherol vitamers (a-, 8-, y-, 6-tocopherol) are effective chain- breaking antioxidants in bulk unsaturated lipids. However, since a-tocopherol is the predominant tocopherol found in human and animal biomembranes, most studies have focused on this vitamer. The commonly accepted theory for initiation and 29 propagation of free radical-mediated oxidation and their inhibition by antioxidants is outlined in Table 2. Table 2. Autoxidation and antioxidant reactions involving vitamin E 1. Initiation (formation of a free radical) LH __umunmm__> L. . 2. Reaction of radical with oxygen 1..+02 >Loz- 3. Propagation LO' +LH > L. + ROOH 4. Antioxiéant reaction LOZ°+E >E+LOOH 5. Regeneration E. + C > E + C. c. + NADPH ElflflflmlJflflflfllllhfllB> c + NADP a. + zcsn M> E. + cssc cssc + NADPH MW> zesn +NADP 6. Termination E. + E. -——————> E-E (dimer) E. + LOZ' ———> £001. (1?) Abbreviation: LH, fatty acid; L', fatty acid radical; LOz', peroxy radical; E, tocopherol; E' , tocopheroxy radical; LOOH, hydroperoxide; C, ascorbic acid; C', ascorbyl radical; GSH, reduced glutathione; GSSG, oxidized glutathione. (Machlin, 1991) Alpha-tocopherol is currently believed to act as a quencher of oxygen- or carbon-centered fatty acyl radicals or active oxygen via donation of a hydrogen atom from the phenyl hydroxyl of the chromanol ring. The rate of reaction of a- tocopherol and other phenols with peroxyl radicals of styrene under conditions of controlled peroxyl radical formation has been reported by Burton and Ingold (1981, 1986) and Burton et al. (1985). Their results demonstrate 30 that the radical quenching (hydrogen donating) capability of a-tocopherol exceeds that of the other tocopherols and synthetic phenolic antioxidants, and thus may explain in part the superior vitamin E activity of the a-vitamer relative to the 8-, 1-, and 8- forms. Apparently, a- tocopherol is a more effective hydrogen donor than the other tocopherols since electron-releasing methyl groups ortho to the phenolic hydroxyl, lacking in the other vitamers, stabilize the phenoxyl radical and increases the rate of the reaction: ROO' + ArOH ---> ROOH + MO“ The number of alkyl radicals with which each phenoxyl group reacts is approximately two (Burton and Ingold, 1981). The efficient antioxidant activity of a-tocopherol, despite its low concentration in biological membranes relative to that of polyunsaturated fatty acids, suggests that the effective half-life of the vitamin in membranes is rather long. Several studies have indicated that the chromanoxyl radical of a-tocopherol may be reduced to regenerate a-tocopherol by ascorbic acid (Packer et al., 1979) or glutathione (Pryor, 1976). Vitamin E is a special antioxidant compared with synthetic antioxidants for the following reasons: (1) a- tocopherol is located primarily in the membrane where free radicals are generated by lipid oxidation (Molenaar et al., 1972); (2) a-tocopherol acts as a free radical scavenger and thus protects against lipid oxidation (Linder, 1985); (3) a- 31 tocopherol could be regenerated by ascorbic acid or glutathione to maintain a longer half life in the membranes. Molenaar et al. (1980) proposed that a-tOCOpherol is situated in the membranes such that the chromanol ring is at the polar surface and the phytol side chain is in contact with the unsaturated fatty acids of the phospholipids in the nonpolar interior. This positioning of a-tocopherol allows effective radical scavenging as it would be adjacent to the membrane-bound enzymes such as NADPH oxidase, which generates free radicals. McCay and King (1980) reported that the superoxide anion (05’) interacts with hydrogen ions to produce hydrogen peroxide, which is distributed in both the aqueous and membrane phases of the cell. Glutathione peroxidase, a selenium-containing enzyme, destroys the hydrogen peroxide in the aqueous phase, thus shifting most of the hydrogen peroxide into the membrane. Any hydrogen peroxide remaining in the membrane may react with superoxide anion to form hydroxy radicals ('OH) , which can react with tocopherol localized in the membrane. If insufficient‘ tocopherol is available to trap 'OH, this extremely reactive radical may initiate peroxidation of the polyunsaturated fatty acid in the membrane. This hypothesis accounts for many experimental observations, including the role of selenium (as part of glutathione peroxidase), in alleviating some of the symptoms of vitamin E deficiency. Some of these concepts are summarized in Figures 3. and 4. 32 OLUTATHION! "BOND”! "STE“ p-oxioarios ”"O’RmLUCONAf‘ e-rocomeaor 694 "some“: can aeoucraee on oeuvonocm,“ I 3 0. : J-R H GLUCOSE-8' M: PEROXIDATION ‘ m ”SPRAY! I \\ UALONALDEHYDI O WATIDYL ITNMOLAMINI 'LUOSESINT PRODUCTS “WORN Figure 3 . Glutathione peroxidase system. RH: Polyunsaturated fatty acid; ROOE: fatty acid hydroperoxide; ROE: hydroxy fatty acid; 68H: reduced glutathione; 0880: oxidized glutathione (From Maohlin, 1984) 33 STAILE COMPOUNDS come auction resets slth 10A OH HALONYL elements! 88” DAMAGE TO sealants GLUTATHIONE in presence e! in essence e! 5e an morals: PEROXIDASE "adequate dietary Se zuzmes mo '. TISSUES «H (b) FREE- . RADICAL . 12.1.,” -'..';"-"'-'5.335:' REACTIONS GLUTATHIONE , PEROXIDASE O superoxide (so, °a "" .....i... 7"”! °"' ’39 N. e/,‘. \ '0" Ive—‘7 h. —;°“ " °I"’°"- OXIDASES -—/ (xanthine ealdese) I" mm”. edeeuele 5e Figure 4 . Protection of membranal li ids a-toco Hachlin, 1984) p by pherol (From 34 _ . . . VA, . 1» . _ ;<.. y, ,y ., .. ,‘ .LL £hl£l£§9£1£§1£l_91_lfini Lipid oxidation plays a major role in the loss of quality of muscle foods during storage (Pearson et al., 1983). Off-flavor development in meat has been attributed to the formation of a complex mixture of carbonyls during the autoxidation of unsaturated fatty acids (Reindl and Stan, 1982; Morrissey and Apte, 1988). Oxidation products ' also adversely affect color, texture, nutritive value (Pearson et al., 1983) and even the safety of meat (Addis, 1986). .Vitamin E is known to be the major lipid antioxidant of nature because of the benefits of its biochemical function which were mentioned in the previous section. Tocopherols are absorbed by the animal, transported, and deposited in body lipids. Deposition is usually relatively uniform throughout the animal body on a lipid basis. Incorporation of a-tocopherol into the muscle prior to slaughter by dietary supplementation with vitamin E has a more beneficial antioxidative effect than addition of a-tocopherol to meats just prior to processing (Faustman et al., 1989b). This is expected, as it is difficult to distribute lipid-soluble antioxidants to the sites of lipid oxidation in intact meats (Watts, 1961; Faustman et al., 1989b). Free radical scavengers are most effective during the initial stages of oxidation so that a build-up of hydroperoxides can be prevented. It is well established that vitamin E 35 supplementation of diets results in increased a-tocopherol concentrations in the tissue and a concomitant increase in the stability of chicken (Sklan et al., 1983; Sheldon, 1984; Lin et al., 1989b), pork (Buckley and Connolly, 1980) and veal (Shorland et al., 1981; Engeseth, 1990). Buckley and Connolly (1980) reported lower thiobarbituric acid reactive substances (TEARS) during the storage of fresh and frozen raw pork from swine which were fed a vitamin E-supplemented diet. Buckley et al. (1989) evaluated the effects of dietary vitamin E (200 IU/kg feed) on the oxidative stability of pork chops and ground pork during refrigerated and frozen storage. They found that the stability of ground pork patties from the pigs fed the short-term (4 weeks) and long-term (10 weeks) vitamin E supplements was not significantly different. However, they did report that the vitamin E-supplemented pork was significantly (p < 0.05) more stable than the control pork. In a further study, Asghar et al. (1991b) evaluated the influence of three levels of dietary vitamin E (10, 100 and 200 IU/kg feed) on the subcellar deposition of a-tocopherol in the muscle and on certain quality characteristics of pork (oxidative stability of lipids, color and drip loss). The concentrations of a-tocopherol in the adipose and muscle tissues increased with increasing levels of dietary vitamin E, with concomitant decreases in TEARS, color loss and drip loss. Erin et al. (1986) proposed that a-tocopherol protects the membrane from the action of phospholipases 36 which remove the fatty acid moiety from phospholipid molecules. The loss of phospholipids from membranes causes a substantial reduction in their fluidity (Ramada et al., 1985) and hence may increase drip loss. Poultry meat is very susceptible to oxidative rancidity during frozen storage (Pearson et al., 1977), although it can be stabilized by dietary vitamin E supplementation (Webb et al., 1972; Uebersax et al., 1978; Eartov et al., 1983). Marusich et al. (1975) found that turkeys required higher levels of vitamin E supplementation than broiler chickens to delay the onset of rancidity. Recently, Lin et al. (1989b) investigated the effects of dietary oil (with different degrees of unsaturation) and vitamin E supplementation on lipid composition and stability of broiler meat. They found the broilers which were fed the diets containing the more highly unsaturated oils had more unsaturated fatty acids in the muscle tissue, while dietary vitamin E significantly improved the oxidative stability of dark and white meat during refrigerated and frozen storage. ’The effect of vitamin E supplementation on pigment and lipid stability of beef has been studied by Faustman et al. (1989a). They reported that steaks from Holstein steers fed 370 IU vitamin E/day maintained their red color longer during storage than those from the control animals. Faustman et al. (1989b) reported more than a two-fold increase in the concentrations of a-tocopherol in the steaks from the supplemented animals. Pigment and lipid stability 37 was improved by the absorption of a-tocopherol and its incorporation into the cellular membranes. Engeseth (1990) also evaluated the effect of vitamin E supplementation on membranal lipid peroxidation and meat lipid stability (including cholesterol). She proposed that the oxidative stability of raw and cooked veal muscle held at 4°C was improved by vitamin E supplementation and cholesterol oxide development was reduced in cooked meat samples from the supplemented animals. WWW Winn Very few reports of growth performance, as affected by vitamin E supplementation, have been published. In an early study, the negative effect of oxidized dietary oil on the growth performance of rats was investigated by Burr and Barnes (1943). Oxidized dietary oil when fed to experimental animals (rats and pigs) causes various types and degrees of abnormalities. Severe oxidation causes a readily detected illness in broilers called encephalomalacia. Century and Horwitt (1959) found that vitamin E can prevent encephalomalacia which is a staxis symptom resulting from hemorrhages and edema within the molecular and granular layers of the cerebellum in chickens by functioning as a biological antioxidant and interrupting free radical-initiated lipid peroxidation. Shermer and Calabotta (1985) reported that broilers are routinely fed 38 diets containing slightly oxidized fat and this can adversely affect the growth performance of the broilers, thereby reducing the profits of the poultry grower. Waldroup et al. (1960) reported that turkeys receiving diets containing the synthetic antioxidant, ethoxyquin, were significantly heavier than birds receiving diets containing no antioxidant. This might be due to the prevention of oxidation of oil in the diet. Shermer and Calabotta (1985) also reported that alpha-tocopherol effectively delayed the symptoms associated with the presence of oxidized oils in the diet. Studies by Lin et al. (1989a) revealed that EHA, BHT and a-tocopherol can improve growth when added to broiler diets. These investigators also reported that .short-term and long-term vitamin E supplementation significantly (P < 0.05) increased body weights of the broilers. 'Asghar et al. (1991a) demonstrated the positive effects 'of supranutritional levels of vitamin E (100 and 200 IU/kg feed) on the performance of growing pigs, and feed conversion efficiency and carcass yield. Their studies confirmed the hypothesis that undesirable lipid peroxidation that possibly occurs in the subcellular membranes and other tissue components during normal growth and development of meat animals can be minimized by vitamin E supplementation of the diet at supranutritional levels. On the other hand, Asghar et al. (1991a) found high vitamin E supplementation may prevent growth enhancement because ingestion of large 39 amounts of vitamin E may inhibit absorption of vitamin A, which is an important vitamin for growth (Eieri et al., 1983). EHPERIIEITAL W Cholesterol oxide standards used in this study were: cholest-S-en-BB-ol (cholesterol), cholest-5-ene-38, 7a-diol (7a-hydroxycholesterol), cholest-S-ene-38, 78-diol (78- hydroxycholesterol), cholest-S-ene-38, ZOa-diol (200- hydroxycholesterol), cholest-S-ene-38, 25-diol (25- hydroxycholesterol), 5-cholesten-38-ol-7-one (7 ketocholesterol), cholestan-38, 5a, 68-triol (triol), cholestan-SB, 68-epoxy-3B-ol (B-epoxide), cholestan-Sa, 6a- epoxy-38-ol (a-epoxide), 50-cholestan-38-ol-6-one (6- ketocholesterol) and Sa-cholestane. These chemicals were obtained from Steraloids Inc., Wilton, NH. Ellfllnil Bis-(trimethylsilyl)-trifluoroacetamide (ESTFA) and pyridine were obtained from Pierce Chemical Co. (Rockford, IL). All other reagents used in the experiments were analytical grade. 40 41 W One hundred and eight, day-old male turkeys (Cooper Hatchery, Oakwood, OH) were randomly divided and placed into nine pens (12 turkeys/pen). The pens of birds were randomly assigned to receive one of three dietary treatments (3 pen/treatment). The three dietary treatments consisted of standard turkey feed formula (Table 3) containing different levels of vitamin E supplementation: (1) 12.5 IU vitamin E (as a-tocopherol acetate, EASF Corp, wyandotte, MI)/kg feed, (2) 50 IU vitamin E/kg feed, and (3) 100 IU vitamin E/kg feed. The turkeys were grown to 18 weeks of age at the Poultry Science Research and Teaching Center, Michigan State University. The birds were weighed initially and at 6, 12 and 18 weeks, and body weights were individually recorded. The feed consumed by each pen of turkeys was recorded to determine feed efficiency. At the end of the feeding trial, four turkeys from each pen were slaughtered in the poultry processing lab of Animal Science, Michigan State University and immediately prepared for analysis as described below. W The carcasses were cut and separated by manual skinning and deboning. Dark (thigh) and light (breast) meat from the individual turkeys were separately wrapped and stored properly for analysis (Figure 5). For the refrigerated study, dark or light meat samples were placed on polystyrene trays, wrapped in oxygen-permeable PVC meat stretch-wrap 42 Table 3. Composition of turkey rations used in the feeding study Pounds/ton of feed Turkey Turkey Turkey Turkey Turkey Ingredient Starter Grower Grower Finisher Finisher 1 2 1 2 Corn, yellow 737 743.5 990 1180.5 1421 Soybean meal 1082 1042 815.5 616 370 (44%) Meat and bone meal 50 50 50 50 50 (50%) Fat 49 86 80 89 92 Dicalcium phosphate 36 36 27 29 31 Limestone 26. 26 22 21 21 Salt 8 8 8 8 8 Vitamin/trace 8 7.5 6.5 6.5 6 minerals Methionine 4 1 1 --- 1 Total 2000 2000.0 2000.0 2000.0 2000 %Protein 28.5 27.5 23.6 20.0 15.7 Turkey Starter; 0-2 weeks; 1.6 lb of feed/bird. Turkey Grower 1; 15-56 days; 16.8 lb of feed/bird. Turkey Grower 2; 57-77 days; 15.5 lb of feed/bird. Turkey Finisher 1; 77-112 days; 28.0 lb of feed/bird. Turkey Finisher 2; 113-126 days; 15.0 lb of feed/bird. 43 .uaooououa Hoowuhausa one .uswuoususuau .usqooou can no Iouuodo soda .n enough .gisiiighigsgotisoc; .texiaua288:5:sirsi.z:5zia§65angli:l§.9§sex.séctaaH 3o. 9.5 J a .63» 5.3.2.6 22.. 355602 J 3.8 l 5:025 .283.on 4 3.222 38:88.... n. £35m 26.2.6 1 $35 2.82.6 I. .32..» :32. ..80 628.. 538a a uwmmmmoc. .....6. ... 62.82 09.2» canoe“. :32“. a 8.2352. 39.2w canoe“. _ _ _ _ .3: Eu... 2a {.5 «33200 68. 95. ..s a. 8. .a 68. 95. ...> 2 cm .w 68. 95. ...) 2 3: .26 .228 .. .383 2.9 “acoESoEcoEBoc 9.68“. 2.9.5... _ 329.0.» 44 (Reynolds Metal Company, Richmond, VA), and kept at 4°C under fluorescent light for 8 days to determine the oxidative stability, color change and drip loss of the raw meat individually during storage. For the frozen study, dark or light meat samples were wrapped in oxygen-permeable PVC meat stretch-wrap, stored at -20°C, and evaluated for oxidative stability after 3 and 6 months. Samples of the frozen meat were removed after 3 and 6 months (thawed at room temperature for one hour), and evaluated for changes in oxidative stability, color and drip loss during refrigerated storage. The oxidative stability of lipids in cooked meat from each turkey (previously frozen raw for 3 months) was determined by grinding the meat, cooking in plastic bags in a water bath at 70°C for 30 minutes, and then storing at 4°C. Dark or light meat from each turkey was vacuum-packed and kept at -20°C for a-tocopherol analysis. Dark and light meat samples from each treatment were pooled for the membranal lipid oxidation studies and stored at -20°C . WWW! Lipid oxidation in the muscle tissues from four turkeys per pen was monitored using a modification of the 2- thiobarbituric acid (TBA) distillation procedure of Tarladgis et al. (1960). Ten grams of finely cut-up muscle tissue from individual turkeys were blended in a tissue homogenizer (Ultra-Turrax, Model #SDF 1810, Tekmar Co., Cincinnati, OH) with 10 ml of antioxidant solution (5% 45 propyl gallate and 5% ethylene-diaminetetraacetic acid (PG/EDTA) in distilled water). The homogenate was transferred to a distillation flask (500 ml), using 87.5 ml of deionized water. To the flask were added 2.5 ml HCl/Hg) (2:1) and 2 drops of antifoam agent (10% silicone emulsion in water, Thomas Scientific, Swedesboro, NJ). The sample was distilled until 50 ml of distillate were collected in a 50 ml screw-capped test tube. A 5 ml aliquot of the distillate was added to 5 ml of TBA reagent (0.2883 g TBA/100 ml distilled water) in a 150 x 18 mm screw-capped test tube. The capped tube was placed in a boiling water bath for 35 minutes. After the tube was cooled to room temperature, the color intensity was quantitated by measuring the absorbance at 532 nm with a Bausch and Lamb Spectronic 2000 spectrophotometer (Bausch and Lamb, Rochester, NY). Absorbances were converted to TEARS (TBA- reactive substances) values by multiplying by a factor of 6.2 (Crackel et al., 1988) and expressing as mg malonaldehyde (MDA)/kg tissue. MW W (a) Plasma: Blood samples were taken from three turkeys per pen at specific times using 10 ml heparinized syringes. The blood was centrifuged (speed 4, IEC Clinical Centrifuge- International Equipment Co., Needham Height, MA) within 30 46 minutes of collection and the plasma fraction stored at - 20°C until required. After thawing, 1 ml of the plasma was transferred to a screw-capped test tube containing 0.5 ml of a 5% aqueous ascorbic acid solution (prepared with distilled water) and the internal standard, 100 ul of an ethanolic solution (0.2 mg/ml) of a-tocopherol acetate (Sigma Chemical Co., St. Louis, MO). After flushing with nitrogen and capping, the tube was vortexed on a Big Vortexer (Glass-Col Apparatus Co., Terre Haute, IN) for 5 seconds. Ethanol (1.5 ml) was then added and the tube was flushed with nitrogen and vortexed for approximately 15 seconds. Bexane (5 ml) was added and the tube was vortexed for 4 minutes after flushing with nitrogen. The tube was centrifuged at 1000 x G until two layers were clearly identified. The hexane (top) layer was transferred by a disposable pipette to another clean test tube. Extraction with another 4 ml aliquot of hexane was repeated, and the hexane layers were combined and evaporated under a stream of nitrogen. When the hexane was completely evaporated, 200 pl of ethanol were added to the tube, which was then quickly flushed with nitrogen, vortexed and frozen until required for HPLC analysis. (b) Muscle tissue: Ground muscle tissue (5 g) from four turkeys per pen was homogenized with 5 ml of distilled water in a 50 ml beaker. One gram of the homogenate was placed in a screw- capped test tube, to which were added 2 ml 1% ethanolic 47 pyrogallol solution and 0.3 ml 50% KOH. The tube was flushed with nitrogen, capped and vortexed for 1 minute. The tube was placed in a water bath (70°C) for 30 minutes. After cooling to room temperature, 4 ml hexane were added to the tube which was recapped and vortexed for 1 minute. After centrifuging at 100 x G for 2 minutes, the upper layer was removed carefully to a clean screw-capped test tube. The hexane extraction step was repeated once more and the hexane extracts combined. After washing two times with distilled water and drying over anhydrous Nazso‘, the hexane extract was evaporated to dryness under a stream of nitrogen. The residue was redissolved in 200 ul ethanol and vortexed for 30 seconds. The tube was flushed with nitrogen, capped and stored at -20°C for HPLC (high performance liquid chromatographic) analysis. (c)Microsomal fraction: The microsomal fractions of control and vitamin E- supplemented turkey muscles were isolated by a centrifugation procedure similar to that reported by Asghar et al. (1990). Samples (100 g) of light or dark meat (pooled from 12 turkeys in each group) were homogenized with one liter of buffer solution (0.1M KCl, 0.2% glycerol, 0.025M Tris-MCl buffer) in a blender for 2 minutes and centrifuged at 1000 x G for 10 minutes in a Sorvall RC2-B centrifuge (Ivan Sorvall Inc. Norwack, Conn.) to isolate the sarcoplasmic components. The sarcoplasmic extracts were then subjected to the differential centrifugation procedure 48 of Schenkman and Cinti (1978) to separate the mitochondrial and microsomal fractions. The mitochondria extracts were centrifuged at 10,000 x G for 15 minutes. Calcium chloride was added to the supernatant to give a final concentration of 8 mM for aggregating the microsomes. The mixture was centrifuged at 13,200 x G for 30 minutes to separate the microsomal fraction. The subcellular fractions were not purified further to remove contaminating myofibrillar proteins and fragments of the plasma membrane. The microsomes were assayed for a-tocopherol in a manner similar to that for muscle, using 0.5 g membrane to which were added 0.5 m1 of deionized water, 2 ml 1% ethanolic pyrogallol and 0.3 ml 50% ROM. (d) HPLC quantitation of alpha-tocopherol: A Waters HPLC system (Water Associates, Milford, MA) with a reverse phase Cu’column (ODS, Beckman Ultrasphere, 5 n, 4.6 mm x 15 cm, Beckman Instruments Inc, Fullerton, CA) and a fixed wavelength detector (Waters,-440 absorbance detector) set at 280 nm was used. The mobile phase was 100% methanol at a flow rate of 1 ml/minutes. Peak areas were integrated using a HP 3380A integrator. Standard curves were prepared using dl-a-tocopherol (Sigma Chemical Co., St. Louis, MD) as described previously by Asghar et al. (1991b). W 'Raw dark or light turkey meat from four turkeys per pen was individually placed in polystyrene petri dishes (100 x 49 15 mm) which were wrapped with oxygen-permeable PVC meat stretch-wrap film (Reynolds Metal company, Richmond, VA). Color changes in the raw dark and light turkey meat during refrigerated storage under fluorescent light were monitored by recording the L (luminance), a (redness) and b (yellowness) values using a Hunterlab, Model 025-2, tristimulus calorimeter (Fairfax, VA). WW Samples (80 g) of dark or light meat from four turkeys per pen was individually placed on polystyrene trays with absorbent pads. The meat was stored at 4°C under fluorescent light. Drip loss was determined by weighing individually the dark and light turkey meat on specific days and recording the decreasing weight (drip loss). The drip loss was expressed as a percentage of the original weight of the meat. After weighing, the absorbent pads in the trays were replaced before repackaging the turkey meat. WWW Cholesterol oxides in cooked muscle (dark and light) samples from six turkeys per treatment were determined following the method of Monahan et al. (1992b). Total lipid extracts were prepared from 5 g cooked muscle samples by the method of Marmer and Maxwell (1981). The lipid extracts, in dichloromethane/methanol (90:10), were evaporated to dryness on a rotary evaporator (Brinkmann Instruments Inc., 50 westbury, NY) and redissolved in 4 ml 9:1 hexane/ethyl acetate. COPS were separated from cholesterol and other muscle lipids using the sample clean-up procedure of Park and Addis (1985b). Prior to sample clean-up, 6-keto- cholesterol was incorporated into the hexane/ethyl acetate extracts as an internal standard. Acetone extracts were evaporated to dryness by rotary evaporation and redissolved in 4 m1 ethyl acetate. Prior to gas chromatographic (GC) analysis, 1 ml of the ethyl acetate solution was evaporated to dryness under nitrogen and trimethylsilyl (TMS) derivatives of cholesterol oxides were prepared by redissolving the COPS in 100 pl BSTFA (Pierce Chemical Co., Rockford, IL) and holding at room temperature for 30 minutes. The TMS other derivatives of the sterols were then evaporated to dryness under nitrogen and redissolved in 100 pl ethyl acetate. A Hewlett Parkard 5890 gas chromatograph (Hewlett Parkard, Avondale, PA) equipped with a flame .ionization detector, HP 5673A automatic injector, and a HP 3392A integrator, was used to quantify COPS. The COPS were separated on a fused silica capillary column DB-1 (15m x0.25mm id, film thickness 0.25 pm, J 8 W Scientific, Folsom CA). Carrier gas (helium) was delivered at 1.2 ml/min. Oven temperature programming was as follows: 170°C to 220°C at 10°C/min and held isothermally at 220°C for 5 min; 220°C to 234°C at 0.4°C/min; 234°C to 255°C at 1.5°C/min and held isothermally at 255°C for 40 min. Injector port and detector temperatures were 275°C and 330°C, respectively. 51 Four pl of the TMS derivatives of the COPS were injected onto the column with a split ratio of 11:1. WW Cholesterol was extracted from cooked meat (dark and light) from six turkeys per group following the direct saponification procedure of Adams et al. (1986). The method involved the accurate weighing of the ground meat sample (3 9) into a flat bottom distillation flask to which 8 ml 50% KOH and 40 m1 reagent alcohol (95:5 v/v ethanol-methanol) were added. The mixture was saponified while refluxing and stirring for 1 hour. Sixty ml of reagent alcohol were added and the mixture was cooled. Toluene (100 ml) was added and the mixture was vigorously stirred for 1 minute. The mixture was extracted with 110 ml 1N KOH in a 500 ml separatory funnel, shaking vigorously for 30 seconds, and then discarding the lower aqueous layer upon separation. To the toluene layer was added 40 ml 0.5N KOH and the separatory funnel was rotated gently. The lower layer was again discarded, and the extract was washed five times with water (100 ml) with increasing degrees of agitation. The toluene extract was dried over anhydrous HazSO‘. A 50 ml aliquot was removed and rotary evaporated to dryness, washed with 5 ml of acetone, dried, and redissolved in 3 ml dimethylformamide. The 3 ml extract was kept frozen in a glass vial (15 x 45 mm, 1 dram) for GC analysis. Cholesterol in the extract was quantified on a Hewlett 52 Packard 5840A gas chromatograph equipped with a flame ionization detector and a packed (1% SE-30 on 100/120 Gas Chrom Q) columns. The carrier gas was helium with a flow rate of 25 ml/minute and the GC was operated isothermally at 230°C. Injector port and detector temperatures were 225°C and 275°C, respectively. WW m1: Membranal lipid oxidation studies were performed by the method reported by Kanner and Harel (1985). Microsomes were solubilized in a buffer solution which contained 0.1M KCl and 0.01M lactic acid and adjusted to pH 5.7 with NaOH. The protein content of the isolated fraction was determined by the method of Lowry et al. (1951). Membrane solutions of equal protein concentrations (0.4 mg/ml) were prepared at a final volume of 9.5 ml in a 150 ml erlenmeyer flask. The flasks were placed in a shaking water bath (GCA/Precision Scientific, Chicago, IL) at 37°C. Metmyoglobin (30 uM) and H202 (30 pH) were immediately added to initiate the oxidation reaction. Aliquots (1 ml) were taken initially (time 0) and after 15, 30, 60, 90, 120, 150 and 180 minutes. The aliquots were added to 2 ml TBA reagent (TBA:trichloroacetic acid:HCl, 1:25:6.25) in 100 x 15 mm screw-capped test tubes which were then capped and vortexed. At the end of the three hour assay, the tubes were placed in a boiling water bath for color development. The tubes were 53 centrifuged for 2 minutes at speed 7 (IEC Clinical centrifuge, Needham Heights, MA) prior to determination of the absorbance at 532 nm with a Bausch and Lomb Spectronic 2000 spectrophotometer. Using an extinction coefficient of 1.56 x 105 M“cm'1 (Harel and Kanner, 1985) , concentrations of malonaldehyde per gram of protein of the membrane solutions were calculated. W The analysis of data for dark and light turkey meat was performed using a three factor (treatment x time x replication) repeat-measure split-plot design for TBARS value, COP concentration, color change, and drip loss. The Improved Bonferronit t-test was used to determine the significance of means for multiple comparisons. The analyses of variance were performed using a SPSS-X statistical program (Computer Service Center, Michigan State University, East Lansing, MI). 1A two factor (treatment x replication) complete, randomized design was used for a- tocopherol concentration. Tukey's test was used to determine the significance of means for multiple comparisons. The analyses of variance were performed using MSTAT-C microcomputer statistical program (Michigan State University, East Lansing, MI, 1989). menu an DISCUSS!“ Effect of Dietary Vitamin E Supplementation on Growth Performance Total feed consumption, body weight and feed conversion data-are presented in Table 4. When the turkeys were fed the vitamin E-supplemented diets (50 and 100 IU/kg), their average body weights tended to be higher (3.7% and 2.2%) than the average body weight of the turkeys fed the control diet (12.5 IU vitamin E/kg feed). There was no significant (p < 0.05) differences, however, between the average body weights and feed conversion ratios for the different groups. These results confirmed the observations of Monahan et al. (1990b) for pigs fed vitamin E-supplemented diets. Similarly, Engeseth (1990) reported no advantage with respect to growth for calves fed vitamin E supplements. Very few reports of growth performance, as affected by vitamin E supplementation, have been published. Popekhina and Tveritnev (1982) observed that c-tocopherol supplementation of swine diets produced pigs which were 10.6% heavier than pigs fed a basal diet. Ashgar et al. (1991b) reported 4 and 6% increases in daily body weight gain in the early growth stages (first 4 weeks) of pigs fed diets supplemented with 100 and 200 IU vitamin E [kg feed, 54 55 Table 4. Effects of dietary vitamin E level on feed consumption, feed conversion and body weights of turkeys Control 50 IU 100 IU vitamin E vitamin E [kg feed [kg feed Total body weight (kg)‘ 460. 6 505.4 498. 1 Total feed consumption 1309.3 1388.5 1362.3 (k9) Feed conversion 2.910.1' 2.810.2' 2.7i0.1' ratio2 Average body 13.511.2' 14.011.0' 13.811.2' weight (kg) 1Total body weight: 36 turkeys for the 50 and 100 IU vitamin E/kg treatments; 34 turkeys for the control (12.5 IU/kg) group 2Ratio = total feed consumption/total body weight gain The values in the table are the group means of data from 36 or 34 turkeys (3 pens) 3The values in the table are the group means of data from 36 or 34 turkeys i standard deviation. 3Mean values in a row with the same superscript are not significantly different (p > 0. 05) 56 respectively. At later stages of the feeding period, the growth of the supplemented animals was parallel to that of the control animals; thus, the advantage persisted. Stuber et a1. (1990), however, found slightly increased gain-to- feed ratios in supplemented pigs (100 and 200 IU/kg feed) as compared to controls (11 IU/kg feed). Lin et al. (1989a) reported that short-term (10 days) and long term (6 weeks) vitamin E supplementation (200 mg/kg diet) significantly increased carcass weights of broilers (p < 0.05). Short-term supplementation produced increases of 1.8% and 2.5% in body and carcass weights, respectively, while long-term supplementation resulted in increases of 1.6% and 3.7%, respectively, when compared to the corresponding weights of the broilers fed a control diet. Effect of Vitamin E Supplementation on Alpha-Tocopherol Concentration in Plasma and Muscle Tissue Turkeys were bled every four weeks in order to determine the a-tocopherol concentrations in the blood plasma. The results are presented in Table 5. Although there were wide variations in the plasma a-tocopherol levels within a group, as is evident from the magnitude of the standard deviations, plasma concentrations of the supplemented animals were significantly higher (p < 0.05) than those from control animals during the entire period. At the 4th, 8th and 12th weeks of the feeding period, the a- 57 Table 5. Alpha-tocopherol content (ug/ml plasma) in plasma samples of turkeys fed three dietary levels of vitamin E Age (weeks) Dietary level of vitamin E (IU/kg feed) Control 50 100 4 8 12 18 2.26 1 0.40c 1.65 1 0.03° 1.78 1 o.15° 1.58 1 0.15b 3.29 1 o.35° 3.62 1 o.2o° 3.51 1 o.os° 3.51 1 o.32° 5.05 t 0.66. 5.09 i 0.22. 4.75 i 0.16. 4.26 t 0.23. Values represent means of data from 9 turkeys :1: standard deviation Mean values having the same superscript in a row are not significantly different (P< 0.05) 58 tocopherol concentrations in the plasma from the different groups were significantly (p < 0.05) different. These results agree with those of Asghar et al. (1991a) and Engeseth (1990) who reported that supplementing the diets of swine and veal calves with vitamin E increased plasma a- tocopherol concentrations relative to those of the control animals. Monahan et al. (1989) also measured plasma a- tocopherol concentrations in pigs at the time of slaughter and observed significantly higher concentrations (p< 0.01) in plasma from supplemented animals. The deposition of a- tocopherol in plasma is different between animal species. In this study, the concentrations of a-tocopherol in the plasma from the control turkeys and those fed 100 IU vitamin .E/kg feed were higher than the plasma concentration in pigs fed comparable levels of vitamin E (Asghar et al., 1991a). The amounts of a-tocopherol deposited in the dark and light meat of turkeys fed the respective diets are shown in Table 6 and Appendix 1. There were significant (p < 0.05) differences in a-tocopherol concentrations between the control and supplemented groups for both light and dark meat. The a-tocopherol concentrations in dark meat from the turkeys fed supplemented diets (50 and 100 IU vitamin E/kg feed) were approximately 4 and 6 times greater than those in meat of the control animals. Similarly, the a-tocopherol concentrations in the light muscle from the turkeys fed the supplemented diets were approximately 4 and 5 times greater than those in the control animals. These results agree with 59 Table 6. Concentration of a-tocopherol (ug/g tissue) in the light and dark meat of turkeys fed three dietary levels of vitamin E Dietary level of vitamin E (IU/kg feed) Muscle Control 50 100 Light 0.44 1 0.07° 1.59 1 0.19° 2.13 1 0.07° Dark 0.83 1 0.20° 3.35 1 0.77' 4.59 1 0.45- ‘Values are the group means of data from 12 turkeys 1 standard deviation Mean values having the same superscript in a row are not significantly different from one another (p < 0.05) 60 those of previous studies (Sheldon, 1984; Lin et al., 1989b; Asghar et al., 1991a) in which it was demonstrated that dietary vitamin E supplementation could increase the a- tocopherol concentrations in muscle tissue. Sheldon (1984) reported that turkeys receiving 275 IU dl-a-tocopherol acetate/kg feed for three weeks before slaughter had mean a- tocopherol concentrations 3 to 3.3 times greater than tissues from turkeys fed control diets (1.63 IU/kg feed). Lin et al. (1989b) also reported that the concentration of a-tocopherol in the light and dark meat from broilers fed 100 IU vitamin E/kg feed were approximately 5 and 9 times greater than corresponding concentrations in the light and dark meat from broilers fed a control diet. Asghar et al. (1991a) reported that the concentrations of a-tocopherol in meat from pigs fed 10, 100, 200 IU vitamin E/kg feed showed sequentially increasing concentrations of tissue a- tocopherol (0.5, 2.6, 4.7 ug/g tissue). The species of animal and muscle type (dark and light) might affect the absorption and deposition of vitamin E. Supplementing broiler diets with 100 IU vitamin E/kg feed resulted in a greater deposition of a-tocopherol (Lin et al., 1989b) into muscle tissue than was observed in the present study. This could be due to the fact the turkey has a less efficient absorption and deposition of vitamin E and/or greater physiological utilization of vitamin E compared to the chicken (Marusich, et al., 1975). In the present study, the extent of deposition of a- 61 tocopherol in the dark and light meat was approximately 68% and 32% of the total deposition in the muscle tissue. Previous studies (Lin et al., 1989b; Sheldon, 1984) indicated similar ratios of a-tocopherol distribution in the dark and light muscle. Lin et al. (1989b) reported that dark meat accumulated approximately 50% more a-tocopherol than did the light meat. Sheldon (1984) also found the distribution of a-tocopherol in dark and light muscle to be approximately 72% and 27% of the total deposition in the turkey muscle. The different rate of deposition apparently results from physiological variations in the vascular network of dark and light muscle tissues (Sheldon, 1984). Dark meat from turkeys and broilers has a more highly developed vascular system and a higher lipid content than light meat. Therefore, a-tocopherol is deposited to a greater degree in dark meat than in light meat. Effect of Vitamin E Supplementation on the Quality of Raw Meat During Refrigerated and Frosen Storage W Raw dark and light meat from turkeys fed various levels of dietary vitamin E was held at 4°C under fluorescent light for 8 days. The degree of oxidation was monitored every two days by the TBA assay and expressed as TBA-reactive substances (TBARS) (Table 7, Appendix 2 and 3). The data indicated that both dark and light meat from the turkeys fed the diets with the higher (50 and 100 IU/kg feed) vitamin E 62 Table 7. The effect of vitamin E supplementation on TBARS1 values of raw dark and light meat during storage at 4°C. Dietary level of vitamin E (IU/kg feed) Control 50 100 Storage Dark meat Time (days) 0 0.26 1 0.04' 0.19 1 0.01° 0.15 1 0.02- 2 0.29 1 0.03' 0.20 1 0.04'” 0.14 1 0.01” 4 0.80 1 0.12' 0.23 1 0.02” 0.22 1 0.02” 6 1.14 1 0.17” 0.42 1 0.08” 0.22 1 0.02c 8 1.38 1 0.18' 0.53 1 0.07” 0.31 1 0.02° Light meat 0 0.21 1 0.02° 0.17 1 0.01° 0.16 1 0.01° 2 0.22 1 0.01' 0.18 1 0.01'” 0.15 1 0.01” 4 0.31 1 0.03' 0.22 1 0.02” 0.18 1 0.03” 6 0.45 1 0.08' 0.24 1 0.01” 0.22 1 0.03” 8 0.49 1 0.03” 0.28 1 0.04” 0.211 0.02c TBARS values in the table are the group means of data from 12 turkeys 1 standard deviation. Mean values in a row with the same superscript are not significantly different (p < 0.05) 1TBARS values are expressed as mg malonaldehyde/kg meat 63 levels had significantly lower (p < 0.05) TBARS values during storage than meat samples from the turkeys fed the control diet. These results confirmed those of previous studies for swine (Buckley et al., 1989; Monahan et al., 1990a; Asghar et al., 1991b), veal (Engeseth, 1990), and broilers (Lin et al., 1989b). The effect of frozen storage on development of TBARS in raw turkey meat is shown in Table 8 and Appendix 4 and 5. Meat from the turkeys fed with the two higher dietary vitamin E levels had lower TBARS values than meat from the turkeys fed the control diet. There was no significant difference (p < 0.05) between treatments for light meat after 3 months of frozen storage, but dark meat. After 6 months, the dark and light meat from the two supplemented groups had significantly lower (p < 0.05) TBARS values than the meat from the control turkeys. These results demonstrate a positive relationship between tissue a- _tocopherol concentration and stability of meat toward lipid oxidation during both refrigerated and frozen storage.’ Sklan et al. (1983) reported that dietary vitamin E supplementation (60 IU/kg feed) resulted in increased tissue a-tocopherol concentrations and decreased oxidation of turkey meat tissues during storage. Bartov et al. (1983) also pointed out that tissue a-tocopherol levels significantly influenced the oxidative stability of poultry meat during frozen storage. Sheldon (1984) stated that tissue e-tocopherol levels and TBARS values during 64 Table 8. The effect of vitamin E supplementation on TBARS values of raw dark and light meat during storage at -20°c‘. Dietary level of vitamin E (IU/kg feed) Control 50 100 Storage Dark meat (months) 0 0.26 1 0.04” 0.19 1 0.01° 0.15 1 0.02' 3 0.65 1 0.15” 0.42 1 0.07”” 0.32 1 0.01” 6 0.99 1 0.14' 0.31 1 0.09” 0.36 1 0.02” Light meat 0 0.21 1 0.02'I 0.17 1 0.01. 0.16 1 0.01' 3 0.26 1 0.03' 0.16 1 0.01' 0.16 1 0.02' 6 0.34 1 0.09' 0.17 1 0.03” 0.16 1 0.02” The values in the table are the group means of data from 12 turkeys 1 standard deviation. . Mean values having the same superscript in a row are not significantly different (p < 0.05) 1TBARS values are expressed as mg malonaldehyde/kg meat) 65 refrigerated and frozen storage were significantly influenced by dietary vitamin E levels. The present study also supports the findings of Lin et al. (1989b), who reported that vitamin E supplementation at the 100 IU/kg feed level significantly (p < 0.05) improved the stability of lipids in both the light and dark meat of broilers during frozen and refrigerated storage. It was also found that dark meat gave consistently higher TBARS values than light meat from all three treatments at the two storage temperatures. These results agreed with those of Sklan et al. (1983), Pikul et al. (1984) and Lin et al. (1989b) who demonstrated that the oxidative changes were much more extensive in dark meat than in light meat, possibly due to the higher phospholipid concentrations. Similarly, the beneficial effects of dietary vitamin E on the oxidative stability of pork products have been demonstrated. Monahan et al. (1990a) reported that supplementing the diets of pigs fed with 200 IU vitamin E/kg feed significantly (p < 0.01) decreased TBARS values of pork during refrigerated storage. Asghar et al. (1991b) demonstrated that TBARS values of raw pork from pigs receiving vitamin E supplements (100 and 200 IU vitamin E/kg feed) were significantly (p < 0.05) lower than those for pork from swine fed a control diet during refrigerated storage. Furthermore, pork samples from the 200 IU vitamin E [kg supplemented group were more stable than pork from pigs receiving the feed supplemented with 100 IU vitamin 66 E/kg. 0:19.122: Fresh raw turkey meat from the three dietary treatments did not exhibit significantly different rates of drip loss (p < 0.05) during the first three days of refrigerated storage (Table 9). After 12 days, however, the dark meat from the 50 and 100 IU vitamin E/kg group had significantly (p < 0.05) less drip loss than meat from the control groups of turkeys. On the other hand, after 12 days, the light meat from the turkeys receiving 100 IU vitamin E/kg supplements had significantly less drip loss than meat from the control group (p < 0.05). In the frozen storage study, the drip losses from the dark meat were not significantly different between the three groups after 3 and 6 months storage (p <0.05) (Table 10). On the other hand, the drip losses from the light meat were significantly (p < 0.05) different between the control and supplemented treatments during frozen storage. The results of both refrigerated and frozen storage showed that vitamin E supplementation had a greater effect on the drip loss of light meat than that of dark meat during storage. This might be due to the fact that light meat had greater concentrations of a-tocopherol per unit of fat, thus decreasing the rate of phospholipid changes than dark meat (Sheldon, 1984; Kameda et al., 1985; Erin et al. 1986). These data confirmed the results of Asghar et al. (1991b) Table 9. Percentage drip loss from meat from turkeys fed vitamin E-supplemented diets when stored under fluorescent light at 4°C Dietary level of vitamin E (IU/kg feed) Storage (days) Control 50 100 Dark meat 3 2.26 0.06' 1.87 i 0.12' 1.80 i 0.25' 7 5.38 0.34° 4.93 1 0.11'” 4.32 1 0.27” 12 10.84 0.64' 8.52 1 0.24” 8.44 1 0.42” Light meat 3 2.31 0.03' 1.87 i 0.41' 1.83 i 0.11' 7 5.71 0.22' 5.05 1 0.55' 5.31 1 0.43' 12 10.66 0.44. 9.92 1 0.38'” 9.14 1 0.62” Drip loss values are the group means of data from 12 turkeys 1 standard deviation. Mean values in a row with the same superscript are not significantly different (p < 0.05) 68 Table 10. Percentage drip loss from turkey meat which had been frozen for 3 and 6 months, and subsequently stored under fluorescent light at 4°C Dietary level of vitamin E (IU/kg feed) Storage (days) Control 50 100 Dark meat (frozen for 3 months) 2 4.97 1 0.30' 4.35 1 0.67' 4.28 1 0.15” 4 6.20 1 0.38' 5.74 1 0.75” 5.42 1 0.22' 6 7.43 1 0.28' 6.99 1 0.77' 7.14 1 0.45° Dark meat (frozen for 6 months) 2 5.11 1 0.68' 5.87 1 0.66' 5.81 1 0.83' 4 7.94 1 0.32' 7.92 1 0.28' 7.26 1 0.32' 6 8.18 1 0.31” 8.60 1 0.83' 8.84 1 0.48' Light meat (frozen for 3 months) 2 7.22 1 0.56° 4.81 1 0.47” 3.66 1 0.24” 4 7.60 1 0.52° 5.89 1 0.21” 4.97 1 0.57” 6 8.32 1 0.42' 6.46 1 0.20” 5.71 1 0.32” Light meat (frozen for 6 months) 2 10.78 1 0.04' 6.72 1 1.32” 7.76 1 0.31” 4 12.11 1 0.23” 8.55 1 0.31” 8.67 1 0.47” 6 12.62 1 0.23' 9.74 1 0.35” 9.24 1 0.54” The values in the table are the group means of data from 6 turkeys 1 standard deviation. Mean values in a row with the same superscript are not significantly different from one another (p < 0.05) 69 who showed that a higher a-tocopherol concentration in pork had a positive effect in minimizing drip losses in meat that had been frozen for 3 months, before thawing and storing at 4°C. Asghar et al. (1991b) also demonstrated that pork from pigs fed 200 IU vitamin E/kg feed exhibited significantly (p < 0.05) different rates of drip loss than meat from the control group (10 IU vitamin E/kg feed). Results of the present study revealed that vitamin E was more effective in controlling drip loss in the fresh dark meat samples than in samples that were frozen and then thawed. This might be due to frozen (long term storage) dark meat requiring a higher vitamin E content to prevent drip loss. As the study of” Asghar et al. (1991b) indicated, dietary supplementation with 200 IU vitamin E/kg supplied enough vitamin E to show a significant difference in drip loss relative to the control diet (10 IU vitamin E/kg feed), but the 100 IU/kg supplementation level did not. In the present study, the light meat exhibited a greater drip loss than did the dark meat. Although the exact reasons for the effect of vitamin E on drip loss are not clear, Asghar et al. (1991b) have suggested several possible explanations. First, a- tocopherol may protect the integrity (fluidity) of cell membranes from freeze injury which increases their permeability and results in leakage of sarcoplasmic fluid. Second, a-tocopherol has been shown to protect the membranes from the action of phospholipases (Erin et al., 1986) which 70 remove the fatty acid moiety from phospholipid molecules. The loss of phospholipids from membranes causes a substantial reduction in its fluidity (Kameda et al., 1985) and hence may increase the drip loss. Third, a-tocopherol may preserve the fluidity of membranes which could otherwise be adversely affected by oxidative changes in the phospholipids. 2219: Changes in color (expressed as L, a, b Hunter values) of turkey meat (dark and light) during storage at 4°C under fluorescent light for 12 days and at -20°C for 6 months are presented in Tables 11, 12, 13 and 14. In the refrigerated samples, L (luminance, whiteness) and b (yellowness) values of dark and light turkey meat of each treatment oscillated or increased very slightly during storage and did not show any apparent change with the length of storage and dietary treatment. This might be due to the difficulty in quantifying the very slight changes in surface color of turkey meat. The redness (a value) of turkey meat apparently decreased with the duration of exposure to fluorescent light. However, changes in redness were slower in the supplemented groups (50 and 100 IU vitamin E/kg feed) compared to those in the control group for both dark and light meat during the first 8 days of storage. There was a significant (p < 0.05) difference in the color of dark meat between the control group and the supplemented groups. 71 Table 11. Changes in the color characteristics of dark turkey meat stored at 4°C for 8 days. Storage Hunter Dietary vitamin E level (days) parameter Control 50 IU/kg 100 IU/kg 0 L-value 39.7 1 0.6” 40.3 1 0.6” 40.9 1 1.0” 2 38.3 1 0.8” 39.2 1 0.5” 40.0 1 1.3” 4 38.0 1 0.3” 38.5 1 0.4” 39.0 1 0.9” 6 38.1 1 0.6” 39.0 1 0.3” 39.1 1 0.8” 8 39.1 1 0.7” 39.3 1 1.0” 38.7 1 0.7” 0 a-value 10.8 1 0.3” 10.9 1 0.2” 10.7 1 0.5” 2 9.4 1 0.4” 10.5 1 0.2” 10.0 1 0.4”” 4 8.8 1 0.4” 9.8 1 0.5” 9.8 1 0.4” 6 8.1 1 0.6” 9.3 1 0.2” 8.8 1 0.3”” 8 6.9 1 0.7” 8.4 1 0.4” 8.6 1 0.2” 0 b-value 8.3 1 0.4” 8.2 1 0.4” 7.4 1 0.2” 2 8.4 1 0.2” 8.1 1 0.2” 7.8 1 0.3” 4 8.2 1 0.3” 8.4 1 0.3” 8.0 1 0.4” 6 8.4 1 0.3” 8.6 1 0.1” 8.2 1 0.2” 8 8.9 1 0.2” 8.6 1 0.2” 8.3 1 0.1” to zero for black. greenness when negative. blueness when negative. The values in the table are the group means of data from 12 turkeys 1 standard deviation. Mean values in a row with the same superscript are not significantly different (p < 0.05) measure lightness and varies from 100 for perfect white measures redness when positive, greyness when zero and measures yellowness when positive, greyness when zero, and 72 Table 12. Changes in the color characteristics of light turkey meat stored at 4°C for 8 days. Storage Hunter Dietary vitamin E level (days) parameter Control 50 IU/kg 100 IU/kg 0 L-value 46.6 1 0.3' 46.1 1 1.4' 45.2 1 0.7' 2 48.2 1 0.4' 47.2 1 1.2' 47.6 1 1.0' 4 48.1 1 0.7' 47.5 1 1.3' 47.7 1 0.8' 6 48.0 1 0.8' 47.3 1 1.7' 47.6 1 0.9' 8 48.1 1 0.5' 47.6 1 1.4' 47.9 1 0.9' 0 a-value 9.0 1 0.5' 9.1 1 0.2'I 9.1 1 0.6'| 2 8.2 1 0.4' 8.9 1 0.3' 8.4 1 0.4' 4 8.0 1 0.5' 8.5 1 0.4' 8.3 1 0.3‘ 6 7.7 1 0.4' 8.2 1 0.5' 8.0 1 0.2' 8 7.2 1 0.7' 7.5 1 0.3' 7.7 1 0.3' 0 b-value 8.4 1 0.4' 8.3 1 0.7' 8.2 1 0.6' 2 9.6 1 0.3' 9.6 1 0.3' 9.5 1 0.5' 4 9.8 1 0.3' 9.8 1 0.3' 9.6 1 0.2. 6 10.1 1 0.3. 10.2 1 0.4' 10.1 1 0.4' 8 10.6 1 0.3' 10.3 1 0.4' 10.2 1 0.5' L: measure lightness and varies from 100 for perfect white to zero for black. a: measures redness when positive, greyness when zero and greenness when negative. b: measures yellowness when.positive, greyness when zero, and blueness when negative. The values in the table are the group means of data from 12 turkeys 1 standard deviation Mean values in a row with the same superscript are not significantly different (p < 0.05) 73 Table 13 . Changes in the color characteristics of dark turkey meat stored at -2 0°C for 3 and 6 months Storage Hunter Dietary vitamin E level (months) parameter Control 50 IU/kg 100 IU/kg 0 L‘Value 39s? 1 Oe6. ‘0e3 t 0.6. 40e9 t 1.0. 3 37.7 1 1.3' 36.7 1 0.5' 36.51 1.0' 6 35.8 1 0.9' 34.9 1 0.9' 35.112.0' 0 a-value 10.8 1 0.3' 10.9 1 0.2' 10.7 1 0.5' 3 10.2 1 0.5' 10.2 1 0.4' 10.11 0.2' 6 8.1 1 0.4' 8.8 1 0.2' 8.910.4' O b-Value 8e3 t Oe4. 8e2 t Oe4. 7e4 i Os? 3 8.7 1 0.3' 7.5 1 0.6‘I 7.610.5' 6 8.0 1 0.4' 7.9 1 0.3' 7.610.6' L: measure lightness and varies from 100 for perfect white to zero for black. a: measures redness when positive, greyness when zero and greenness when negative. b: measures yellowness when positive, greyness when zero, and blueness when negative. The values in the table are the group means of data from 12 turkeys 1 standard deviation. Mean values in a row with the same superscript significantly different (p < 0.05) are not 74 Table 14. Changes in the color characteristics of light turkey meat stored at -20°C for 3 and 6 months. Storage Hunter Dietary vitamin E level (months) parameter Control 50 IU/kg 100 IU/kg 0 L-Value 46.6 1 0.3' 46.1 1 1.4' 45.2 1 0.7' 3, 46.9 1 1.5' 43.9 1 1.6' 46.4 12.1' 6 40.5 1 1.6' 41.2 1 2.2' 40.61 0.2' 0 a-value 9.0 1 0.5' 9.1 1 0.2' 9.11 0.5' 3 10.3 1 0.5” 12.2 1 0.3” 9.510.5” 6 7.1 1 0.2' 7.5 1 0.2' 7.810.5' 0 b-value 8.4 1 0.4' 8.3 1 0.7' 8.2 1 0.6' 3 10.6 1 0.1' 10.3 1 0.4' 10.61 0.3' 6 8.1 1 0.1' 8.0 1 0.2' 8.510.4' L: measure lightness and varies from 100 for perfect white to zero for black. a: measures redness when positive, greyness when zero and greenness when negative. b: measures yellowness when.positive, greyness when zero, and blueness when negative. The values in the table are the group means of data from 12 turkeys 1 standard deviation. Mean values in a row with the same superscript are not significantly different (p < 0.05) 75 The L and b values of the turkey meat during frozen storage also did not show apparent changes with length of storage and treatment. The "a” value decreased after 6 months of frozen storage, but was not related to the vitamin E level in the diet of turkeys. This was possibly due to the fact that the level of vitamin E supplementation (50 and 100 IU/kg feed) was not high enough to deposit sufficient c- tocopherol in the meat to protect the pigments during 6 months of frozen storage. Haurowitz et al. (1941) revealed that hemoglobin was destroyed in the presence of unsaturated fatty acids and oxygen. Lin and Hultin (1977) found that the oxidation of oxymyoglobin in vitro was enhanced by the addition of linoleate hydroperoxide; butylated hydroxyanisole (BHA) and glutathione plus glutathione peroxidase were both effective as antioxidants. Hutchins et al. (1967) also reported a positive correlation (r-0.73, p< 0.005) between metmyoglobin , accumulation and malonaldehyde production in ground beef. Lipid and pigment oxidation reactions are closely coupled in beef; an increase in one of these processes results in a similar increase for the other (Hutchins et al., 1967; Greene, 1969; Faustman et al., 1989b). The basis for this relationship is not clearly understood. From the viewpoint of meat color, it is possible that radical species produced during lipid oxidation act directly to promote pigment oxidation, and/or indirectly by damaging pigment reducing systems (Faustman and Cassens, 1990). 76 Color stability is generally enhanced by the addition of antioxidants (ascorbic acid, butylated hydroxyanisole, propyl gallate and vitamin E) to meat (Greene, 1969; Greene et al., 1971; Govindarajan et al., 1977; Faustman et al., 1989a,b; Asghar et al., 1991b). Faustman et al. (1989a,b) reported that sirloin steaks from vitamin E-supplemented Holstein steers (370 IU/animal/day) had greater (p < 0.01) chroma and Hunter "a" values than steaks from control animals. Fresh ground beef from vitamin E-supplemented Holstein steers was more stable to lipid oxidation, and this may be responsible for the associated increased pigment stability. Asghar et al. (1991b) also demonstrated that changes in redness (a value) during the first 6 days of refrigerated storage under light were relatively slow in pork from vitamin E-supplemented pigs (100 and 200 IU vitamin E/ kg feed) compared to the pork from control group (10 IU vitamin E/kg feed). In addition to inhibiting lipid oxidation and minimizing changes in color, a-tocopherol is also an efficient quencher of singlet oxidation which is normally initiated by fluorescent light (Foote, 1985). Alpha- tocopherol can maintain the reduced form of heme iron for a long time depending on its concentration in the meat system. 77 Effect of Vitamin E Supplementation on Lipid Stability and Cholesterol Oxidation in Cooked Meat mm Data in Table 15 show the influence of the different dietary levels of vitamin E on the oxidative stability of cooked ground turkey meat stored at 4°C under fluorescent light for 48 hours. TBARS values increased with storage time in all cases. The TBARS values of the cooked ground dark meat from the supplemented groups were slightly lower than the control group, but the differences in the treatments during storage were not significant (p < 0.05). The cooked ground light meat, however, did show significantly (p < 0.05) different results between the control group and the supplemented groups after 48 hours of refrigerated storage. That the vitamin E supplementation decreased the rate of lipid oxidation in light meat slightly more than in dark meat might be possibly due to the differences in concentrations of tocopherol per unit of fat between dark and light meat (Sheldon, 1984). Monahan et al. (1992b) reported that the formation of lipid oxidation products was significantly higher (p< 0.01) in pork from pigs fed a basal level of c-tocopherol acetate (10 mg/kg) compared to the pigs fed the supplemented levels (100 and 200 mg/kg feed). Differences in TBARS between the two supplemented groups were only significant (p < 0.05) after 4 days of storage. Vitamin E supplementation in the present study was not as effective in preventing lipid oxidation as 78 Table 15. The effect of dietary vitamin E supplementation on TBARS values of cooked turkey dark and light meat during storage at 4°C1 . Dietary level of vitamin E (IU/kg feed) Control 50 100 Storage Dark meat time (hr) 0 3.06 0.20' 3.18 1 0.46' 3.14 1 0.08' 24 8.60 0.90' 7.37 1 1.11' 7.25 1 0.94' 48 11.83 1.15' 11.53 1 0.81' 9.81 1 1.17' Light meat 0 3.22 0.60' 2.97 1 0.48' 2.50 1 0.38' 24 5.94 0.64' 5.25 1 0.53' 4.80 1 0.28' 48 8.07 0.20' 7.40 1 0.47' 6.90 1 0.43' The values in tables are the group means of data from 12 animals 1 standard deviation. Mean. values in a row with the same superscript are not significantly different from one another (p < 0.05) 1TBARS values are expressed as mg malonaldehyde/kg meat 79 was reported by Monahan et al. (1992b). This might be due to the fact that turkey lipids are more highly unsaturated than pork lipids and that the turkeys were fed less vitamin E than were the pigs. The susceptibility of muscle tissue from different species to oxidize is related to the degree of unsaturation of muscle lipids (Allen and Foegeding, 1981; Tichivangana and Morrissey, 1985). The polyunsaturated fatty acid content of muscle varies between species and decreases in the order: fish > poultry > pork > beef > lamb (Pearson et al., 1977). Higher vitamin E supplementation might be necessary to supply a more beneficial antioxidative effect in those meats containing a higher proportion of unsaturated lipids (Rhee, 1988; Frigg et al., 1990; Monahan et al., 1992c). The present study also showed that the TBARS values of cooked dark meat were higher than the cooked light meat values in all cases during refrigerated storage. Jacobson and Koehler (1970) reported that cooked dark meat had higher TBARS values than cooked light meat because the former contains more heme pigment which can catalyze or enhance the lipid oxidation process. They also demonstrated a marked increase in TBARS value in the two types of meat during refrigeration. WM Four major COPS were consistently present in detectable quantities in all turkey samples: cholesterol c-epoxide, 80 cholesterol B-epoxide, 7B-hydroxycholesterol and 7- ketocholesterol (Table 16). The other minor cholesterol oxidation products were 7a-diol (7a-OH), 25- hydroxycholesterol and cholestane-triol. COPS in the cooked turkey samples increased during refrigerated storage (Table 16). The principal oxidation product was 7-ketocholesterol and constituted over 50% of the total COPS, as was reported earlier by Zubillaga and Maerker (1991). The secondary oxysterols did not increase over the 48 hour period in the manner reported by Pie et a1. (1991). These investigators demonstrated that there was a greater increase in primary oxysterols (i.e, those formed by oxidation at carbon 7 or the side chain) compared to the secondary derivatives (cholesterol epoxides and cholestanetriol) during the cooking of fresh meat. The concentration of oxysterols doubled in the commercially available precooked frozen meals after 3 months at -20°C with the secondary oxysterols showing the highest increase (Pie et al., 1991). Data in Table 17 and Appendix 6 and 7 show that the concentration of COPS, expressed as a percentage of total cholesterol in cooked turkey meat, increased during refrigerated storage, thus confirming the observation of Pie et al. (1991) that the COPS in cooked meat increase with storage time. In this study, total COPS were calculated as the sum of the concentrations of the four major COPS and the three minor COPS mentioned previously. Pie et al. (1991) demonstrated that all oxidized derivatives increased 81 Auacaficc my enema macho on» use case» on» ma enac> 0mm 055.0 NOH.H th.H v0n.0 050.0 0Hh.0 Oz 02 02 bmH.0 0Hn.0 000.0 00 N0 H0 H50: 0c ¢¢O.H 000.H HNO.H mmN.0 000.0 Hmm.0 00H.0 bdn.0 HON.0 HNH.0 00H.0 00H.0 MU NU HU H00: 0v N0¢.0 500.0 mNO.H wHN.0 th.0 0¢v.0 HHH.0 me.0 000.0 «00.0 N0d.0 mon.0 06 N6 H0 H508 #N H00.0 000.0 0H0.H 00H.0 50v.0 000.0 H¢H.0 HOH.0 00H.0 ##0.0 00H.0 NOH.0 00 N0 #0 H00: vN 0N0.0 .fihd.0 0nd.0 050.0 hNH.0 0mH.0 Oz e00.0 000.0 000.0 000.0 500.0 mm NU HU Moon o 000.0 0HN.0 wmn.0 0H0.0 000.0 006.0 hd0.0 000.0 000.0 0H0.0 0¢0.0 Hv0.0 00 N0. H0 H50: 0 ouoxlb moses msg#00018 ocwxonenm mmOU HauaIHQMHA ouoxlh nouns ocwaQOIc coaxomouo mmOU .mocnoum coucuomwnuou asauso Home hogan» un0HH use xuco ossouo 003000 cw Honounoaono Acuou on oowxo Honouuoaoco uo omcusoouom 0:» so :o«ucvsoaoanmsm Houmsuooouuc hhcyowu uo voouuu .wH manna 82 Table 17. Effect of vitamin E supplementation on the development of total cholesterol oxidation products in cooked dark and light turkey meat during storage at 4°C. Total COP concentration (expressed as a percentage of initial cholesterol) Dietary Vitamin E 12.5 50 100 Level (control) (IU/kg feed) Storage Time Dark meat (hr) 0 0.73 1 0.16” 0.59 1 0.06'” 0.23 1 0.04” 24 2.38 1 0.26” 1.79 1 0.13” 1.00 1 0.20c 48 3.12 1 0.24” 2.44 1 0.21” 1.53 1 0.23c Light meat 0 0.55 1 0.03” 0.35 1 0.04” 0.21 1 0.03” 24 1.88 1 0.32” 1.19 1 0.07” 0.91 1 0.17” 48 2.49 1 0.18” 2.22 1 0.18” 1.17 1 0.15” The values in the table are group means of data from 6 turkeys per treatment 1 standard deviation. Mean values in a row with the same superscript are not significantly different (p < 0.05). 83 progressively in meat samples stored for 3 months at -20°C in both raw and cooked meat. Table 17 also shows that the concentration of COPS in dark meat were slightly greater than those in light meat. Park and Addis (1987) reported that total COPS formed in dark turkey meat after 3 days of refrigerated storage at 4°C represented 1.20% of the total cholesterol. A slightly higher concentration of COPS (3.12%) in the dark meat after 2 days at 4°C were observed in the present study and can be explained by the fact that the meat had been stored at -20°C for 3 months before cooking. The formation of COPS in cooked turkey meat during storage was significantly (p < 0.05) influenced by dietary vitamin E. Cholesterol oxide concentrations in the cooked ground dark turkey meat from the three groups were significantly (p < 0.05) different after 24 hours. Cholesterol oxide formation in cooked ground light meat from the control group was significantly (P < 0.05) different from that in meat from the higher vitamin E (100 IU/kg feed) supplemented group over the same storage period. There was no significant difference between the data (2.22% and 1.17%) of the two vitamin E (50 and 100 IU/ kg feed) supplemented groups or the data (2.49% and 2.22%) of the control group and lower vitamin E (50 IU/kg feed) supplemented group. The results of this study agree with those of previous studies (Engeseth, 1990; Monahan et al., 1992b) in that COP formation in cooked meat is significantly influenced by 84 dietary vitamin E. Engeseth (1990) reported that the COP concentration in cooked veal from calves fed a basal diet and one containing 500 IU vitamin E/day were 1.53 and 0.53% of the total cholesterol after 4 days of refrigerated storage, respectively. Monahan et al. (1992b) also demonstrated that the total COPS present in cooked pork from pigs fed a basal level of vitamin E (10 IU/kg feed) represented 2.7% of the total cholesterol after 2 days of refrigerated (4°C) storage. In the case of pork from pigs fed diets containing 200 IU vitamin E, total COPS accounted for 1.6% of the total cholesterol. The relationship between lipid oxidation, as estimated by the TBA method, and COP formation was examined (Figures 6 and 7). The data indicate correlations of r- 0.79 and r- 0.55 between TBARS values and total COPS (expressed as a percentage of the total cholesterol) in cooked dark and light turkey meat stored for 2 days at 4°C, respectively. Park and Addis (1987) noted greater cholesterol oxide development in beef and turkey with increasing rancidity (as determined by TBARS values). Engeseth (1990) also showed that cholesterol oxidation followed similar trends as TBARS development. De Vore (1988) reported that TBARS values and 7-ketocholesterol were correlated for raw (r-0.91) and cooked (r-0.99) beef. Monahan et al. (1992b) also Concentration of COPS (73) 85 TBARS value, mg malonaldehyde/ kg Figure 6. Relationship between TBARS and total COP formation in cooked ground dark turkey meat stored for up to 3 days at 4°C. 86 Concentration of COPS (%) 'Figure 7. TBARS value, mg malonaldehyde/ kg Relationship between TBARS and total COP formation in cooked ground light turkey meat stored for up to 3 days at 4°C. 87 demonstrated a strong correlation (r- 0.88) between TBARS and total COP concentration in cooked pork stored at 4°C for 4 days and the correlation coefficients for B-epoxide, 78-OH and 7-keto with TBARS were +0.75, +0.78 and +0.84, respectively. These studies demonstrated that the rate of cholesterol oxidation in meat is greatly accelerated by cooking and appears to follow the same trend as lipid oxidation in general. Polyunsaturated lipids are susceptible to oxidation and to the formation of highly reactive free radicals. Igene et al. (1980) established that membrane phospholipids are the major source of polyunsaturated fatty acids (PUFA). The structural membranes in lean beef muscle contain the major share of the cholesterol (Rhee et al., 1982b). PUFA and cholesterol are integral components of the membrane structure, free radicals from phospholipid oxidation may initiate cholesterol oxidation in the tissue membranes of ground beef. Dietary c-tocopherol supplementation has been shown to significantly increase the a-tocopherol content of muscle mitochondrial and microsomal cell membranes in pigs (Monahan et al., 1990b) and chickens (Asghar et al., 1989b). Thus, the localization of c- tocopherol at the proposed site of initiation of lipid oxidation provides a means of retarding lipid and cholesterol oxidation in meats through dietary supplementation with vitamin E. Effect of Vitamin E Supplementation on Microsomal Lipid Stability Alpha-tocopherol concentrations in the microsomal fraction isolated from the light and dark meat from turkeys receiving the vitamin E-supplemented diets were approximately 3-5 fold greater than those in comparable fractions from meat of the control animals (Table 18). The vitamin E concentration in the microsomes increased with increasing dietary vitamin E level. Several researchers have investigated the deposition of a-tocopherol in the membranes as a consequence of feeding vitamin E-supplemented diets.. Asghar et al. (1990) reported that the microsomes from the dark meat of control broilers and those receiving a vitamin E diet (100 IU vitamin E/kg), contained 3.3 and 126.0 ug a-tocopherol/g membrane, respectively. The microsomes from the light meat of the .control group and the vitamin E group contained 0.9 ug/g membrane and 2.2 ug/g membrane, respectively. Microsomal a- tocopherol concentrations in the dark meat of turkeys were much lower than those reported in chicken microsomes by Asghar et al. (1990), however, light meat concentrations were similar. The results of studies with pigs (Menahan et al., 1990b; Asghar et al., 1991b) and calves (Engeseth, 1990) also agreed with those of the present study in that vitamin E supplementation increased the c-tocopherol concentration 89 Table 18. Concentration of c-tocopherol in the microsomes of dark and light meat from turkeys fed control, and vitamin E-supplemented diets Treatment Alpha-tocopherol (pg/g membrane, wet basis) Microsome Vitamin E7 dietary level Dark Light (IU/kg feed) 12.5 1.53 1 0.84 0.68 1 0.14 50 6.29 1 0.54 2.32 1 1.20 100 7.70 1 2.72 2.51 1 0.10 The values in the table are the means of data from the duplicate pooled sample (12 turkeys from each treatment) 1 standard deviation. 90 of microsomes. Monahan et al. (1990b) reported that the microsomal a-tocopherol concentrations in the meat of pigs which were fed a basal diet (<40 IU vitamin E/kg feed) and 160 IU/kg feed were 62.4 and 164.8 ug/g protein. Asghar et al. (1991b) demonstrated that the deposition of c-tocopherol in the microsomes (12, 29.4, 53.1 ug/g protein) increased linearly with the level (10, 100, and 200 IU/kg feed) of vitamin E in the diet. Engeseth (1990) reported microsomal c-tocopherol concentrations in control and vitamin E- supplemented (500 IU/day) veal calves of 139 and 707 ug/g protein, respectively. Microsomal lipid oxidation in turkey dark and light meat is graphically presented in Figures 8 and 9, respectively. Generally, oxidative changes were more extensive in the microsomal fractions isolated from dark meat than those in the comparable fraction from light meat. These results confirm the findings of Harel and Kanner (1985) and Asghar et al. (1989b) who reported a higher rate of lipid peroxidation in microsomes from the dark muscle tissues of turkeys and broilers, respectively, compared to the light muscle microsomes. The reason for these differences was not explained by these investigators. However, greater total lipid contents may contribute to the larger TBARS values for the membranes from the dark meat (Lin, 1988). TBARS (nmol MDA/ mg protein) 91 30 0—0 Control diet 0 — O 50 IU vitamin E/ kg feed A— A 100 IU vitamin E/kg feed 20 -- / r,// 10 -~ ’ O : : : : : O 30 60 90 120 150 180 Time (min) Figure 8. Metmyoglobin/hydrogen peroxide-catalyzed lipid peroxidation in the microsomal membranes from dark meat of turkeys fed a control and vitamin E- supplemented (50 and 100 IU/kg feed) diets TBARS (nmol MDA/ mg protein) 92 00 O O —0 Control diet “ 0—0 50 IU vitamin E/kg feed A —A 100 IU vitamin E/kg feed N O 10 ._ . = ' r 5 M1: .../2 ‘ 0 .L I. 4 : : . 0 30 60 90 120 150 180 Time (min) Figure 9. Metmyoglobin/hydrogen peroxide-catalyzed lipid peroxidation in the microsomal membranes from light meat of turkeys fed control vitamin E- supplemented (50 and 100 IU/kg feed) diets 93 Membranal oxidation in the supplemented groups (50 I0 and 100 IU vitamin E/kg feed) were slower than the control group. Asghar et al. (1989b) demonstrated that membrane lipids from broilers receiving 200 IU vitamin E/kg showed significantly (p < 0.01) different rates of peroxidation to membrane lipids from control group. In the case of broiler microsomal lipids showed more significant difference between the supplemented group and control group than those of turkey membranal lipids did. The difference might be due to the fact that the broilers were fed higher levels of vitamin E than the turkeys in the present study. It is quite likely that turkeys need higher levels of dietary vitamin E than broilers to increase the a-tocopherol concentration of the microsomes of turkey meat in order to decrease the rate of oxidation (Harel and Kanner, 1985). In our study, the microsomes of the dark and light meat from the control group were the least stable, while the higher level of vitamin E supplementation (100 IU/kg feed) produced the more stable microsomes when reacted with the metmyoglobin/hydrogen peroxide catalytic system. These results confirmed that a- tocopherol can inhibit in vitro microsomal lipid peroxidation catalyzed by hydrogen peroxide-activated metmyoglobin as reported by Harel and Kanner (1985), Asghar et al. (1989b; 1991b), Buckley et al. (1989) and Monahan et al. (1990a). The latter group demonstrated that vitamin E stabilized the membrane-bound lipids in pork against metmyoglobinlfifb-catalyzed oxidation and also significantly 94 (p<0.05) improved the oxidative stability of rendered fat. Asghar et al. (1991b) reported that the differences in the concentration of c-tocopherol in the mitochondrial and microsomal fractions from L. dorsi muscle of the pig fed with 10, 100 and 200 IU vitamin E/kg feed were clearly reflected in the enhanced stability of the membrane-bound lipids. sauna! an MOB!“ The influence of three levels of dietary vitamin E (12.5, 50 and 100 IU/kg feed) on the deposition of a- tocopherol in the muscle, on selected quality characteristics of raw turkey meat (oxidative stability of lipids, color, drip loss) during refrigerated and frozen storage, and on the oxidative stability of lipid and cholesterol in cooked meat during refrigerated storage was studied. The effect of vitamin E supplementation on the oxidative stability of turkey microsomal lipids was also investigated. Results indicated that the level of dietary vitamin E influenced the concentration of.a-tocopherol in the muscle membranes and the resultant oxidative stability of turkey lipids. Dietary supplementation at the higher levels (50 IU and 100 IU/kg feed) resulted in 3-5 fold increases in muscle and microsomal a-tocopherol concentrations and enhanced the oxidative stability of muscle and microsomal lipids. Concentrations of a-tocopherol in the dark and light meat from turkeys fed 50 IU and 100 IU vitamin E/kg feed were 3.35 ug and 1.59 89/9, and 4.59 ug and 2.13 ug/g, respectively. These values were significantly higher (p 95 96 <0.05) than those for the control group. Growth performance was not significantly (p < 0.05) improved by dietary supplementation with vitamin E. Dietary vitamin E significantly (p < 0.05) influenced the oxidative stability of raw meat during refrigerated and frozen storage. The increased oxidative stability of turkey meat through dietary vitamin E supplementation also improved color stability and decreased drip loss during refrigerated storage. During frozen storage, however, vitamin E supplementation did not produce any significant differences in the color of turkey meat. TBARS values of cooked dark meat were not significantly influenced by dietary treatment. However, vitamin E supplementation had a positive effect on the stability of cooked light meat. The microsomes of the dark and light meat from the control group were the least stable while vitamin E supplementation (100 IU/kg feed) produced the more stable microsomes when reacted with the metmyoglobin/hydrogen peroxide catalyst. The oxidative changes were much more extensive in the microsomes from dark meat compared to those from light meat. Cholesterol oxidation occurred in cooked turkey meat stored for 3 days at 4°C. The formation of COPS in cooked turkey meat during storage was significantly (p < 0.05) influenced by dietary vitamin E. The relationship between lipid oxidation indicated correlations of rs +0.79 and r- +0.55 between TBARS values and cholesterol oxide 97 concentrations in cooked dark and light turkey meat, respectively. m-ICIB 98 6 Darkmeat - 5- Light meat 3’ g .... '6' 56 3.- 1: §‘ ZJ ’\ a b \ I \ (I: 0 1 \IV 1\\ \ Control 50 IU 100 IU Dietary Vitamin E level/ kg feed Appendix 1 . Concentrations of c-tocopherol (pg/g meat) in dark and light meat from turkeys fed diets containing three levels of vitamin E. TBARS value, mg MDA/ kg 99 213 O — 0 Control diet 0 -—— O 50 IU vitamin E/ kg feed A — A 100 IU vitamin E/kg feed 1.5 - 1.0 ” 0.5 :- 0.0 I 1 1 1 1 O 2 4 6 8 Storage time (days) Appendix 2. The effect of vitamin E supplementation on TBARS values (mg malonaldehyde/kg sample) of raw dark turkey neat stored at 4°C for 8 days. TBARS value. mg MDA/ kg 100 2.0 0—0 Control diet 0— O 50 IU vitamin E/ kg feed A— A 100 IU vitamin E/kg feed 1.5 - 1.0 - 0 5 - #9 00 4 1 L 1 2 4 6 8 0 Storage time (days) Appendix 3. The effect of vitamin E supplementation on TBARS values (mg malonaldehyde/kg sample) of raw light turkey meat stored at 4”C for 8 days. TBARS value, mg MDA/ kg 101 1.6 O— 0 Control diet 0 — O 50 IU vitamin E/kg feed A—A 100 IU vitamin E/kg feed Storage time (months) Appendix 4. Effect of different levels of dietary vitamin E on TBARS values (mg malonaldehyde/kg sample) of raw dark meat stored at -20”C for 6 months TBARS value, mg MDA/ kg 102 1.6 0—0 Control diet e — O 50 IU vitamin E/kg feed A—A 100 IU vitamin E/kg feed 1.2 - 0.8 *- 04 - y) ._%§__ 1 . 3 0.0 ‘ ‘ 0 3 6 Storage time (months) Appendix 5. Effect of different levels of dietary vitamin E on TBARS values (mg malonaldehyde/kg sample) of raw light meat stored at -20”C for 6 months Total COPS (75) 103 4 O— 0 Control (12. 5 IU vitamin E/kg feed) 0 — O 50 IU vitamin E/ /kfi feed A—A 100 IU vitamin E/ g feed 3 ..- 3 2 ._ 1 ~~ - O . i % 0 24 48 Storage time (hours) Appendix 6. Effect of vitamin E supplementation on the deve10pment of cholesterol oxidation products (expressed as a percentage of the initial cholesterol concentration) in cooked ground dark turkey meat during storage at 4°C. Total COPS (75) 104 4 0—0 Control diet L O -— O 50 IU vitamin E/kg feed A —A 100 IU vitamin E/kg feed 3 L ’ e 2 ” ‘_ 1 - _ ‘ 4'. 0 L 1 0 24 48 Storage time (hours) Appendix 7. Effect of vitamin E supplementation on the development of cholesterol oxidation products (expressed as a percentage of the initial cholesterol concentration) in cooked ground light turkey meat during storage at 4°C. 105 Appendix 8. Analysis of variance for TBARS values of raw dark turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T - Treatments 2 2.05 0.01 174.32 < 0.05 8(1) -pens/T 6 0.04 1.02 P 8 periods 4 1.71 0.43 168.06 < 0.05 TP = interaction 8 0.96 0.12 47.30 < 0.05 E(2) - residual 24 0.06 0.00 error Appendix 9. Analysis of variance for TBARS values of raw light turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T - Treatments 2 0.21 0.10 78.91 < 0.05 3(1) -pens/T 6 0.01 0.00 P I periods 4 0.16 0.04 45.53 < 0.05 TP 8 interaction 8 0.08 0.01 11.27 < 0.05 E(2) - residual 24 0.02 0.00 error 106 Appendix 10. Analysis of variance for "a" values of color of raw dark turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T - Treatments 2 8.14 4.07 5.04 < 0.05 3(1) -pens/T 6 4.85 0.81 P - periods 4 44.14 11.03 111.92 < 0.05 TP 8 interaction 8 3.11 0.39 3.94 < 0.05 E(2) a residual 24 2.37 0.10 error Appendix 11.Analysis of variance for ” a” values of color of raw light turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability variation Freedom Square Square value T I Treatments 2 1.46 0.73 0.71 > 0.05 E(l) -pens/T 6 6.12 1.02 P - periods 4 12.47 3.12 44.32 < 0.05 TP - interaction 8 0.46 0.06 0.82 > 0.05 E(2) - residual 24 1.69 0.07 error 107 Appendix 12.Analysis of variance for "L" values of color of raw dark turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 6.44 3.22 1.04 > 0.05 3(1) Ipens/T 6 18.66 3.11 P I periods 4' 17.42 4.36 17.76 < 0.05 TP I interaction 8 4.02 0.50 2.05 > 0.05 3(2) I residual 24 5.89 0.25 error Appendix 13.Analysis of variance for ”L" values of color of raw light turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 4.02 2.01 0.28 > 0.05 2(1) Ipens/T 6 43.07 7.13 P I periods 4 22.60 5.65 53.09 < 0.05 TP I interaction 8 2.10 0.26 2.46 < 0.05 3(2) I residual 24 2.55 0.11 error 108 Appendix 14. Analysis of variance for "b" values of color of raw dark turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 2.16 1.08 4.17 > 0.05 3(1) Ipens/T 6 1.55 0.26 P I periods 4 2.39 0.60 8.76 < 0.05 TP = interaction 8 0.70 0.09 1.29 > 0.05 3(2) I residual 24 1.64 0.07 error Appendix 15. Analysis of variance for "b" values of color of raw light turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 0.23 0.12 0.11 > 0.05 3(1) Ipens/T 6 6.48 1.08 P I periods 4 23.41 5.85 159.76 < 0.05 TP I interaction 8 0.22 0.03 0.76 > 0.05 3(2) - residual 24 0.88 0.04 error 109 Appendix 16. Analysis of variance for total cholesterol oxidation product concentrations in cooked dark turkey meat (cooked study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 6.10 3.05 53.52 < 0.05 3(1) Ipens/T 6 0.34 0.06 P I periods 2 15.87 7.94 162.33 < 0.05 TP I interaction 4 0.99 0.25 5.07 < 0.05 3(2) I residual 12 0.59 0.05 error Appendix 17. Analysis of variance for total cholesterol oxidation product concentrations in cooked light turkey meat (cooked study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 4.30 2.15 30.67 < 0.05 3(1) Ipens/T 6 0.42 0.07 P I periods 2 11.70 5.85 62.22 < 0.05 TP I interaction 4 1.27 0.32 3.37 < 0.05 3(2) I residual 12 1.13 0.09 error 110 Appendix 18. Analysis of variance for TBARS values of cooked dark turkey meat (cooked study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 5.43 2.71 1.71 > 0.05 3(1) Ipens/T 6 9.52 1.59 P I periods 2 285.65 142.83 170.32 < 0.05 TP I interaction 4 5.03 1.26 1.50 > 0.05 3(2) I residual 12 10.06 0.84 error Appendix 19. Analysis of variance for TBARS values of cooked light turkey meat (cooked study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 4.62 2.31 4.35 > 0.05 3(1) Ipens/T 6 0.42 0.07 P I periods 2 93.89 46.94 210.89 < 0.05 TP I interaction 4 0.25 0.06 0.29 > 0.05 3(2) I residual 12 2.67 0.22 error 111 Appendix 20. Analysis of variance for drip loss of raw dark turkey meat (refrigerated study). Source of Degree of Sun of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 8.59 4.30 17.30 < 0.05 3(1) Ipens/T 6 1.49 0.25 P I periods 2 242.57 121.28 1131.06 < 0.05 TP = interaction 4 4.59 1.15 10.69 < 0.05 3(2) I residual 12 1.29 0.11 error Appendix 21. Analysis of variance for drip loss of raw light turkey meat (refrigerated study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 3.17 1.58 2.80 < 0.05 3(1) Ipens/T 6 3.40 0.57 P I periods 2 283.30 141.65 1952.08 < 0.05 TP I interaction 4 1.42 0.35 4.88 < 0.05 3(2) I residual 12 0.87 0.07 error 112 Appendix 22. Analysis of variance for drip loss of raw dark turkey meat (3 months frozen study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 1.81 0.91 0.96 > 0.05 3(1) Ipens/T 6 5.65 0.94 P I periods 2 31.66 15.83 210.07 < 0.05 TP I interaction 4 0.26 0.06 0.85 > 0.05 3(2) I residual 12 0.90 0.08 error Appendix 23. Analysis of variance for drip loss of raw light turkey meat (3 months frozen study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 40.25 20.12 32.41 < 0.05 3(1) Ipens/T 6 3.73 0.62 P I periods 2 11.62 5.81 74.88 < 0.05 TP I interaction 4 0.95 0.24 3.06 > 0.05 3(2) I residual 12 0.93 0.08 error 113 Appendix 24. Analysis of variance for drip loss of raw dark turkey meat (6 months frozen study). Source of Degree of Sun of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 3.84 1.92 4.71 > 0.05 3(1) Ipens/T 6 2.45 0.41 P I periods 2 45.82 22.91 86.35 < 0.05 TP I interaction 4 4.14 1.04 3.90 < 0.05 3(2) I residual 12 3.18 0.27 error Appendix 25. Analysis of variance for drip loss of raw light turkey meat (6 months frozen study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 69.15 34.57 49.57 < 0.05 3(1) Ipens/T 6 4.19 0.70 P I periods 2 20.68 10.34 32.14 < 0.05 TP I interaction 4 2.00 0.50 1.56 > 0.05 3(2) I residual 12 3.86 0.32 error 114 Appendix 26. Analysis of variance for TBARS values of raw dark turkey meat (frozen study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 0.89 0.44 24.30 < 0.05 3(1) Ipens/T 6 0.11 0.02 P I periods 1 0.03 0.03 4.47 < 0.05 TP I interaction 2 0.15 0.07 10.37 < 0.05 3(2) I residual 6 0.04 0.01 error Appendix 27. Analysis of variance for TBARS values of raw light turkey meat (frozen study). Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 0.08 0.04 12.64 < 0.05 3(1) Ipens/T 6 0.02 0.00 P I periods 1 0.00 0.00 1.87 < 0.05 TP I interaction 2 0.01 0.00 1.30 < 0.05 3(2) I residual 6 0.01 0.00 error Appendix 28. 115 Analysis of variance for ”a” value of color of raw dark turkey meat (frozen study). Source of variation T I Treatments 13(1) =Pen8/T P I periods TP I interaction 3(2) I residual error Degree of Sum of Mean F Probability Freedom Square Square Value 2 0.55 0.28 1.47 > 0.05 6 1.13 0.19 1 11.38 11.38 47.68 < 0.05 2 0.71 0.35 1.48 > 0.05 6 1.43 0.24 Appendix 29. Analysis of variance for ”a" values of color of raw light turkey meat (frozen study). Source of Variation T I Treatments 3(1) Ipens/T P I periods TP I interaction 3(2) I residual error Degree of Sum of Mean F Probability Freedom Square Square value 2 5.18 2.59 37.88 < 0.05 6 0.41 0.07 1 45.25 45.25 119.25 < 0.05 2 6.98 3.49 9.19 < 0.05 6 2.28 0.38 Appendix 30. 116 Analysis of variance for "L" value of color of raw dark turkey meat (frozen study). Source of Variation T I Treatments 3(1) Ipens/T P I periods TP I interaction 3(2) I residual error Degree of Sum of Mean F Probability Freedom Square Square Value 2 3.74 1.87 0.87 > 0.05 6 12.94 2.16 1 12.92 12.92 6.69 < 0.05 2 0.15 0.07 0.04 > 0.05 6 11.60 1.93 Appendix 31. Analysis of variance for "L" values of color of raw light turkey meat (frozen study). Source of Variation T I Treatments 13(1) 'Pensl'l‘ P I periods TP I interaction 3(2) I residual error Degree of Sum of Mean F Probability Freedom Square Square Value 2 4.55 2.28 0.61 > 0.05 6 22.48 3.75 1 111.50 111.50 25.01 < 0.05 2 11.64 5.82 1.30 > 0.05 6 26.75 4.46 Appendix 32. 117 Analysis of variance for "b" value of color of raw dark turkey meat (frozen study). Source of Variation T I Treatments 3(1) Ipens/T P I periods TP I interaction 3(2) I residual error Degree of Sum of Mean F Probability Freedom Square Square Value 2 1.87 0.93 1.92 > 0.05 6 2.92 0.49 1 0.05 0.05 0.39 > 0.05 2 1.07 0.53 3.91 > 0.05 6 0.82 0.14 Appendix 33. Analysis of variance for "b” values of color of raw light turkey meat (frozen study). Source of variation T I Treatments 3(1) Ipens/T P = periods TP I interaction 3(2) I residual error Degree of Sum of Mean F Probability Freedom Square Square Value 2 0.57 0.29 2.98 > 0.05 6 0.57 0.10 1 23.80 23.80 182.53 < 0.05 2 0.10 0.05 0.37 > 0.05 6 0.78 0.13 118 Appendix 34. Analysis of variance for a-tocopherol contents in plasma samples of turkey during feeding period. Source of Degree of Sum of Mean F Probability Variation Freedom Square Square Value T I Treatments 2 53.31 26.65 217.31 < 0.05 3(1) Ipens/T 6 0.74 0.12 P I periods 3 0.87 0.29 2.22 > 0.05 TP I interaction 6 1.47 0.24 1.88 > 0.05 3(2) I residual 18 2.34 0.13 error Appendix 35. Analysis of variance for body weights of turkey during feeding period. Source of Degree of Sum of Mean F Probability Variation Freedom Square Square value T I Treatments 2 1.56 0.78 1.23 > 0.05 3(1) Ipens/T 6 3.81 0.64 119 Appendix 36. 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