..1 1" ‘ 310(03):)“011 LIBRARY Michigan State University This is to certify that the dissertation entitled LIPID PEROXIDATION IN POULTRY MEAT AND ITS SUBCELLUIAR MEMBRANES AS INFLUENCED BY DIETARY OILS AND ANTIOXIDANT SUPPIJSMEIN'I‘ATION presented by Charles Fley-Fung Lin has been accepted towards fulfillment of the requirements for _EhLll._ degree in EmiScience fiaWd/W’ 6’ 3% fl Major professor ' W Date 1/ LOW/z, /é} /Q&F MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LlBRARlES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date {stamped below. LIPID PEROXIDATION IN POULTRY HEAT AND ITS SUBCELLULAR MEMBRANES AS INFLUENCED BY DIETARY OILS AND ANTIOXIDANT SUPPLEMENTATION BY Charles Fley-Fung Lin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1988 1" 1‘ DO. ‘4. I 1:: ABSTRACT LIPID PEROXIDATION IN POULTRY MEAT AND ITS SUBCELLULAR MEMBRANES AS INFLUENCED BY DIETARY OILS AND ANTIOXIDANT SUPPLEMENTATION BY Charles Fley-Fung Lin Experiments were designed to study the influence of dietary oils varying in degree of unsaturation, oxidized oil, and dietary supplements of butylated hydroxyanisole (BHA)/butylated hydroxytoluene (BHT) and alpha-tocopherol on membranal lipid stability and its relationship to the oxidative stability of broiler meat during refrigerated and frozen storage. The fatty acid composition of the broiler depot fat as well as intramuscular (membrane-bound) lipids was influenced by the fatty acid composition of dietary oils. This, in turn, influenced the rate of NADPH/ADP/ferric iron-induced lipid peroxidation in mitochondria and microsomes. Similarly, oxidized oil in the broiler diet tended to increase the susceptibility of subcellular membranal lipids to metmyoglobin/hydrogen peroxide- initiated lipid peroxidation and produced rapid oxidative changes in raw and cooked meat during storage. The 5% U“ eht. “ooh “A“; VV..\ 5}. cue ‘L. b.‘r: Sta; ‘1 presence of a synthetic antioxidant such as BHA or BHT in the broiler diet significantly (P < 0.05) lowered the thiobarbituric acid-reactive substances (TEARS) numbers of raw meat but had a limited effect on the oxidative stability of cooked meat. Supplementation of the broiler diets with alpha- tocopherol significantly (P < 0.01) increased the concentrations of this antioxidant in the dark and white meat, and in the microsomal and lipoprotein fractions of the dark meat as well as in the lipoprotein fraction of the white meat. This antioxidant stabilized the membranal lipids toward peroxidative changes initiated by nonenzymic processes, and produced a longer shelflife for raw and cooked broiler meat during storage. Results of this study support the hypothesis that lipid peroxidation in raw meat is initiated in the membrane-bound lipids. Stabilization of the membrane lipids, either by alteration of the fatty acid composition or by the incorporation of alpha-tocopherol into them through diet, had a positive influence on the oxidative stability of raw and cooked meat during storage. 45- v “Age. thUI A’:‘; 5...... H: '- “ea-.3 ‘ lAv' Asae. ACKNOWLEDGEMENTS The author wishes to express his sincere gratitute to Dr. J. I. Gray for his effective guidance, advice and encouragement throughout this research program, and for his critical review of this manuscript. My appreciation is extended to Dr. D. M. Smith., Dr. J. R. Giacin., Dr. Cal Flegal., Dr. A. M. Booren., and Dr. J. F. Price for serving on the guidance committee. Further acknowledgement is due Dr. Ali Asghar for his technical advice. Finally, my deepest gratitude is extended to my family, Diana, Angy and Frank, for their moral support and continual love. ’9‘ to. 0‘0. fio‘. TABLE OF CONTENTS Page LIST OF TABLES ......... ................................ vii LIST OF FIGURES ............... ..... .................... xi INTRODUCTION ..... ...................................... 1 LITERATURE REVIEW ....... ............................... 5 Lipids in Meat Systems ...... ...................... 5 Adipose Lipids of Broilers ................... 5 Membrane-Bound Lipids ........................ 5 Membrane Bound Cholesterol .. ................. 6 Membrane-Bound Alpha-Tocopherol .............. 7 Influence of Diet on Fatty Acid Composition of Meat Lipids .............OOOOOOOOOOIOOOOOOO. ....... 9 Adipose Lipid ... ...... .. .................... . 9 Intrmuscularlipids . ..................... ..... 10 1) Marbling Fat ............................. 11 2) Membrane-Bound Lipid ......... ............ 11 Effects of Dietary Fats on Growth Performance .... 13 Dietary oil Supplementation .................. 14 Oxidized Dietary Oil ......................... 15 Lipid Peroxidation in Meats ...................... 17 Catalysts of Lipid Peroxidation in Meat Systems .. ........... ..... ................... 21 Initiation of Lipid Peroxidation in Meat systems ......OOOOOOOOOOOO ..... O. OOOOOOOOOOOOO 21 1) Nonenzymic Initiators ................... 21 2) Enzymic Initiators ...... . ................ 26 Inhibition of Lipid Oxidation in Meat Systems .... 27 Direct Addition of Antioxidants to Meat Products .... ................................. 27 1) Synthetic Phenolic Antioxidants ......... 27 2) Natural Antioxidants .................... 28 3) Maillard Reaction Products .............. 29 Introduction of Antioxidant into Meats by Dietary Supplemenmtation ..................... 30 1) Supplementation of Poultry Diets with Alpha-Tocopherol ........................ 30 2) Supplementation of Broiler Diets with Synthetic Antioxidants ........... ....... 32 Yv-M a i -r. “ A‘\\ \ --- . I ‘n‘t.-. ““Vv‘ V'\ ”‘5... ”v \. C» J\ (I) (I) r ‘ ii Alpha-Tocopherol Activity and Biochemical Function ......OOOOOOOO......OOOOOOOOOOOOOO0...... 33 REFERENCES ......OOOOOOOOOOOOOOOOO....OOOOOOCOOOOOOOOOO 38 CHAPTER I LIPID PEROXIDATION IN POULTRY MEAT. I: EFFECTS OF DIETARY OILS AND ALPHA-TOCOPHEROL SUPPLEMENTATION ON BROILER GROWTH PERFORMANCE AND MEAT STABILITY.. 51 ABSTRACT ......OOOOOOOOOOOO......OOOOOOOOOOOOOO0...... 52 INTRODUflION ......OOOOOOOOOOOOOOOO ..... O ...... 0...... 53 MATERIALS AND METHODS ................. ..... . ......... 55 Source of Oils ................ ....... . .......... 55 Broiler Feeding Regimens .. ..... ..... ....... ..... 55 Sample Preparation .............................. 57 Lipid Extraction ................................ 58 Fatty Acid Analysis ............................. 58 Analysis of Alpha-Tocopherol in Muscle Tissue ... 59 Separation and Estimation of Phospholipids ...... 60 2-Thiobarbituric Acid (TBA) Test ................ 60 Statistical Analyses ........ ... ................. 61 RESULTS AND DISCUSSION ...... ..................... .... 61 Carcass Characteristics ............. ............ 61 Lipid Component in Broiler Meat ....... ......... . 64 Fatty Acid Composition of Neutral Lipids ........ 66 Fatty Acid composition of Phospholipids ......... 68 Changes in Muscle Lipids During Storage ......... 71 Concentration of Alpha-Tocopherol in Meats ...... 75 Oxidative Stability of Dark and White Meat . ..... 78 SUMMARY AND CONCLUSIONS ... ........ .. .......... . ...... 83 CHAPTER II LIPID PEROXIDATION IN POULTRY MEAT. II: EEFFECTS OF DIETARY OILS AND ALPHA-TOCOPHEROL SUPPLEMENTATION ON MEMBRANAL LIPID STABILITY .... 88 :v U) (I "’\."" .0“ ‘- on... '11 i - I (ll) iii ABSTRACT ......OOOOOOOOOOOOOOO.... OOOOOOOOOOOOOOOOOOOO INTRODUflION ......OOOOOOOOOOOOOOO........OOOOOIOOOOOOO MATERIALS AND METHODS .......................... ...... Broiler Feeding Regimens ........................ Extraction of Sarcoplasm from Meat and Isolation of Mitochondria and Microsomes .................. Lipid Extraction ................................ Fatty Acid Analysis ............................. Quantitation of Alpha-Tocopherol in Membranes ... Measurement of Subcellular Membrane Protein ..... Measurement of NADPH-Induced Lipid Peroxidation in Membranes .................................... Statistical Analyses of Experimental Data ....... RESULTS AND DISCUSSION ......... ...................... Membranal Lipid Composition ..................... Fatty Acid Composition .......................... Concentration of Alpha-Tocopherol in Subcellular Membranes ....................................... 89 9O 92 92 92 93 93 94 94 95 96 96 96 99 105 Oxidative Stability of Membrane Bound lipids ..... 107 SUMMARY AND CONCLUSION ..... .......................... REFERENCES .......... ................................. CHAPTER III LIPID PEROXIDATION IN POULTRY MEAT. III: EFFECTS OF OXIDIZED DIETARY OIL AND ANTIOXIDANT SUPPLEMENTATION ON BROILER GROWTH AND MEAT STABILITY ................... ....... ........ ..... ABSTRACT ....... ..... ......... ................. . ...... INTRODUCTION ............ ............................. MATERIALS AND METHODS ......... ....................... Broiler Feeding Regimens ................. ...... . Oxidation of Sunflower Oil ...................... Sample Preparation for Analysis ................. Lipid Extraction and Fatty Acid Determination ... Separation of Phospholipids ..................... Quantitation of Alpha-Tocopherol in Muscle Tissue 124 125 126 127 128 128 128 129 AA “ \er «C :x \w. iv Analysis of ERA and BHT in Muscle Tissues ........ 129 Lipid Oxidation in Meat systems ........... ...... . 130 Statistical Analyses ........... .................. 131 RESULTS AND DISCUSSION .......................... 131 Carcass Characteristics .... ...................... 131 Lipid Component in Broiler Meat .................. 134 Fatty aCids comPOSition 00.0000.0.000000000000000. 136 Concentration of Antioxidants in Broiler Tissues . 139 Oxidative Stability of Dark and White Meat ....... 144 CONchIONS 00000000000000.000000...0.0.0.0000... ..... 0 149 REFEMNCES 0.0.0.0.... 000000000000000000000000000000000 150 CHAPTER IV LIPID PEROXIDATION IN POULTRY MEAT. VI: EFFECTS OF OXIDIZED DIETARY OIL AND ANTIOXIDANT SUPPLEMENTATION ON MEMBRANAL LIPID STABILITY ..... 153 ABSTRACT ....................... ................. ...... 154 INTRODUCTION ............... ......................... .. 155 MATERIALS AND METHODS ....... .......................... 157 Broiler Feeding Regimens .. ........... . ........... 157 Extraction of Sarcoplasm from Meat and Isolation of Mitochondrial, Microsomal and Lipoprotein Fractions .............. .......................... 157 Lipid Extraction .. ........... . ...... ...... ....... 157 Fatty Acid Analysis ........... ................... 158 Quantitation of Alpha-Tocopherol in Membranes .... 158 Idenfication of BHA and BHT in membranes ......... 158 Measurement of Subcellular Membrane Protein ...... 158 Measurement of Metmyoglobin/Hydrogen Peroxide- Induced Lipid Peroxidation in Membranes .......... 159 Statistical Analyses of Experimental Data ........ 159 RESULTS AND DISCUSSIONS ......... ...................... 160 Membranal Lipid Composition ...................... 160 Fatty Acid Composition of Membranal Neutral Lipids 162 Fatty Acid Composition of Membranal Phospholipids 166 Concentration of Alpha-Tocopherol in Subcellular membranes ......0.0.000000000000000000000.0.000... 166 Oxidative Stability of Membrane Bound Lipids ..... 173 SUMMARY AND CONCLUSION ............. ....... ...... ...... 181 REFERENCES . ........................................... 182 CHAPTER V LIPID PEROXIDATION IN POULTRY MEAT. V: THE INFLUENCE OF DIETARY OILS AND ANTIOXIDANT SUPPLEMENTATION ON THE OXIDATIVE STABILITY OF COOKED MEAT DURING STORAGE ....................... 185 ABSTRACT ..... ......................................... 186 INTRODUCTION ..... ..................................... 187 MATERIALS AND METHODS .... ............................. 189 Broiler Feeding Regimens ...... ................... 189 Cooking Procedure ................................ 189 Lipid Extraction from cooked meat ........ . ...... . 189 Measurement Lipid Oxidation in Cooked Meat ....... 190 Hexanal Determination ... ............... . ......... 190 Thiobarbituric acid (TBA) Procedure ..... ......... 191 Sensory Evaluation of Cooked Meat ................ 191 Analysis of Experimental Data .................... 192 RESULTS AND DISCUSSION 0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 OOOOOOOOOOOOOOOOO 192 Lipid Component of Cooked Meat ................... 192 Oxidative Stability of Cooked Dark and White Meat During Refrigerated Storage . ......... .... ........ 194 Hexanal Contents in Cooked Meat ..... ............. 198 Sensory Panel Scores for Cooked Meat ............ . 203 REFERENCES ......... ................................... 209 SUMMARY CONCLUSIONS ........ ..... .. ................... . 212 PROPOSAL FOR FUTURE RESEARCH ......................... . 216 APPENDICES 0.00.00.00.0000000000 00000000000000000000000 217 Percent fatty acid composition of the neutral lipids isolated from lipoprotein fraction of dark (D) and white (W) meat of broilers fed oxidized dietary oil and antiOXj-dants 0.00.00.00.00000000 ...... 0.... Percent fatty acid composition of phospho- lipids isolated from lipoprotein fraction of dark (D) and white (W) meat of broilers fed oxidized oil and antioxidants ........ Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in lipOproteins from dark meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/ BHT supplementation ...................... Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in lipoproteins from white meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementation .... ...................... The evaluation procedures and scoresheet for sensory analysis of cooked meat. ...... 217 218 219 220 221 TABLES LIST OF TABLES CHAPTER I 1. 2. Composition of the experimental all-mesh broiler diets used in the feeding study .... Effect of dietary oils and alpha-tocopherol on the feed consumption, feed conversion, body weight and dressing percentage of broilers ........... ..... .......... ......... Lipid composition of dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol .. ....... Percent fatty acid composition of neutral lipids isolated from dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol ................. Percent fatty acid composition of phospho- 1ipids isolated from dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol ................. Percent fatty acid composition of neutral lipids isolated from dark 3D) and white (W) broiler meat stored at -20 C for 8 months . Percent fatty acid composition of phospho- lipids isolated from dark 8D) and white (W) broiler meat stored at -20 C for 8 months.. Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat) of dark (D) and white (W) meat (stored at 4°C from broilers fed different dietary oils and alpha-tocopherol ................. Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat) of dark (D) and white (W) meat (stored at -20°C from broilers fed different dietary oils and alpha-tocopherol ................. 56 62 65 67 69 73 74 79 80 v9“ Iv . II U“ a..\ "T: be 1 0 1.3 O ”{J In. viii CHAPTER II 1. Lipid composition of the subcellular membranes isolated from the dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol . ..... ... 97 Percent fatty acid composition of the neutral lipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha—tocopherol ..................... 100 Percent fatty acid composition of phospho- lipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha- tocopherol ......... ........... . ......... 101 Percent fatty acid composition of the neutral lipids isolated from microsomal membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherool .. ................. 102 Percent fatty acid composition of phospho- lipids isolated from microsomal membranes of dark (D) and white (W) of broilers fed differeant dietary oils and alpha- tocopherol ............. .......... .. ..... 103 Levels of alpha-tocopherol in dark and white meat from broilers fed different dietary oils and alpha-tocopherol, one day post slaughter ...................... 106 Analysis of variance of thiobarbituric acid-reactive substances (TBARS numbers, nmole malonaldehyde/mg membrane protein) in subcellular membranes of dark and white meat from broilers fed different dietary oils and alpha-tocopherol ........... .... 113 Oxidative stability of subcellular membranes isolated from dark and white meat of broilers fed different dietary oils and alpha-tocopherol as expressed by TBARS numbers (nmole malonaldehyde/mg membrane protein) ....................... 114 #19 9*": Lu“. L .— K) ()1 Ofl‘im EIZJ'U O ix CHAPTER III 10 Effect of oxidized dietary oil and antioxidant suupplementation of the feed consumption, feed conversion, body weight and dressing percentage of broilers ...... Lipid composition of dark (D) and white (W) meat from broilers fed oxidized dietary oil and antioxidants ............ Percent fatty acid composition of neutral lipids isolated from dark (D) and white (W) meat of broilers fed oxidized dietary oil and antioxidants .............. ...... Percaent fatty acid composition of phospho- lipids isolated from dark (D) and white (W) meat of broilers fed dietary oil and antioxidants .. .......................... Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat) of dark (D) and white (W) meat from broilers fed oxidized dietary oil and antioxidants . ....................... .... Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat) of dark (D) and white (W) meat from broilers fed oxidized dietary oil and antioxidatns .... ........................ CHAPTER IV 1. Lipid composition of the subcelluilar membranes isolated from the dark (D) and white (W) meat of broilers fed oxidized dietary oil and antioxidants ........... Percent fatty acid composition of the neutral lipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers ........... .................. Percent fatty acid composition of the neutral lipids isolated from microsomal membranes of dark (D) and white (W) meat of broilers ............................ 132 135 137 138 145 146 161 163 165 Percent fatty acid composition of phospho- lipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers ....... ................ Percent fatty acid composition of pholpho- lipids isolated from microsomal membranes of dark (D) and white (W) meat of broilers fed oxidized dietary oils and antioxidants Concentration of alpha-tocopherol in the subcellular membranes of dark (D) and white (W) meat from broilers fed different dietary regimens .............. Oxidative stability of subcellular membranes isolated from dark and white meat of broilers fed oxidaized dietary regimens as expressed by TBARS numbers (nmole malonaldehyyde/mg membrane protein) CHAPTER V 1. Lipid composition of cooked dark (D) and white (W) meat from broilers fed dietary oils and antioxidants .................... Effect of storage at 4°C on thiobarbituric acid-reactive substances (TBARS) numbers of cooked dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol (EXperiment #1) .... ..... Effect of storage at 4°C on thiobarbituric acid-reactive substances (TBARS) numbers of cooked dark (D) and white (W) meat of broilers fed oxidized dietary oil and antioxidants (Experiment #2) ............. Hexanal concentrations (mg/kg met) in cooked dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol ..... ...... ............ Hexanal concentrations (mg/kg meat) in cooked dark (D) and white (W) meat from broilers fed oxidized dietary oils and antioxidants . ...... ..... ..... . ......... 167 168 170 179 193 195 196 199 201 xi Sensory panel scores of cooked dark (D) and white (W) meat from broilers fed different dietary oils and alpha- tocopherol ................... ........... Sensory panel scores of cooked dark (D) and white (W) meat from broilers fed oxidized dietary oils and antioxidants .. 204 206 LIST OF FIGURES FIGURES REVIEW OF LITERATURE 1. Schematic diagram depicting the lipid bilayer organization of a eukaryotic plasma membrane .............. .......... 2. Diagrammatic representation of events which appear to be involved in enzyme- catalyzed lipid peroxidation and its control ..... ............................ 3. Interrelationships of alpha-tocopherol (vitamin E) in protecting membranes ..... CHAPTER I 1. Alpha-tocopherol concentration (mg/kg meat) in the dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol .... ........ .. ..... CHAPTER II 1. NADPH-initiated lipid peroxidation in microsomes from white meat of broilers fed different dietary oils varying in degree of unsaturation and alpha- tocopherol supplementation ............. NADPH-initiated lipid peroxidation on microsomes from dark meat of broilers fed different dietary oios varying in degree of unsaturation and alpha- tocopherol supplementation ............. NADPH-initiated lipid perocidation in mitochondria from white meat of broilers fed different dietary oils varying in degree of unsaturation and alpha- tocopherol supplementation ............ xii 35 36 76 108 109 110 nov ‘ l .- Era-:1: N:\ v v.41: L.‘ h) L.) .‘L xiii NADPH-initiated lipid peroxidation in mitochondria from dark meat of broilers fed different dietary oils varying in degree of unsaturation and alpha- tocopherol supplementation ............ CHAPTER III 1. Alpha-tocopherol concentration (mg/kg meat) in the dark (D) and white (W) meat from broilers fed oxidized dietary oils and antioxidants ....................... CHAPTER IV 10 Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in mitochondria from dark meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/ BHT supplementation .................... Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in mitochondria from white meat of broilers fed oxidixed dietary oil, alpha-tocopherol and BHA/ BHT supplementation ......... ........... Metmyoglobin/hydrogen perxide-initiated lipid peroxidation in microsomes from dark meat of broilers fed oxidized dietary oil, alpha—tocopherol and BHA/ BHT supplementation ................... Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in microsomes from white meat of broilers fed oxidized dietary uoil, alpha-tocopherol and BHA/ BHT supplementation ................ ... 111 140 174 175. 176 177 4U v. INTRODUCTION Lipid peroxidation in muscle foods is a major degradative process responsible for the deterioration in quality of meat and meat products during storage (Pearson et al., 1983). Poultry meat is more susceptible than red meat to the development of rancidity during frozen storage in the raw state (Wilson et al., 1976) and to the development of "warmed-over" flavor (WOF) during refrigerated storage of the cooked meat (Tims and Watts, 1958). Oxidation of muscle lipids has been recognized as being contributory to the development of rancidity and/or WOF in meat and meat products. It is generally accepted that lipid peroxidation in meat is initiated at the membrane level, and that the phospholipids in the membranes are the primary centers for the initiation of peroxidation (Gray and Pearson, 1987; Asghar et al., 1988). The localization of relatively large amounts of polyunsaturated fatty acids in the membranes of mitochondria, microsomes and lipoproteins makes them highly vulnerable to peroxidative changes (Buege and Aust, 1978). Prooxidants such as oxygen, peroxidase enzymes (Vladimirov et al., 1980), heme and nonheme iron (Igene et al., 1979), hydrogen peroxide, and superoxide radicals (Kanner and Harel, 1985) are normally present in muscle cells. These C" VA :16: ‘ .8 “e in. C21“. a» .‘A an an.— Gd 6 ‘ AV uh u a SIM H ~A PM hi. a 1‘1‘ <\ C F \ k .. e a» D. C. D. endogenous compounds play an important role in the formation of the primary pool of biological catalysts in muscle tissues. Since these endogenous initiators are not easy to control, alteration of the fatty acid composition of the membranes and incorporation of an antioxidant into the membranes by dietary means appear to be viable methods in controlling muscle lipid oxidation. Dam and Granados (1945) postulated that alpha-tocopherol could function as an antioxidant in 1129. In the biological system, alpha- tocopherol is located primarily in the mitochondrial, microsomal, and lipoprotein portions of the cell (Machlin, 1984). As a structural component of the membrane, alpha- tocopherol is believed to be located adjacent to membrane- bound enzymes such as nitcotinamide adenine dinucleotide phosphate (NADPH) oxidase, that generate superoxide anions and other free radicals. In the cytosol of a cell, the superoxide anion interacts with hydrogen ions to produce hydrogen peroxide, which is then distributed in both the aqueous and membrane phases of the cell. In the aqueous phase, glutathione peroxidase and catalase destroy most of the hydrogen peroxide formed. The remaining hydrogen peroxide may move into the membranes and react with superoxide anion to form the hydroxy radical (OH'). Membrane-bound alpha-tocopherol ...- can trap hydroxy radicals as quickly as they are formed. Otherwise, this extremely reactive radical may initiate peroxidation of membrane lipids. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are usually added to oil or feed to prevent oxidative changes. Unfortunately, little is known about their ability to stabilize membranal lipids when introduced into the muscle through dietary supplementation. Dietary-induced changes in lipid composition have been observed in muscle triacylglycerols (Sklan et al., 1983) as well as in mitochondrial and microsomal membranes upon feeding animal diets supplemented with elevated levels of saturated or unsaturated fatty acids (McMurchie et al., 1986). The foremost element of this study was to provide evidence to support the hypothesis that lipid peroxidation in raw meat is initiated in the membrane-bound lipids and that stabilization of the membrane lipids, either through alteration of the fatty acid composition or by the incorporation of an antioxidant into them, may influence the oxidative stability of meat during storage. (1) (2) Specific objectives of this study were: To determine the effect of different dietary oils including oxidized oil, BHA/BHT, and alpha-tocopherol supplementation on the fatty acid composition of, and the antioxidant concentrations in the subcellular membranes of broiler meat: To evaluate the influence of these dietary treatments on the peroxidative stability of membranal lipids and its relationship to the stability of broiler meat during storage. nezbran-e v. :,. , hie... C~ .. x . 2‘5319 ¢ LITERATURE REVIEW Lipids in Meat Systems Lipids in meat systems are composed namely of adipose lipids (depot fat) and intramuscular or tissue lipids, including membrane-bound lipids. Other meat lipids include membrane-bound alpha-tocopherol and cholesterol, although their concentrations are low relative to those of the other muscle lipids. .QLA'oseLieidesfM The adipose lipids of broiler muscles consist mainly of triacylglycerols which are largely composed of straight- chain, even-numbered carbon fatty acids, typically containing 16 or 18 carbon atoms (Dugan, 1971). Generally, the predominant fatty acids of broiler depot fat are saturated or contain only one or two double bonds, i.e., C16:0 (22.8%), Cl8:1 (37.0%), C18:2 (23.7%), C18:0 (6.5%) and C16:1 (5.7%). Polyunsaturated fatty acids with three or more double bonds comprise less than three percent of the total triacylglycerol fatty acids found in broilers (Pearson et al., 1977). Membrane-Bound Lipids Membrane-bound lipids are part of the structural components of subcellular membranes such as mitochondria, (Holman and Widmer, 1969), microsomes (Macfarlane et al., 1960), sarcoplasmic reticulum (Newbold et a1, 1973), and muscle fiber sarcolemma (Kono and Colowick, 1961). The membrane-bound lipids are composed mainly of phospholipids and lipoproteins (Rothfield and Finkelstein, 1968), the contents of which tend to be constant in the membranes, even when the fat content of muscles is highly variable. However, there is still considerable variation in the phospholipid content among species (Kaucher 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 (Bloor, 1943; Acosta et al., 1966). Most of the phospholipids located in the membranes contain highly unsaturated fatty acids. They are susceptible to oxidation (Buege and Aust, 1978) and are assumed to be the site of the initiation of lipid peroxidation (Asghar et al., 1988). e e-Bo nd C o esterol Cholesterol is the most abundant sterol in animal tissues, occurring in both the free and combined forms. It is present as the free alcohol, or as long-chain fatty acids esterified with the C-3 hydroxl group of the A ring of the cholesterol molecule (Smith et al., 1983). 'n fist *4. :1 It) '-6 C) '1 "6. 1“1 5L. “‘4‘ ‘L 5“( The specific function of cholesterol in membranes is to maintain the lipid bilayers in the fluid state (Smith et al., 1983). At the same time, cholesterol reduces the mobility of the fatty acyl chains and increases the microviscosity in the interior of the bilayer (Smith et al., 1983). The presence of cholesterol in membranes could influence the availability of the active lipid components in animal membranes toward oxidation (Pearson et al., 1977). A schematic diagram depicting the lipid bilayer organization and the position of cholesterol in a eukaryotic plasma membrane is presented in Figure 1. Me ne- 0 d A h - oco herol Molenaar et a1. (1980) proposed that alpha-tocopherol is located adjacent to membrane-bound enzymes such as reduced nicotinamide adenine dineucleotide phosphate (NADPH) oxidase. In membranes, the arachidonyl residue of the phospholipids forms a complex with the phytyl chain of the tocopherol molecule. Thus, the chromanol ring of the alpha-tocopherol is in contact with the NADPH oxidase. The unsaturated fatty acids surrounding the oxidases are exposed to free radical attack, but are protected by the redox function of the chromanol ring of the alpha- tocopherol molecule. Because the range of action of a free . unfit. .fl:fl.o.\u ._/?.._>..:.._:<.. 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Influence of Diet on the Fatty Acid Composition of Meat Lipids Dietary fats/oils are known to influence the total lipid content and fatty acid composition of depot fats and tissue lipids (intramuscular lipids) including marbling fat and membrane-bound lipids of poultry. Depot fats (adipose tissue) are localized mainly in the abdominal and subcutaneous areas of the animal body. Tissue lipids are localized not only between muscle fibers but also in the membranes. mm Past studies have concentrated primarily on dietary- induced changes in the fatty acid distribution of either the total lipids or the depot fat from unfractionated tissues. The effects of diet on the total lipid content and composition of depot fat of pigs have been investigated extensively. Ellis and Isbell (1926) demonstrated that different levels of corn oil (4.1 and 11.5%) in the diet affected not only the amount of lipid, but also the composition of the depot fat of the pig. At the 11.5% leve love fat‘. eat: in c h e. \i N \t L; a 10 level, corn oil produced a fat with a lower melting point, lower firmness grade, and greater proportion of unsaturated fatty acids. Soft pork which is largely due to the consumption of a high content of unsaturated lipids by pigs has a much softer fat and is more susceptible to autoxidation (Ellis, 1933). Shorland (1955) reported that the depot fats of ruminants were more resistant than non-ruminants to changes in composition by dietary oils. In order to have a similar response for ruminants, it is necessary to bypass the rumen using a duodenal fistula, or to protect the dietary oil from the action of rumen microorganisms (Ogilvie et al., 1961). In poultry, dietary oils influence not only the total lipid content but also the fatty acid composition of the adipose tissue (Edward et al., 1973). Poultry can readily incorporate the unsaturated fatty acids of the diet into depot fats (Marion and Woodroof, 1963; Salmon and O’Neil, 1973; Deaton et al., 1981). Highly unsaturated dietary fats will increase the unsaturation, thus increasing the potential for rapid oxidation of poultry depot fats. V‘ It" that than 11 WW Intramuscular lipids or tissue lipids are composed of deposits of triacylglycerols in fat cells localized between muscle fibers (commonly called marbling), and of phospholipids or lipoproteins located in the membranes. 1. Marbling EB; Orme et al. (1958) reported that the composition of the marbling fat in beef is relatively constant as compared to the depot fat which is highly variable. Koch et al. (1968) studied the fatty acid composition of back fat (adipose tissue) and of tissue lipids (intramuscular fat) in pigs during different fattening periods. They reported that the change in the amount of intramuscular fat was less than that of the depot fat. It has also been reported that dietary oil affects mainly the fatty acid composition of triacylglycerols, and, to a lesser extent, that of the phospholipids in turkey tissue lipids (Sklan et al., 1983; Sheldon, 1984). However, feeding broilers with poultry grease, beef tallow and lard resulted only in small differences in the fatty acid composition of the triacylglycerols of tissue lipids (Hulan et al., 1984). 56')? ch ages zezbrane Tahin e: 1931a,b: composit 12 2. Membranal-Bound Linus Several studies have focused on dietary-induced changes in the fatty acid composition of subcellular membranes such as microsomes (Kurata and Privett, 1980a,b; Tahin et al., 1981) and mitochondria (Innis and Clandinin, 1981a,b; Tahin et al., 1981). Changing the fatty acid composition of membranes can influence the specific role of membranal lipids, thereby modulating the functions of the membranes (Gibson et al., 1984). Physicochemical properties and biochemical functions of membranes depend extensively on both the phospholipids at the membrane surface and the fatty acyl chains contained within the bilayer matrix (Fourcans and Jain, 1974; Cronan and Gelman, 1975). Innis and Clandinin (1981a,b) demonstrated that the fatty acyl composition of phospholipids in the inner membranes of cardiac mitochondria of monkey is influenced by dietary oil. Although membrane-bound lipids, namely phospholipids, can be altered by diet (Innis and Clandinin, 1981a,b; McMurchie and Raison, 1979; McMurchie et al., 1986), the changes are relatively small compared to those of the depot fats. The ratio of saturated/unsaturated fatty acids in membrane phospholipids generally remains C02! Q ...u: have aris effec Effec Sourcl 13 constant, regardless of the dietary treatment (Gibson et al., 1984; McMurchie et al., 1986). In order to keep a fluid state and a constant rate of permeability, membranes have the capacity of buffering abrupt changes which could arise from the nature of the dietary lipid. Tahin et al. (1981) reported that each organelle has its own specific fatty acid composition. The composition may be influenced by diet, but to different degrees in different subcellular membranes. Changes in the fatty acid composition of the phospholipids of the mitochondria and microsomes can be induced in rats by diets having different contents of unsaturated fatty acids (Tahin et al., 1981). Even though it is apparent that a number of fatty acid profiles exist for different membrane lipids (McMurray and Magee, 1972), little attention has been paid to diet- induced modification of membranal phospholipids and its effect on the peroxidative stability of meat systems. Effects of Dietary Oils on Growth Performance of Broilers Dietary oils are widely supplemented in poultry feed to meet the high energy requirement of the fast growing broilers (Brue and Latshaw, 1985). Oils from different sources have different metabolic energy and exhibit different efficiency for growth of broilers (Fuller and Rendon, 1977). The utilization of dietary oil by broilers acid cxid. fact: 14 depends on its absorbability (Hulan et al., 1984), fatty acid composition (Corino et al., 1980), and its level of oxidation in the feed (Shermer and Calabotta, 1985). These factors all affect the growth performance of broilers. WW The addition of fat/oil to the diet of growing broilers has been reported to increase energy intake and improve growth performance as measured by growth and feed utilization (Harms, 1986). There is considerable evidence which indicates that increasing the energy level of the diet will also increase the intake of other nutrients by broilers (Aliken et al., 1954; Leong et al., 1959; Hurwitz et al., 1978) Harms (1986) reported a significant increase in broiler body weight and a linear improvement in feed-to-gain ratio (feed conversion) upon the addition of oil to the diet. A similar phenomenon in rats was first observed by Forbes and Swift (1944) who termed it the associative dynamic action of fats/oils. The digestibility of fats and oils by broilers has been shown to vary widely and depends upon many factors such as the condition of upper intestinal mucosal cells, the amount of pancreatic lipase and bile salt, and type of fat/oil (Young, 1982). The formation of a lipid-bile salt micelle is an important physiochemical prerequiste for 9.3] .k‘ ‘4 b l ‘a 0 0 \ die 1315?: 111188 15 maximium fat absorption. Several studies have demonstrated that oils from different sources vary in absorbability and this can significantly influence growth performance (Mateos et al., 1982; Hulan et al., 1984). The fatty acid composition of dietary oils also influences the utilization of the oils by broilers, with polyunsaturated fatty acids being more effectively utilized than the more saturated ones (Corino et al., 1980; Brue and Latshaw, 1985). Sklan et al. (1983) and Sheldon (1984) reported that fatty acid composition of the tissue lipids reflected dietary levels in broilers. The higher the degree of unsaturation of oils in the broiler diets, the more unsaturated fatty acids are deposited in the muscle tissue. Rapid oxidative changes have been observed in meat from broilers fed diets supplemented with polyunsaturated fatty acids (Salmon and O’Neil, 1973; Sklan et al., 1983). Med _;___r¥D'eta 9_il The effect of oxidized dietary oil on the growth performance of rats was initially investigated by Burr and Barnes (1943). Poor growth rate for rats was observed in this experiment. Oxidized dietary oil when fed to experimental animals (rats and pigs) causes various types and degrees of abnormalities. Restricted growth (Anglemier and Oldfield, 1957), reduced efficiency of feed utilization Vita H u wen RAJ fifia l I L'sL'lCl 16 (Oldfield and Anglemeir, 1957), and the deposition of soft fat susceptible to oxidation (Barnes et al., 1943; Lea, 1953) are among symptoms reported. The symptoms observed in the rats and pigs in these studies were induced by the destruction of selected vitamins in the feed or in the intestine (Anglemier and Oldfield, 1957). Rasheed et al. (1963) confirmed the findings of Anglemier and Oldfield (1957), who reported that highly unsaturated marine oil, when fed at only 10% of the diet, caused steatitis and enlarged livers. In addition, high levels of malonaldehyde were detected in the blood and excreta of the rats. Antioxidants such as ethoxyquin or alpha-tocopherol effectively negated most of the symptoms attributed to the presence of the oxidized oils in the diet. Severe feed oxidation significantly decreases the vitamin E content in the feed and can cause a well known, readily detected illness in broilers called encephalomalacia. This is an ataxia symptom resulting from hemorrhages and edema within the molecular and granular layers of the cerebellum (Century and Horwitt, 1959). Vitamin E prevents encephalomalacia in chicks by functioning as a biological antioxidant and interrupting free radical-ininiated lipid peroxidation. It is quite likely that broilers are routinely fed diets containing slightly oxidized fat and this can reduce 17 the profits of the poultry grower (Shermer and Calabotta, 1985). Peroxide levels of 2 meq/kg feed is a threshold level of oxidation in the poultry feed. The hydroperoxides can undergo decomposition to produce secondary products that can react with other ingredients in the feed such as lysine and certain vitamins, to reduce its nutritive value (Shermer and Calabotta, 1985). Lipid Peroxidation in Meats Lipid peroxidation is one of the major causes of deterioration in the quality of frozen meat or precooked meat products during storage. This results in undesirable changes in color, flavor, texture and nutritional value of the meat products. Lipid peroxidation generally involves a three-step series of reactions as shown in the scheme below: I. Initiation: Initiators RH ---------- > R‘ + H' (unsaturated fatty acid) (fatty free radical) II. Propagation: R' + 02 ----> ROO' (peroxy radical) ROO' + RH ---> ROOH + R' 18 ROOH ---> R0‘ + OH' R0’ + RH ---> ROH + R' III. Termination: n- + n° ---> n-n n; + n00° ---> noon noon + noo- ----- > noon + 02 Oxidation in polyunsaturated fatty acids takes place at one of the reactive methylene groups located between two non-conjugated double bonds. Upon uptake of oxygen, a hydroperoxide (ROOH) is initially formed which may then break down to carbonyl compounds (Gray, 1978). The hydroperoxides will also decompose to yield more free radicals and these new radical species can then propagate the peroxidative process. The formation of non-radical products at the termination step leads to the end of the chain reaction. Catalysts g; Lipid Peroxidation in Meat Systems Peroxidation of food lipids is promoted by heat, light and trace metal catalysts, especially copper and iron (Ingold, 1967; Waters, 1971). The rate of oxidation of fatty acids varies according to their degree of unsaturation and the influence of catalytic agents such as heat, enzymes, metal catalysts and hydrogen peroxide I“ 19 Lipid peroxidation in meats was initiately attributed to the presence of heme catalysts such as myoglobin, hemoglobin and cytochromes (Tappel, 1962a), mainly because of their high concentrations in meat systems. The catalytic effect of iron porphyrins on the oxidative deterioration of polyunsaturated fatty acids was first described by Robinson (1924). Tarladgis (1961) attributed the catalytic activity of ferric hemoproteins to the paramagnetic character of the porphyrin-bound iron. He suggested that the presence of five unpaired electrons in metmyoglobin produces a strong magnetic field that would favor the formation of free radicals. Catalysis by iron porphyrins is characterized by the rapid initiation and propagation of the lipid oxidation process (Tappel, 1962a). Homolytic cleavage of the RO-OH bond of the hydroperoxide is a generally accepted mechanism for hematin catalysis. According to Tappel (1962a), the hematin compound combines with the lipid hydroperoxide to form an activated compound. Following the scission of the peroxide bond, a lipid radical (RO‘) and a heme radical (Fe-O“) are produced. These free radicals have the ability to abstract a hydrogen atom from an unsaturated fatty acid (RH), thus regenerating the hematin compound and producing a fatty acid radical (R'). Fe-O' + RH ---------- > Fe-OH + R' In 3.5 in DCF’. \ H «(Ems 20 Fe-OH + noon --------- > Fe-OOR + 2 H+ Fe-OOR ---------------- > Fe-O' + no; There is also considerable evidence that indicates that nonheme iron is the active catalyst of lipid oxidation in cooked meat. Sato and Hegarty (1971) reported that nonheme iron, rather than heme iron, was the iron species responsible for the rapid oxidation of cooked meat. Love (1972) confirmed the observations of Sato and Hegarty (1971) and reported that metmyoglobin did not influence the level of oxidation in cooked meat. However, levels of ferrous iron as low as 1 mg/kg effectively increased lipid oxidation in meat systems. Igene et al. (1979) demonstrated that cooking increased the level of free iron in meat and concluded that heme pigments undergo decomposition during cooking. Schricker et al. (1982) and Schricker and Miller (1983) also reported increased levels of nonheme iron in cooked meat, slow heating producing more nonheme iron than fast heating. Tichivangana and Morrissey (1985) studied the activity of ferrous iron as a prooxidant in a model meat system and obtained similar results. Recently, Rhee et al. (1987) studied the prooxidant activity of the total extracted pigments, myoglobin and nonheme iron in raw and cooked beef, pork and chicken 21 muscles. They demonstrated that total pigment and myoglobin concentrations were the most important factors which affected the TBA values of stored, raw muscles among the three species. These investigators concluded that the heme pigment content of meat may be the most important of the three variables affecting the stability of lipids in raw lean meats. Initiation a; Lipig Peroxidation ia Biological Systams Lipid peroxidation in biological systems may be initiated by nonenzymic (Igene et al., 1979; Rhee et al., 1987) and/or enzymic reactions (Lin and Hultin, 1976; Rhee et al., 1984; German and Kinsella, 1985; McDonald and Hultin, 1987). Recently, Asghar et al. (1988) and Kanner et al. (1987) have reviewed the various mechanisms of enzymic and non-enzymic initiation of lipid peroxidation. (1) Noaenaymic Initiators Lipid oxidation in meat was originally considered to be a nonenzymatic reaction (Pearson et al., 1977). Initiation of lipid peroxidation was proposed to take place through the abstraction of a methylene hydrogen from a labile fatty acid, resulting in the formation of alkyl or allyl radicals (Dugan, 1961). Heme pigments (Tappel, 1962a; Liu and Watts, 1970; Rhee et al., 1987) and nonheme iron (Sat 1974; Ige 1935) ha neat lipi these cat in cookec 22 iron (Sato and Hegarty, 1971; Love, 1972; Love and Pearson, 1974; Igene et al., 1979; Tichivangana and Morrissey, 1985) have been recognized as catalysts of oxidation of meat lipids. However, 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 peroxidative process (Asghar et al., 1988). In recent years, much effort has been devoted to identifying the initiators of lipid peroxidation in raw meat and other biological systems. In this review, only those theories receiving the most attention will be addressed. These include those involving the hydroxyl radical, perferryl or ferryl radical, di-iron-oxygen- bridged radical, and the porphyrin cation radical. Hydroxyl radicals in biological tissues are extremely active compounds and possess the ability to abstract hydrogen from unsaturated fatty acids. Several research groups (Fong et al., 1973, 1976; Gutteridge, 1984) studied NADPH-dependent microsomal lipid peroxidation and concluded that hydroxyl radicals (OH') generated in living cells by the superoxide anion-driven, iron-catalyzed Haber-Weiss reaction can initiate lipid peroxidation in membranes. a furthc ion fe 23 o? + Fe3+ ------- > Fe2+ + o 2 2 . + -_ 202 + 2H ----- > H2 02 + 02 Fe2+ + H2 02 ------- > Fe3+ + OH’ + OH— The initiation of lipid peroxidation in membranes by perferryl and ferryl radicals has been extensively studied by Aust and his associates. Svingen et al. (1979) and Tien and Aust (1982) studied lipid peroxidation in liver microsomes involving NADPH-cytochrome P-450 reductase and xanthine oxidase. Cytochrome P-450 reductase catalyzes the transfer of an electron from NADPH to ferric ion to generate ferrous ion in the system. Oxygen in the system can react with ferric ions to produce perferryl and ferryl radicals. Tien et al. (1981) demonstrated that ADP- chelated ferric iron can be indirectly reduced by superoxide anion (product of xanthine oxidase catalysis) to form the perferryl ion. The perferryl ion may undergo further reduction by superoxide anion to give the ferryl ion. ADP-Fe3+ + 02 ----------- > ADP-Fe2+ 02 Aust and Svingen (1982) represented the dioxygen- ferrous comples by the following equivalent structures: or Syste: 24 ADP-Fe2+ o --------- > Abp-Fe3+ o2 Perferryl and ferryl radicals have the ability to abstract hydrogen from polyunsaturated lipids and initiate the peroxidation process (Tien and Aust, 1982). In these systems, chelation of iron by ADP or a similar nucleotide or pyrophosphate was needed to enhance the solubility of iron in solution (Aust and Svingen, 1982). ADP-Fe3+ o; + RH ------ > ADP-Fe3+ 02 H + n- or ADP-Fe3+ 05' + Fez + RH ------ > ADP-Fe2+ (Fe3 n ) + HO' Although most of the studies with the microsomal systems have indicated that iron and oxygen are required to form a nonenzymic initiator, the ratio of ferric and ferrous iron in the system has not been determined. Minotti and Aust (1987) proposed that the actual species responsible for the initiation reaction is the ferrous- dioxygen—ferric complex. The structure of this initiator was first proposed by Aust and Svingen (1982) as indicated previously. This system can be prepared by using NADPH, superoxide anion, ascorbate or thiol compounds to reduce ferric ions, or by utilizing hydrogen peroxide to oxidize ferrous ions. The presence of both ferrous and ferric ions in D. . C a,» a} 25 a 1:1 ratio is required for initiating activity. Therefore, factors such as concentration of iron and the presence of reducing or oxidizing agents and chelating compounds all play important roles in the initiation process. Recently, Kanner and his associates (Kanner and Harel, 1985; Harel and Kanner, 1985a,b) have extensively studied the capacity of hydrogen peroxide and superoxide radical to initiate lipid peroxidation in muscle membranes. Hydrogen peroxide and the superoxide radical are normal metabolites in cells and can react with each other to produce the hydroxyl radical. Hydrogen peroxide can also activate metmyoglobin to an active species which can initiate lipid peroxidation. This initiator is called the porphyrin cation radical (PT- Fe4+ = O ). Initiation of lipid peroxidation by the porphyrin cation radical proceeds via the two electron-reduction of the catalyst (Kanner and Harel, 1985): PT- Fe4+ = o + RH ------- > P - Fe4+ = o + n- + H+ R' + 02 ------- > ROO' n00; + RH ------- > noon + n° P - Fe4+= o + noon ----- > P - Fe3+ + noo- + on— noo- + RH ------- > noon + n- Porphyrin cation radicals possess the capacity to abstract hydrogen from the methylene group of the L1 26 unsaturated fatty acids and produce free radicals. (2) WM Initiation of lipid peroxidation in rat liver microsomes by an enzyme system was first described by Hochstein and Ernster (1963). This system required NADPH, ferric or ferrous iron, oxygen and chelators including pyrophosphate, ADP or ATP. Evidence of enzymic lipid peroxidation in chicken muscle microsomes has been presented by Hultin and his associates (Lin and Hultin, 1976, 1977; McDonald et al., 1979; Apgar and Hultin, 1982; McDonald and Hultin, 1987; Luo and Hultin, 1986). Microsomal fractions prepared from chicken dark or white muscle produced TBA-reactive substances in the presence of NADPH, ADP and ferric ions. NADPH or NADH is the electron donor in the chicken microsomal system. Rhee et al. (1984, 1987) also reported the presence of an enzymic peroxidation system in beef and concluded that the enzyme systems could catalyze lipid peroxidation in raw beef during storage. The reaction required NADPH or NADH, ADP, and iron in either the ferric or ferrous forms. natu: 27 Svingen et al. (1979) and Tien and Aust (1982) reported that cytochrome P-450 reductase and xanthine oxidase also can catalyze microsomal lipid peroxidation through the action of perferryl or ferryl radicals. Detail of this mechanism has already been presented in the section on non-enzymic initiators. Inhibition of Lipid Peroxidation in Meat Systems Lipid peroxidation in meat systems can be inhibited, or at least reduced, by the direct addition of antioxidants to meat products or by the introduction of an antioxidant into the meat through the diet of the animal. Lipid oxidation in meat and meat products can be controlled by a number of compounds including synthetic phenolic antioxidants (Chastain et al., 1982), extracts of natural food products (Pratt, 1972; Rhee et al., 1983) and products of the Maillard reaction (Sato and Hegarty, 1971; Sato and Herring, 1973). (1) Synthetic Phenolic Antioxidants A phenolic antioxidant functions by donating a 28 hydrogen to a lipid radical, thus interrupting the free radical chain mechanism. Phenolic antioxidants include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyll gallate (PG) and tertiarybutyl hydroquinone (TBHQ). Greene (1969, 1971) reported that BHA and PG effectively inhibited lipid oxidation in raw ground beef. Chastain et al. (1982) utilized BHA and TBHQ in the manufacture of restructured beef/pork steaks and reported a decrease in TBA numbers and an increase in sensory scores for flavor and acceptability. Crackel et al. (1988) also reported that TBHQ effectively retarded lipid oxidation in restructured beef steaks. (2) NQEBIQI Antioxidants Compounds possessing antioxidative activity have been found in a variety of natural products such as oats, soybeans, tea leaves, coffee beans, spices, citrus waste, tree barks, hydrolyzed plant and single cell proteins (Dugan, 1980). Aqueous extracts of onions, potato peelings, green peppers (Watts, 1962) and plant extracts (Pratt and Watts, 1964) have been shown to effectively retard oxidation in cooked meat. The antioxidant activity was found to be related to the content of flavonoids, a major group of plant phenols. Soybean protein hydrolyzates, glandless cottonseed, and defatted flours and by (U \f) '1 U) 29 protein concentrates from peanuts and soybeans possess the ability to retard lipid oxidation in cooked ground beef during refrigerated storage. Many spices and herbs have been shown to function as antioxidants in fats/oils amd in model food systems (Bishov et al., 1977). Rosemary oleoresin, for example, contains carnosol, rosmanol, rosmariquinone and rosmaridiphenol. The oleoresin effectively reduced lipid oxidation in turkey breakfast sausage (Barbut et al., 1985), while rosmariquinone and rosmaridiphenol stabilized lard under accelerated test conditions (Houlihan et al., 1984, 1985). (3) Maiiiagg Reaggion Pgoducts Zipser and Watts (1961) reported that retorting of ground beef reduced lipid oxidation during subsequent storage of the beef. The substances responsible for the antioxidative activity result from the heat-catalyzed interaction between amino acids or proteins with carbohydrates. Sato et al. (1973) reported that reductic acid, maltol and products of the amino/sugar reaction were effective inhibitors of the development of warmed-over flavor in cooked ground beef during storage. Reductic acid had been previously shown to retard lipid oxidation in frozen minced red salmon and herring tissues (Tarr and Cooke, 1949). '5’) 'i 4 IH Ifn 30 Recently, Bailey et al. (1987) reviewed the inhibition of warmed-over flavor by Maillard reaction products. They concluded that such products might prove useful in preserving the desirable flavor of cooked meat during refrigerated or frozen storage. WefAM'OX'd tsintsz—tsMea muster! Suppiamantation Animals including mammals and birds require exogenous antioxidants for the stabilization of muscle lipids, and in particular, the cellular and subcellular membrane-bound lipids (Marusich, 1980). As a naturally occurring antioxidant, alpha-tocopherol has been widely supplemented in the diets of animals, particularly meat animals. (1) Suppiementation a: Poultgy Diets with Alpaa- Igggphergi As increasing numbers of precooked/frozen poultry products are marketed for consumer convenience, the stabilization of meat lipid has become increasingly important. Laksesvela (1960) reported that tocopherol (Vitamin E) supplementation significantly reduced undesirable flavors in meat from poultry fed fish oil. When broilers were fed 36.7 mg vitamin E/kg feed, significant improvements in the taste and odor of fresh 31 meat were observed. The quality of broiler meat and its oxidative stability are related to the tissue concentration of vitamin E. Webb et al. (1973) studied the effect of feeding fish meal, with and without supplemental vitamin E (22 mg/kg feed), and concluded that dietary vitamin E reduced the degree of oxidation of the broiler meat. Crawford et al. (1975) reported that 200 mg/kg alpha- tocopheryl acetate were required to afford optimum protection against the development of fishy flavors in turkey when 2% tuna oil was fed. Dam and Granados (1945) observed that body fat from vitamin E-deficient chicks underwent rapid oxidation. Supplementation of the diet with vitamin E prevented hydroperoxide formation i vivo. Prevention of hydroperoxide accumulation in the fat of turkeys and broilers fed tocopherol-supplemented diets was also reported by Kummerow et al. (1948) and Hood et al. (1950). These findings were later confirmed by Mecchi et al. (1953, 1956a,b) who reported that the stability of chicken fat was increased when 100 mg/kg alpha-tocopheryl acetate was added to the diet. Mecchi et al. (1956a,b) also found that feeding vitamin E for at least one and two weeks increased tissue tocopherol levels and significantly improved the stability of broiler meat. Similar data were reported by ant. inc] ins“ H: tul’m 32 Marusich et al. (1975). The positive effect of tissue alpha-tocopherol in retarding the oxidative deterioration of the carcass lipids of broilers has been reported by several investigators (Webb et al., 1972, 1974; Sklan et al., 1983). Feeding lower concentrations (40 mg/kg) of alpha-tocopherol for several weeks (Marusich et al., 1975) or higher levels (220 mg/kg) for one week (Webb et al., 1972) prior to slaughter will produce a tissue (breast muscle) alpha-tocopherol concentration of approxidately 5 mg/kg meat. This concentration of alpha-tocopherol will significantly delay oxidative changes in uncooked broiler meat when held at refrigerator temperatures. (2) Supplemegtation 9f Broiler Diets with Synthatig Aatioxidants Krishnamurthy and Bieri (1962) reported that synthetic antioxidants possess some physiological limitations. These include inefficient absorption from the intestine, insufficient deposition in the meat, and/or extremely rapid turnover in the tissue. Webb et al. (1972) feed broiler diets containing BHT at levels of 0.01, 0.02, or 0.04% and reported that rancidity development in precooked and frozen broiler meat was not significantly reduced. Supplementation of broiler diets with a combination of 0.02% ethoxyquin and 0.02% BHT r4 diph m Stab] 33 significantly reduced TBA numbers in precooked broiler meat. Alpha-Tocopherol Activity and Biochemical Function The biological function of alpha-tocopherol in animals has been a topic of research ever since the discovery of its antioxidant activity (Olcott and Mattill, 1941). Many early investigators recognized that alpha-tocopherol could function as an antioxidant ia yiyg, and this is believed to be its most important function (Olcott and Matill, 1941; Dam and Granados, 1945; Dam, 1957; Lundberg, 1961). Tappel (1962b) proposed the antioxidant mechanism in which alpha- tocopherol protects tissue lipids from free radical attack. This mechanism has been detailed and expanded by Molenaar et al. (1980) and McCay and King (1980). It is now recognized that:(1) alpha-tocopherol is located primarily in the membrane portion of the cell; and (2) alpha- tocopherol is only part of the membrane’s defense against free radicals generated by enzymatic and nonenzymatic reactions in the cells (Machlin, 1984). Packer et al. (1979) proposed that the ability of alpha-tocopherol to penetrate to a precise site within the membrane may be an important feature in its ability to 34 prevent free radical attack. It activity as a free radical scavenger may very well take place within the membrane lipid bilayer as it is able to move very rapidly through the nonpolar portion of the membrane (Tinberg and Barber, 1970; and Molenaar et a1, 1972). In addition, it has been proposed by Molenaar et al. (1972) that alpha-tocopherol is located adjacent to membrane-bound enzymes such as NADPH oxidase. Free radicals generated by this enzyme can be terminated immediately. Superoxide anions are formed in the cytosol of a cell through the oxidation of NADPH (Figure 2) which, in turn, interacts with hydrogen ions to produce hydrogen peroxide (Figure 3) (Machlin, 1984). Hydrogen peroxide is then distributed in both the aqueous and membrane phases of the cell. In the aqueous phase, hydrogen peroxide is destroyed by hydrophilic enzymes such as glutathione peroxidase and catalase (Figure 3). Any hydrogen peroxide remaining in the membrane may react with the superoxide anion to form hydroxy radicals, which can initiate membranal lipid oxidation, unless prevented by the presence of alpha- tocopherol in the membrane (Figure 3). Many of the biochemical effects of alpha-tocopherol may be explained based on the protection of membranes from free radical attack (Machlin, 1984). Although the antioxidant role of alpha-tocopherol has “Exc “LNG “ATM: N l ‘ «Sure 2 35 ENZYME-DEPENDENT SUPEROXIOE ANION puooucno~ 05 1r O neouceo ' 3 ° 2 * 3“ "mp orumrmoue \ H o . o HEXOSE 3 3 3 MONOI’HOSPHATE PATHWAY m oxmuzeo _- 3‘ "ADP" ownrmone "0' t 0" t 02 “2° mnoxiunms GLUTATHIONE GLUTATHIONE FREE RADICAL ATTACK REDUCTASE PEHOXIDASE ON LIPIDS AND OTHER CE L LULAR COMPONENTS Figure 2 Diagrammatic representation of events which appear to be involved in enzyme-catalyzed lipid peroxidation and its control (from Machlin, 1984). a. i.\ Ni, ‘Sure 3 36 STAILE coueouuos am” "new“ ’reacts with TBA ON NALONYL DIALDENYOE 6880 NHAGE TO * HENDRANES GLUTA‘IHIONE in presence at - in absence of Se ..QLPBQTEL'YE _ PEROXIDASE adequate dietary 5e ENZYMES AND Se '\ tissues 03" (bl ‘éi was: ,. name 5’3}? Pnooucme ._ .. .. ... ' neacnous ' GLUTATHIONE adeaaale 5e PEROSXIDASE SUDCIOIICC4 H O dismutose ' 3 3\. OXIDASES ——/ (xanthine oxidase) superoxide ion ? + 0g 92" Figure 3 Interrelationships of alpha-tocopherol (vitamin E) in protecting membranes (from Machlin, 1984) 37 been accepted by almost all investigators, unequivocal evidence has not been presented for the existence of peroxides in tissues from vitamin E-deficient animals. There is no evidence of alpha-tocopherol oxidation products in oxidized tissues. It is now recognized that adequate levels of glutathione peroxidase in such tissues may destroy peroxides as quickly as they are formed. It is also possible that alpha-tocopherol can be regenerated from a tocopherol radical, either by reduced glutathione (Pryor, 1976) or by ascorbic acid (Packer et al., 1979), with the generation of either oxidized glutathione or an ascorbate radical. The interrelationship of alpha-tocopherol in protecting membranes against free radical-mediated membranal lipid peroxidation is presented in Figure 3. Bai REFERENCES Acosta, S. 0., Marion, W. 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L. Scott & Associates, Inc. Itaca, New York. Zipser, M. W. and Watts, B. M. 1961. Oxidative rancidity in cooked mullet. Food Technol. 15:318. CHAPTER 1 Lipid peroxidation in poultry meat. I: Effects of dietary oils and alpha-tocopherol supplementation on broiler growth performance and meat stability 51 52 ABSTRACT Broilers were fed diets containing oils of varying degrees of unsaturation, viz., coconut oil, olive oil, linseed oil, and partially hydrogenated soybean oil (HSBO), with and without alpha-tocopherol-supplementation. The types of oils influenced body weight gain, with olive oil and linseed oil producing the greatest and smallest body and carcass weights, respectively. The different oils significantly (P < 0.01) affected the fatty acid composition of the neutral lipids and, to a lesser extent, the fatty acid composition of the phospholipids. Fatty acid composition, in turn, influenced the oxidative stability of the meat during refrigerated and frozen storage. Meat from broilers fed olive oil or coconut oil was consistently more stable than meat from the linseed oil group. Alpha-tocopherol concentrations in the dark and white meats reflected dietary levels. Dietary supplementation of alpha-tocopherol significantly (P < 0.01) improved the oxidative stability of the dark and white broiler meat during refrigerated and frozen storage as compared to meat from the broilers fed HSBO. 53 INTRODUCTION Oils are widely supplemented in poultry feed in order to meet the high energy demand of the fast growing broilers (Brue and Latshaw, 1985). Although Volker and Amich-Galli (1967) observed no differences in growth rate and feed efficiency of broilers fed different dietary oils, other studies have demonstrated that oils from different sources significantly varied in absorbability and in their influence on growth performance (Sibbald and Kramer, 1980; Mateos et al., 1982; Hulan et al., 1984). It has also been shown that utilization of dietary fats by broilers depends on fatty acid composition (Corino et al., 1980; Brue and Latshaw, 1985), with polyunsaturated fatty acids being more effectively utilized than the more saturated ones. With regard to the influence of dietary oils on carcass composition, most of the attention has been centered on the fatty acid composition of body fat (Marion and Woodroof, 1963; Jen et al., 1971; Deaton et al., 1981). It is generally believed that dietary oils influence mainly the composition of triacylglycerols and, to a lesser extent, the phospholipids in poultry meat (Sklan et al., 1983). However, Hulan et al. (1984) noted only small differences in the fatty acid composition of the neutral lipids of chicks fed poultry grease, beef 54 tallow, and lard. as increase in the degree of unsaturation of carcass fat has also been related to a decrease in oxidative stability of poultry meat ( Marion and Woodroof, 1963; Salmon and O’Neil, 1973; Bartov et al., 1974; Sklan et al., 1983). The poultry carcass is comprised of dark and white meats which differ in certain biochemical and physiological characteristics ( Asghar et al., 1984). The inclusion of alpha-tocopherol in the diet of broilers has long been accepted as a feasible means of delaying the onset of lipid oxidation and extending the shelf-life of meat (Marusich et al., 1975; Bartov et al., 1983; Sklan et al., 1983). The concentration of tocopherol in poultry meat depends on the diet of the birds (Marusich et al., 1975; Sheldon, 1984, Sklan et al., 1983; Lillard, 1987), and on the length of time the broilers are fed tocopherol-supplemented diets (Marusich et al., 1975; Sheldon, 1984). However, more information is needed as to how the neutral lipids and phospholipids in the dark and white meats of broilers respond to dietary oils of varying degrees of unsaturation and to alpha-tocopherol supplementation, and how these ante-mortem management practices influence postmortem oxidative stability of meat during refrigerated and frozen storage. The present study was designed to address these questions. [(1) .iet and ‘ Vithc The b QIOUp Tedchj and fe broneI COMEIC 55 MATERIALS AND METHODS Sgazce g; Qiia Coconut oil (PVO International Company, St. Louis, Missouri), olive oil (Pompeian Co., Baltimore, Maryland), linseed oil (Mason Packaging Co., Holt, Michigan), partialiy hydrogenated soybean oil (Holsum Foods Company, Waukesha, Wisconsin) and alpha-tocopheryl acetate (Hoffman- LaRoche Inc. Nutley, N.Y.) were used in the broiler diets. Broiie; Feeding Regimen One hundred and seventy five, day-old White Mountain broilers (all male) were obtained from Fairview Farms, Inc. Remington, Indiana, and randomly divided into five groups. Each group was raised on a standard broiler feed formula (Table 1), supplemented at a level of 5.5% with one of the dietary oils, namely, coconut oil, olive oil, linseed oil, and partially hydrogenated soybean oil (HSBO), with or without added alpha-tocopheryl acetate (100 mg/kg feed). The broilers were raised under controlled conditions in group confinement pens at the Poultry Science Research and Teaching Center, Michigan State University. Body weight and feed consumption data were recorded weekly. All broilers were slaughtered and dressed according to standard commercial practices at seven weeks of age. The carcasses 56 Table 1 Composition of the experimental all-mash broiler diets used in the feeding study Ingredients (%) Starter Finisher diet diet Ground yellow corn 47.59 53.34 Soybean meal 44 42.75 37.03 Oil supplement 5.50 5.50 Limestone 1.15 1.12 Dicalcium phOSphate 1.81 1.81 Salt 0.50 0.50 DL methionine 0.20 0.20 Vitamin/trace mineral 0.50 0.50 TOTAL 100.00 100.00 Calculated Analysis Crude protein (%) 23.00 21.00 Fat (%) 7.47 7.66 Fiber (%) 4.10 3.89 Calcium (%) 0.93 0.91 Available phosphorus (%) 0.46 0.46 Alpha-tocopherol (mg) 0.57 0.72 Metabolic energy 3038.00 3106.00 (Kcal/kg feed) tn 57 were kept in ice water overnight, then drained of excess water and transferred to a cold room at 4 C for further sampling and processing. Sampia Egapagation The carcasses were cut into seven parts, i.e. leg, breast, wing, back, feet, neck and head and weighed with the skin. The meat was separated by manual skinning, deboned and wrapped in Nasco Whirl-Pak bags (VWI Scientific Co., Bapavia, IL). Dark (leg) and white (breast) meat from each group were separately pooled for the storage study and other analytical procedures. Packets of dark and white meat from each group were randomized and kept at 4°C and - 20°C to study the storage stability of the raw meat. Samples were withdrawn at appropriate time intervals for different analyses. The meat was ground by passing twice through a food grinder with a 7mm plate before analysis (Kitchen Aid Stand Mixer KS-A, Hobart Corp, Troy, OH). Lipig Eztgacgion Lipids were extracted from the ground meat using the dry column method of Marmer and Maxwell (1981). Meat (5g), anhydrous sodium sulfate (209) and Celite 545 (15g) (Fisher Scientific Co., Fairlawn, NJ) were ground together in a ceramic mortar and transferred to a glass column (3.5 x 30 58 cm) with light tamping to obtain a uniform bed. The rmeatltral lipids were eluted with 150 ml dichloromethane, todtnjile the phospholipids (polar fraction) were eluted with 150 ml dichloromethane-methanol (9:1 v/v) , sequentially. Each lipid fraction was collected in a flask immersed 45.1:1 ice to minimize possible oxidation of the lipids during E3)<1:raction. The extract was concentrated to 10 ml in a Buchi Rotovapor R rotary vacuum evaporator (Buchi Inc. , SWitzerland) with a water bath setting of 30°C, and 'QDJiantitatively transferred to a 5 dram vial. The vial and cOntents were flushed with nitrogen and subsequently stored at —2o°c. EQELY Agig Aaalysis The method of Morrison and Smith (1964) was used for the preparation of fatty acid methyl esters for gas chromatographic (GC) analysis. The methylated fatty acids were analyzed using a Hewlett Packard 5840A gas chromatograph (Hewlett Packard, Avondale, PA), equipped with a flame ionization detector and a glass column (3m x 2mm id) packed with 10% SP 2330 on 100/120 Supelcoport (Supelco, Inc., Bellefonte, PA). Nitrogen was used as the carrier gas at a flow rate of 26 ml per min. The oven temperature was programmed from 150°C to 220°C at a rate of 1.5°C per min. The temperatures of the detector and r). 59 injection port were maintained at 300°C and 200°C, respectively. Fatty acid methyl esters were identified by comparison of the retention times with those of known standards (Supelco Inc., Bellefonte, PA). mam—malt a-Toco o intiuscleliseue Quantitation of the alpha-tocopherol levels in dark and white meat of broilers was achieved by a combination of the methods of Itoh et al. (1973) and Slover et al. (1983). Aqueous potassium hydroxide (1N) was used to saponify the neutral lipid fraction and the non-saponifiables were extracted with di-isopropyl ether. The non-saponifiable fraction was derivatized by adding 50 ul bis-trimethylsilyl trifluoroacetamide (BSTFA) containing 1% trimethylchloro- silane (TMCS) and 100 ul silylation-grade pyridine (Pierce Chemical Co. Rockford, IL) to a portion of the extract and leaving for 30 min at room temperature. The trimethylsilyl derivative of alpha-tocopherol was quantitated using a Hewlett Packard 5890A gas chromatograph equipped with a flame ionization detector and a 30m x 0.2mm o.d. methyl silicone fluid capillary column (Hewlett Packard). The chromatograph was temperature programmed from 180°C (held initially for 10 min) to 260°C at a rate of SOC/min, and then held at the final temperature for 30 min. Alpha- tocopherol was identified using the relative retention time Cu L4 of meat 60 of a silylated alpha-tocopherol standard (Henkel Corp., Minneapolis, MN). wmwgw The phospholipid components were separated on silica gel H plates (Sigma Chemical Co.,St. Louis, MO)(0.5 mm thickness) using a solvent system composed of chloroform— methanol-water, 65:25:4 v/v, (Parker and Peterson, 1965). The spots on the plates were visualized by spraying with sulfuric acid/chromic acid (95:5 v/v) and charring at 150°C for 20 min. Quantitation of the spots was achieved using a Shimazu densitometer (Shimadzu Dual-Wavelength Thin Layer Chromato Scanner Model CS-930) by computing the area of each phospholipid component e.g., lyso- phosphatidylcholine (lyso-PC), sphingolipid (SL), phosphatidylcholine (PC), phosphatidylethanolamine (PE). These were identified by using known phospholipid standards (Supelco Inc., Bellefonte, PA). Each component was expressed as a percentage of the total phospholipid content. Thiobarbiguzic Acid TBA Test The distillation method of Tarladgis et al.(1960) was used to quantitate the extent of lipid oxidation in the meat samples. The thiobarbituric acid-reactive substances 61 (TBARS) numbers were calculated by multiplying the mean absorbance by 6.3 (Crackel et al., 1988) and reported as mg malonaldehyde/kg meat. Sighting—1W $8 The analysis of variance of data for dark and white broiler meat was performed using a complete randomized design for the lipid components and a complete randomized block design for TBARS (Steel and Torrie, 1980). The Duncan's multiple range test was used for testing the significance of the difference between mean values (Duncan, 1955). The least square difference (LSD) test was used to determine the significance of differences in the unsaturated and saturated fatty acid content in neutral and phospholipids isolated from dark and white meat. RESULTS AND DISCUSSION Caggass Chagacteristics The total feed consumption, body weight, feed conversion (ratio of feed consumption/body weight gain), and dressing percentage data are presented in Table 2. Supplementation of the diets with different oils affected the body and carcass weights of broilers. When olive oil 62 Table 2 Effect of dietary oils and alpha-tocopherol on the feed consumption, feed conversion, body weight and dressing percentage of broilers Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol Total feed 189.2 189.6 186.7 187.1 186.5 consumption (kg) Total 84.9 89.2 87.0 87.3 85.4 body weight (kg) Feed conversion1 2.23 2.13 2.15 2.15 2.18 ratio Average 2.42 2.55 2.48 2.49 2.44 body weight (kg) Dressing 71.9 69.9 67.3 70.7 70.0 percent Average 1.74a 1.78a 1.67b 1.76a 1.71ab carcass weight (0.31) (0.35) (0.34) (0.37) (0.34) (kg) Leg 618.3a 634.2a 617.2a 625.3a 616.3a weight (g) (55.8) (62.7) (58.0) (58.1) (58.4) Breast 563.6a 590.4a 534.1b 555.4ab 550.3b weight (g) (62.7) (75.3) (78.8) (78.7) (66.2) Abdominal fat 27.3b 10.4C 11.5C 30.7b 41.5a weight (g) (23.2) (13.8) (10.5) (19.2) (17.2) a,b,c Average carcass weight within rows bearing different superscripts differ significantly at P <0.05. 1 Ratio = total feed consumption/total body weight gain The numbers in parentheses represent the standard deviation 63 was fed, broilers tended to have higher body (4.5%) and carcass (4.1%) weights than broilers fed the HSBO diet. The linseed oil-fed group had smaller average carcass weights (2.3%) than the HSBO group. Data in Table 2 also indicate that the carcass weight of the broilers fed olive oil was the highest among the five treatments, whereas the coconut oil-and linseed oil-fed broilers had the lowest body and carcass weights (P < 0.05), respectively. These results, along with the feed conversion data (Table 2), suggest that the olive oil-fed broilers tended to regulate their caloric intake better than those fed coconut oil, linseed oil or HSBO. These results confirm the observations of Brue and Latshaw (1985) who indicated that broilers tended to regulate their caloric intake when different fats/oils were fed. Supplementation of the diet with alpha-tocopherol also appeared to produce broilers having 2.2% more body weight and 3.2% more carcass weight than the broilers in the HSBO group. However, the feed conversion ratios for the control group and the alpha-tocopherol group were not significantly different (P > 0.05). There is a paucity of literature data reflecting the effect of alpha-tocopherol on the growth performance of broilers. However, Popekhina and Tveritnev (1982) observed that alpha-tocopherol supplementation of swine diets 64 produced pigs which were 10.6% heavier than the pigs fed a basal diet. More studies are required to substantiate the observation that alpha-tocopherol influences the growth performance of broilers. Linidmmoensnmilerltefi. The lipid composition data for dark and white meat are presented in Table 3. The total lipid content of the dark muscles did not vary significantly among the five treatments. This is in agreement with the findings of Hulan et al. (1984) who reported that the total carcass lipid content of broilers was not affected by the addition of poultry grease, beef tallow, and lard, singly and in combination with rapeseed oil, to both starter and finishing diets. The total lipid contents of white meat from the olive oil-and linseed oil-fed groups were significantly higher (P < 0.05) than the lipid contents from the HSBO and coconut oil groups. The total lipid content of the alpha-tocopherol-supplemented group was similar to that of the HSBO group. Furthermore, the percentage of neutral lipids or phospholipids isolated from dark and white meats was not affected by alpha-tocopherol supplementation. In contrast, the data in Table 3 do indicate that the relative proportions of phospholipid components i.e., lyso- PC, SL, PC and PE in both dark and white meats were 65 Table 3 Lipid composition of dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol Total1 8.6a 8.2a 7.9a 7.3a 7.5a lipid w 2.2ab 2.5a 2.4ab 1.9d 2.1c Neutral2 D 81.4b 84.7a 86.4a 84.4a 85.4a lipid w 61.0b 67.3a 63.2ab 61.6b 61.4b Phospho-2 D 18.6a 15.3b 13.6b 15.6b 14.6b lipid b b w 39.0a 32.7 36.8a 38.4a 38.6a Lyso-Pc3 D 6.7b 7.7a 7.7a 6.7b 7.4a w 6.1d 7.5b 6.8Cd 7.1bc 8.3a Lyso-PE3 D 8.0a 7.8a 6.1b 6.7b 7.3ab w 7.3a 7.2a 6.0b 7.2a 6.2b Sphingo-3 D 7.0b 8.2a 8.2a 7.0b 7.9ab lipid w 6.9b 8.4a 7.2b 7.1b 8.2a Pc3 0 50.4a 46.1C 48.4b 50.4a 48.2b w 52.0a 47.6C 52.2a 50.3b 50.2b PE3 0 18.9b 20.2a 21.0a 20.1a 19.5b w 19.2b 20.1a 20.5a 19.2b 18.2b a,b,c The data within rows bearing different superscripts 1,2,3 differ significantly at P < 0.05. Expressed as a percent of meat (1), total lipid (2) and total phospholipids (3)“ *r1 'r1 DC, C) l:\? 66 significantly (P < 0.05) influenced by dietary treatments. The dark meat from the alpha-tocopherol-supplemented-and coconut oil-fed broilers had lower lyso-PC contents but higher levels of PC (P < 0.05) than the other groups. The dark meats from the olive oil-fed group were lower in PC content but higher in PE than the coconut oil-and HSBO-fed groups. Different dietary oils, with different degree of unsaturation and energy levels, may influence the enzyme activity ia yiyg and, in turn, affect the synthesis of PC, PE, and other components in the phospholipid fractions. McMurchie and Raison (1979) demonstrated that diets rich in polyunsaturated fatty acids increased the proportion of polyunsaturated fatty acyl chains in mitochondrial phospholipids and decreased the Arrhenius activation energy of membrane-bound enzymes. Faggy Agig Qomposition 9; Neutral Lipiga The fatty acid profiles of the neutral lipids of the dark and white broiler meat are presented in Table 4. Nineteen fatty acids in the neutral lipids from dark and white meat were identified. Generally, C16 fatty acids (palmitic C16:0 and palmitoleic C16:1) and C18 fatty acids (stearic C18:0, oleic C18:1, and linoleic C18:2) were most prevalent and contributed approximately 95% of the total HSBO alpha- tocopherol Linseed HSBO + oil 67 lipids isolated from dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol Olive oil Percent fatty acid composition of neutral Coconut oil Fatty acid Table 4 4“ 1.. 1 1. 1. 1 591..24200.884..37 nw~/.3nw51.330.0.0.nu. 1. 33 A495585—J5226 nU.8404.3nU.30.nw0nw 1. 33 564207861438 083062932000 1. 32 5.1..4..4..7.69484..2nU. 0.84..0.4..3832nU.nU.1. 1. 32 461185999327 nu.4..3nU.54..84.nw0.00w 1.. 21.2 4..34.1.65974..224“ nU.74..048~/.3nwnwnU.AU. 1. 21.2 35nU.1..9~59941..24” 0.85nU.4..961.00.nwnU. 1.. 4.1. 340574796326 0.1.40.3698.1n00.unv 1 4.1 4,o:~1.1130.44;5243 7nu.4unw6nu.32nw001 31.. 370244196431. .00 0 794061410001. 3 53.0 77.5 76.5 77.2 78.2 73.5 72.6 75.7 75.2 45.3 46.0 22.5 23.5 22.8 21.8 26.5 27.4 24.3 24.3 54.7 Fatty acid data underlined are significantly (P < 0.05) frcm other fatty acid data. ‘ I:rillf'lasat. Sat. (‘1‘ Path V 68 fatty acids in the neutral lipids. These results agree with those reported by previous workers (Igene and Pearson, 1979; Jantawat and Dawson, 1980; Sahasrabudhe et al., 1985). As expected, the composition of the dietary oils was reflected in the fatty acid composition of the muscle triacylglycerols (Sklan et al., 1983). For example, the neutral lipids from broilers fed linseed oil contained much higher amounts of linolenic acid (C18:3) than the lipids from the other broiler groups. There were much higher levels of C12:0 (11%) and C14:0 (7%) in the lipids from the coconut oil group, in contrast to the neutral lipids from the other groups where only trace amounts of these short chain fatty acids were observed (Table 4). The neutral lipids from the olive oil group contained a relatively high percentage (50%) of oleic acid, while those from the HSBO group had a higher percent (30.5-33.8%) of linoleic acid. However, alpha-tocopherol supplementation had no effect on the fatty acid composition of neutral lipids. The results agreed with the findings of Salmon (1976) who stated that dietary oils affected the composition of triacylglycerides in poultry. Fatty Acid Composition of Phospholipids The fatty acid data in Table 5 indicate that the ) O H *3 0“ N O O r) H ox OHOQ r) H on U T ”E3 0 N O .0 o. .0 o. O. .0 o. .0 o. .0 0. .0 .9 O. .. O‘MU‘l-‘bLJNO-‘D-LJNOWNHO (I . 69 Table 5 Percent fatty acid composition of phospholipids isolated from the dark (D) and white (W) meat of broilers fed different dietary oils and alpha- tocopherol Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D W D W D W D W D W C12:0 0.9 0.8 - - - - - - - - C14:0 1.9 2.3 - - - - - - - - C16 01415.“:1 3.0 4.9 3.4 2.9 4.0 3.0 3.9 4.1 4.3 4.1 C16:0 15.7 20.6 16.3 19.3 16.3 17.5 16.9 18.0 16.5 17.4 C16:1 0.9 1.5 0.8 1.5 1.1 1.0 0.8 0.8 0.8 0.6 C17:0 0.9 2.0 0.8 1.0 0.9 0.7 1.0 1.0 1.4 1.0 C18 DMAa 0.4 0.9 1.1 1.4 0.5 0.7 0.7 1.1 0.7 1.0 C18:0 14.2 9.7 12.4 9.9 13.0 9.6 12.5 10.1 13.1 11.7 C18:1 15.7 19.3 20.0 20.1 16.3 14.5 15.6 14.9 15.9 14.8 C18:2 17.2 18.7 15.0 14.8 15.2 14.4 20.2 18.2 19.1 18.5 C18:3 4.5 5.0 5.0 5.2 5.1 4.7 5.4 4.8 7.2 7.0 C20:0 - - - - 2.4 4.2 - - - - C20:2 0.7 0.4 0.4 0.7 1.7 1.7 0.4 0.7 0.6 1.3 C20:3 0.7 0.5 0.4 0.8 - - - - - - C20:4 11.7 7.8 12.1 10.1 5.3 6.6 11.7 11.9 12.6 12.4 C22:0 1.9 1.9 1.3 2.0 1.4 2.7 1.0 1.7 1.0 1.0 C22:2 2.2 0.4 1.6 0.5 3.6 5.2 1.4 1.7 0.4 0.7 C22:3 0.4 0.3 0.4 1.2 0.5 0.2 2.1 1.2 0.7 0.8 C22:4 2.9 1.9 4.3 2.9 1.9 1.7 2.5 2.2 1.5 3.0 C22:5w6 1.3 0.8 1.6 1.1 0.6 0.3 0.6 1.8 1.3 0.8 C22:5w3 1.4 1.5 1.7 1.3 5.3 5.0 2.8 2.6 1.6 1.9 C22:6 1.7 0.8 1.6 1.4 5.1 6.4 1.8 2.6 1.7 1.3 Unsat. 61.2 58.8 65.7 64.6 62.2 62.7 64.8 64.7 63.0 63.1 Sat. 35.8 36.3 31.2 32.6 34.9 34.7 31.3 31.2 32.7 32.8 a DMA = Dimethylacetal derivatives of octadecanal derived from acid hydrolysis of plasmalogens. hexadecanal and DMAs are not counted as fatty acids. 7O predominant fatty acids in the phospholipids of dark and white broiler meat were palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and arachidonic acids (C20:4). These fatty acids accounted for approximately 82% of the total fatty acids. The polyunsaturated fatty acids (i.e., fatty acids with two or more double bonds) represented 38 to 49 % of the total fatty acids. The arachidonic acid content in the phospholipids isolated from the broilers fed linseed oil was much lower than the corresponding data from the other groups (Table 5). The phospholipids in all tissue samples contained higher levels of the long-chain polyunsaturated (20 to 22 carbon) fatty acids than did the neutral lipids. This characteristic is typical of phospholipids in muscle tissue and has been previously reported by various workers (Katz et al., 1966; Jantawat and Dawson, 1980). The composition of the dietary oils was again reflected in the fatty acid composition of the tissue phospholipids, although not to the same extent as was observed for the neutral lipids. This observation agrees with the results reported by Sklan et a1. (1983). Meat from the linseed oil-fed broilers contained relatively high amounts of the C22:2, C22:5w6, C22:5w3 and C22:6 fatty acids, while the C12:0 and C14:0 fatty acids were found 71 only in the coconut oil group. Alpha-tocopherol supplementation had no effect on the fatty acid composition of polar lipids in broiler dark and white meat. During these analyses, two small peaks were observed and were subsequently identified using GC-mass spectometry (Crackel, 1986) as being the dimethylacetal (DMA) derivatives of hexadecanal and octadecanal produced by the acid hydrolysis of the plasmalogens present in the phospholipids of muscle tissue (Grigor et al., 1972). The phospholipids from skeletal muscles are particularly rich in ethanolamine plasmalogens and these will undergo acid hydrolysis during the methylation process to produce the DMA derivatives of hexadecanal and octadecanal. Grigor et al. (1972) and Gardner et al. (1972) reported the presence of similar levels (3-4%) of DMA derivatives of hexadecanal and octadecanal in the fatty acid phospholipids isolated from broilers. Since C16 and C18 DMA derivatives possess similar retention times to C16:0 and C18:O fatty acid methyl esters, respectively, DMAs could easily be mistaken for fatty acids. Therefore, it is recommended that the fatty acid composition of muscle phospholipids be determined using a base-catalyzed methylation procedure such as described by Glass (1971) and Maxwell and Marmer (1983). 72 Changes in Muscle Lipids During Storage Significant (P < 0.05) changes occurred in the phospholipid components during storage of meat at different temperatures. The lyso-PC and lyso-PE contents increased with concomitant decreases in the PE and PC contents of the white and dark meat when stored at 4°C for 8 days. The presence of the lyso compounds indicated lypolytic activity in the meat systems and confirmed the earlier observations of Igene et al. (1979) who reported a similar decrease in PE content during frozen storage of chicken dark and white meat. The different storage temperatures influenced the pattern of fatty acid changes. In both the neutral lipid and phospholipid fractions of meat stored at 4°C for 8 days, the proportion of those fatty acids with carbon numbers less than 18, including C18:1, increased and those with carbon numbers higher than 18, i.e., fatty acids with two or more double bonds, decreased (Tables 6 and 7). During long term frozen storage (8 months at -20°C), C18:3 in the neutral lipids slightly decreased, whereas in the phospholipids it exhibited a marked decrease. Long chain polyunsaturated fatty acids such as C20:2, C20:3, C22:2, C22:3, C22:5w3, and C22:5w6 almost disappeared from the phospholipids of dark and white meat during frozen storage (Table 7), and presumably underwent oxidation during 73 Table 6 Percent fatty acid composition of neutral lipids isolated from dark 5D) and white (W) broiler meat stored at -20 C for 8 months Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D W D W D W D W D W C1230 12.3 10.5 - - - - - - - - C14:0 7.0 6.4 0.3 0.4 0.2 0.5 0.5 0.5 0.6 0. C16:0 25.4 25.5 20.6 20.4 18.4 19.5 21.1 21.7 20.3 21. C16:1 6.7 6.2 5.0 4.9 4.5 4.9 4.5 4.9 5.5 4. C18:0 5.5 12.8 5.0 5.5 6.1 6.5 7.2 6.2 9.3 6. C18:1 31.1 27.9 50.2 50.5 31.4 30.5 35.1 35.9 35.7 35. C18:2 11.5 11.5 16.3 16.0 18.3 18.3 28.2 27.5 26.9 30. C18:3 1.2 0.1 1.2 1.4 21.1 20.6 3.2 3.0 3.0 2. C20:4 - - 0.7 0.5 - - 0.2 0.7 - - C22:5 W3 - - 0.6 0.7 - - 0.1 0.2 - - Unsat. 49,8 44.8 74.1 73.7 75.3 73.5 72.2 71.6 69.8 71.7 Sat. 50,2 55.2 25.9 26.3 24.7 26.5 28.8 28.4 30.2 28.3 Percent unsaturated and saturated fatty acids bearing with or without underline differ significantly at P < 0.05. Table 7 74 Percent fatty acid composition of phospholipids isolated from dark (D) and white (W) broiler meat stored at -20°C for 8 months Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D W D W D W D W D W C12:0 1.8 1.3 - - - - - - - - C14:0 3.5 3.4 2.0 1.8 0.5 0.4 0.2 0.3 0.2 0.3 C16DMAa 0.1 0.1 1.5 1.1 0.9 0.3 0.8 0.6 0.7 0.5 C16:0 25.5 27.5 26.0 26.8 29.2 30.1 22.7 23.8 25.4 25.7 C16:1 1.7 3.7 0.4 0.5 1.5 1.5 1.8 1.7 1.3 1.1 C17:0 - - - - - - 0.4 0.3 0.4 0.3 C18:0 19.1 19.2 19.7 18.9 21.7 19.7 18.1 14.7 20.4 16.2 C18:1 24.4 22.3 24.1 24.2 22.6 22.0 22.1 22.0 21.8 22.3 C18:2 16.0 13.5 16.7 16.9 16.9 16.7 21.3 21.5 21.8 22.3 C18:3 - - - - 2.2 3.4 1.4 1.3 1.6 1.4 C20:0 - - - - - - 0.5 0.5 0.4 0.4 C20:2 - - - - - - 0.8 0.6 0.6 0.5 C20:3 - - - - - - 0.5 0.3 0.5 0.1 C20:4 7.9 7.0 9.3 9.7 3.7 4.5 6.6 9.3 5.7 9.0 C22:4 - - 0.3 0.1 0.8 1.4 0.9 1.1 0.3 0.2 C22:5w3 - - - - - - 1.0 1.0 0.9 0.8 C22:6 - - - - 1.9 2.3 0.9 1.0 0.7 0.9 Unsat. 50.0 48.5 50.8 51.4 47.7 49.5 57.3 59.8 53.5 58.5 Sat. 49.9 51.4 47.7 47.5 51.4 50.2 41.9 49.6 45.8 41.0 a DMA = Dimethylacetal derivatives of hexadecanal derived from acid hydrolysis of plasmalogens. counted as a fatty acid. DMA is not 75 storage. Decreases in the long chain polyunsaturated fatty acids may be also due to hydrolytic decomposition, lipid- browning reactions or to lipid-protein co—polymerization as suggested by Igene and Pearson (1979). The results from the present study and those of previous studies with turkey (Jantawat and Dawson, 1980) and broilers (Igene and Pearson, 1979) suggest that the polyunsaturated fatty acids of the phospholipids are quite susceptible to oxidation and may be the site of initiation of lipid oxidation in muscle systems (Asghar et al., 1988). Concentration of Alpha-Tocopherol in Meats The amounts of alpha-tocopherol deposited in the dark and white meat of broilers fed the respective diets are summarized in Figure 1. Feeding different dietary oils varying in degree of unsaturation to broilers appeared to have little effect on alpha-tocopherol deposition in meats. The dark and white meat from broilers fed HSBO contained 1.5 mg and 1.3 mg alpha-tocopherol/kg meat, respectively. Similar levels of alpha-tocopherol were found in dark and white meat from broilers fed coconut oil, olive oil and linseed oil (Figure 1). The small amounts of alpha- tocopherol found in the dark and white meat probably originated from the feed which contained the minimum requirement of alpha-tocopherol for growth of the broilers 76 w”."wwwwwwwwwwwummuwwwwwww.uwmm”n»».»wumuwwwnmmuwwwwwwwv”.3? C.............. . ... ....99.9...... .... .9.%§§§% 4 HSBO + Alpha-Tocopherol 1 l ...a. w .1. .1 e M nu.unv ”M t 01d .m u e .m mm nee 0 v.5 no nu mum ... E... H a D 1.923 R. - d ‘ d I J G I 1 d 4 I - d I 0 oo 6 4 2 0 l .82 $580588.-.“an we Ikamy'htmnwnm the dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol Figure 1 Alpha-tocopherol concentrations (mg/kg meat) in V 77 (Table 1). These values are similar to those reported by Marusich et a1. (1975) for breast muscle (1.5-1.6 mg/kg meat) of broilers fed only a basal diet. The concentrations of alpha-tocopherol in the dark and white meat from broilers fed the alpha-tocopherol supplement were 9.9 mg and 5.6 mg/kg meat, respectively. These values were approximately nine and five times higher than corresponding levels in the dark and white meat from broilers fed HSBO. The overall deposition of alpha- tocopherol in the muscle tissues was about 0.88% of the total alpha-tocopherol consumed by the broilers, of which the dark and white meat accounted for 0.56% and 0.32%, respectively (Figure 1). These values indicate that dark meat accumulated approximately 50% more alpha-tocopherol than did the white meat. The different rate of deposition apparently results from physiological variations in the vascular network of dark and white muscle tissues (Sheldon, 1984). Dark meat from broilers have a more highly developed vascular system and a higher lipid content than white meat. Therefore, alpha-tocopherol is deposited to a greater degree in dark meat than in white meat. Values obtained in this study generally agree with those reported by Marusich et al. (1975) and Sheldon (1984). Marusich et al. (1975) noted that when broilers were fed alpha-tocopherol continuously for 8 weeks at [(3 011 PCStz 78 levels of 20, 30, 40, and 60 mg/kg feed, the tissue alpha- tocopherol concentrations increased to 2.7 mg, 3.9 mg, 5.0 mg, and 6.2 mg/kg breast meat, respectively. When broilers were fed a diet containing 160 mg alpha-tocopherol/kg feed for only the last 5 days before slaughter, the breast meat had an alpha-tocopherol concentration of 5.3 mg/kg meat. Sheldon (1984) reported alpha-tocopherol concentrations of 2.9 mg and 0.7 mg/kg meat in thigh and breast meat, respectively, when turkeys were fed 55 mg alpha-tocopherol/ kg feed for two weeks before slaughter. With a higher supplement (275 mg/kg feed), the alpha-tocopherol concentrations in the thigh and breast tissues increased to 7.0 mg and 2.7 mg/kg, respectively. Oxidative Stability gt Qgtt gag White Mgat The oxidative stability of dark and white meat from the various groups of broilers was evaluated using the TBA test. The data in Tables 8 and 9 indicate that not only the dietary oils (P < 0.01) but also the storage conditions significantly (P < 0.05) affected the oxidative stability of dark and white meat tissues. Both dark and white meat from the broilers fed linseed oil had higher TBARS numbers (P < 0.05) than the corresponding samples from the HSBO group, even at 48 hr postmortem. These high values are reflective of the higher 79 Table 8 Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat) of dark (D) and white (W) meat stored at 4°C from broilers fed different dietary oils and alpha-tocopherol Days of Coconut Olive Linseed HSBO + HSBO storage oil oil oil alpha- tocopherol Mean 2 0 0.19 0.15 1.40 0.08 0.19 0.40Y 3 0 0.68 0.53 2.28 0.27 0.49 0.85XY 4 0 1.14 0.68 3.98 0.3 1.40 1.50x 6 0 1.47 1.29 4.70 0.49 1.56 1.90x Mean 0.87b 0.66b 3.09a 0.29C 0.91b Mean 2 w 0.21 0.12 0.64 0.09 0.22 0.262 3 w 0.40 0.24 1.64 0.12 0.47 0.57y 4 w 0.90 0.46 2.28 0.15 0.94 0.95xy 6 w 1.43 1.03 2.74 0.24 1.51 1.39X Mean 0.74b 0.46C 1.83a 0.15d 0.79b a,b,c,d TBARS numbers within rows bearing different superscripts differ significantly at P < 0.001. x,y,z TBARS numbers within columns bearing different superscripts differ significantly at (P < 0.05) in dark meat and at (P < 0.01) in white meat. MI. I T“ ‘4 st: 80 Table 9 Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat of dark (D) and white (W) meat stored at —20°C from broilers fed different dietary oils and alpha-tocopherol Time of Coconut Olive Linseed HSBO + HSBO storage oil oil oil alpha— tocopherol Mean 2 days 0 0.19 0.15 1.40 0.08 0.19 0.402 2 months D 0.84 0.78 3.31 0.42 1.13 1.30Y 6 months 0 1.38 1.24 4.36 0.78 1.43 1.84x Mean 0.80b 0.72b 3.02a 0.43c 0.92b Mean 2 days w 0.21 0.12 0.64 0.09 0.22 0.262 2 months w 0.66 0.56 1.73 0.36 0.71 0.79y 6 months w 0.81 0.71 2.89 0.51 1.11 1.22x Mean 0.56bc 0.48C 1.75a 0.35d 0.68b a,b,c,d TBARS numbers within rows bearing different superscripts differ significantly at P < 0.001. x,y,z TBARS numbers within columns bearing different superscripts differ significantly at (P < 0.001) in dark meat and at (P < 0.05) in white meat. 81 polyunaturated fatty acid concentrations in both the neutral lipids and the phospholipids from the broilers fed the linseed oil diet. It is well established that malonaldehyde arises from the oxidative degradation of fatty acids with three or more double bonds (Dahle, 1964). As expected, the higher content of polyunsaturated fatty acids in the dark and white meat from the linseed oil-fed broilers produced high TBARS numbers. 0n the other hand, due to their smaller polyunsaturated fatty acid contents (Tables 4 and 5), the dark and white meat samples from the coconut oil-fed group were relatively more stable toward oxidation during refrigerated and frozen storage than was the meat from the HSBO group. White meat from the olive oil-fed group was consistently more stable than meat from the HSBO group. This again can be partly explained by the fact that oleic acid in the neutral lipids and phospholipids comprised approximately 50% (Table 4) and 20% (Table 5) of the total fatty acids present, respectively. Since malonaldehyde is derived principally from fatty acids with three or more double bonds, the high content of C18:1 in the olive oil-fed broilers would not be particularly susceptible to oxidation. It was also found that dark meat gave consistently higher TBARS numbers than white meat from broilers fed olive oil, linseed oil and HSBO (Tables 8 and 9). These *4» r .l. 82 results agree with those of Sklan et al. (1983) and Pikul et al. (1984) who demonstrated that the oxidative changes were much more extensive in dark meat than in white meat, possibly due to the higher phospholipid concentrations. In contrast, the white meat from broilers fed coconut oil had slightly higher TBARS numbers than the dark meat from the same group during both refrigeratured and frozen storage (Tables 8 and 9). Results in Tables 8 and 9 also demonstrated a positive relationship between tissue alpha-tocopherol concentration and the stability of meat toward lipid oxidation. Meat from the broilers fed the alpha-tocopherol-supplemented feed (100 mg/kg feed) deposited the highest tissue alpha- tocopherol and was more stable toward oxidation (P < 0.05) than meat from the other four dietary treatments. Similar relationships have been recently observed by a number of investigators. Sklan et al. (1983) reported that dietary tocopherol levels of (60 mg/kg feed) resulted in increased tissue tocopherol concentrations and decreased oxidation of meat tissue during storage. Bartov et al. (1983) also pointed out that tissue tocopherol levels significantly influenced the oxidative stability of poultry meat. Sheldon (1984) stated that tissue tocopherol levels and TBA numbers were significantly influenced by dietary tocopherol ‘Concentrations. The latter investigator also concluded 83 that tocopherol-supplemented poultry diets are effective in reducing lipid oxidation of meat during storage. SUMMARY AND CONCLUSIONS The results from this study confirm previous findings that alpha-tocopherol concentrations in dark and white meat of broilers reflect dietary levels. The fatty acid composition of muscle lipids were significaly affected by the dietary oils. Both dietary oils and alpha-tocopherol supplementations significantly influenced the oxidative stability of dark and white meat during refrigerated and frozen storage as determined by TBARS numbers. However, alpha-tocopherol appears to be a more cost-effective way of increasing lipid stability in poultry meat than feeding the more expensive coconut oil and olive oil. REFERENCES Asghar, A., Morita, J., Samejima, K. and Yasui, T. 1984. Biochemical and functional characteristics of myosin from red and white muscles of chicken as influenced by nutritional stress. Agric. Biol. Chem. 48:2217. Asghar, A., Gray, J. I., Pearson, A. M., Booren, A. M. and Buckley, D. J. 1988. Perspectives in warmed-over flavor. Food Technol. (in press). Bartov, I., Bornstein, S. and Lipstein, B. 1974. Effect of calorie to protein ratio on the degree of fatness in broilers fed on practical diets. Br. Poultry Sci. 15:107. Bartov, I,. Basker, D., and Angel, S. 1983. Effect of dietary vitamin E on the stability and sensory quality of turkey meat. Poultry Sci. 62:1224. Brue, R. N. and Latshaw, J. D. 1985. Energy utilization by the broiler chicken as affected by various fats and fat levels. Poultry Sci. 64:2119. Corino, C., Dell’orto, V. and Pedron, O. 1980. Effect of the acid composition of fats and oils on the nutritive efficiency of broiler feeds. Rev. Zootec. Vet. 2:94. Crackel, R. L. 1986. Stability of lipids in restructured beef steaks. M.S.Thesis, Michigan State University. East Lansing, MI Crackel, R. L., Gray, J. I., Booren, A. M., Pearson, A. M. and Buckley, D. J. 1988. Effect of antioxidants on lipid stability in restructured beef steaks. J. Food Sci. 53:656. Dahle, L. R., Hill, E. G. and Holman, R. T. 1962. The thiobarbituric acid reaction and the autoxidation of polyunsaturated fatty acid methyl esters. Arch. Biochem. Biophys 98:253. Deaton, J. W., McNaughton, J. L., Reece, F. N. and Lott, B. D. 1981. Abdominal fat of broilers as influenced by dietary level of animal fat. Poultry Sci. 60:1250. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics. 11:1. 84 85 Gardner, H. R., Huber, C. S., Bourland, C. T. and Smith, Jr. M. C. 1972. Identification and quantitation of hexadecanal and octadecanal in broiler muscle phospholipids. Poultry Sci. 51:1056. Glass, L. 1971. Alcoholysis, saponification and the preparation of fatty acid methyl esters. Lipids 6:919. Grigor, M. R., Moehl, A. and Snyder, F. 1972. Occurrence of ethanolamine and choline-containing plasmalogens in adipose tissue. Lipids 7:766. Hulan, H. W., Proudfoot, F. G. and Nash, D. M. 1984. The effects of different dietary fat sources on general performance and carcass fatty acid composition of broiler chickens. Poultry Sci. 63:324. :3[::<§;ywene, J. 0. and Pearson, A. M. 1979. Role of phospholipids and triglycerides in warmed-over flavor development in meat model systems. J. Food Sci. 44:1285. Igene, J. 0., King, J. A., Pearson, A. M. and Gray, J. I. 1979. Influence of heme pigments, nitrite and non-heme iron on development of warmed-over flavor (WOF) in cooked meat. J. Agric. Food Chem. 27:838. :JIZL-it:;oh, T., Tamura, T. and Matsumoto, T. 1973. Methylsterol composition of 19 vegetable oils. J. Am. Oil Chem. Soc. 50:300. ‘:Jr‘4E=Lntawat, P. and Dawson, L. E. 1980. Composition of lipids from mechanically deboned poultry meat and their composite tissues. Poultry Sci. 59:1043. <3r’crrison, W. R. and Smith, L. M. 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron-trifluoride methanol. J. Lipid Res. 5:600. .];.iEigrker, F. and Peterson N. F. 1965. Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J. Lipid Res. 6:455. IP'J'LIkul, J., Leszczynski, D. E. and Kummerow, F. A. 1984. Relative role of phospholipids, triacylglycerols, and cholesterol esters on malonaldehyde formation in fat extracted from chicken meat. J. Food Sci. 49:704. Popekhina, P. and Tveritnev, M. 1982. Premixes for replacement hogs. Svinovodstov (Moscow) 1982:(1) 34. In "Vitamin E Abstracts 1982" pp 124. Horwitt, M. K. (ed). Distributed by Henkel Corporation. Minneapolis, MN. 87 Sahasrabudhe, M. R., Delorme, N. F. and Wood, D. F. 1985. Neutral and polar lipids in chicken parts and their fatty acid composition. Poultry Sci. 64:910. Salmon, R. E., and O’Neil, J. B. 1973. The effect of dietary fat and storage temperature on the storage stability of turkey meat. Poultry Sci. 52: 314. :EE=hialllmon, R. E. 1976. The effect of age and sex on the rate of change of fatty acid composition of turkeys following a change in fat source. Poultry Sci. 55:201. 8 heldon, B. W. 1984. Effect of dietary tocopherol on the oxidative stability of turkey meat. Poultry Sci. 63:673. S ibbald, I. R. and Kramer, J. K. G. 1980. The effect of the basal diet on the utilization of fat as a source of true metabolizable energy, lipid, and fatty acids. Poultry Sci. 59:316. =555:3I-I::Zlan, D., Tenne, Z. and Budowski, P. 1983. Simultaneous lipolytic and oxidative changes in turkey meat stored at different temperatures. J. Sci. Food Agric. 34:93. 8 lover, H. T., Thompson, R. H. and Merola, G. V. 1983. Determination of tocopherols and sterols by capillary gas chromatography. J. Am. Oil Chem. Soc. 60:1524. S“";eel, R. G. D. and Torrie, J. H. 1980. "Principles and Procedures of Statistics. A Biological Approach." 2nd ed. McGaw-Hill Book Co., New York, NY flz'iiagrladgis, B. G., Watts, B. M., Younathan, M. T. and Dugan, Jr. L. R. 1960. A distillation method for the quantitative determination of malonaldehyde in rancid foods. J. Am. Oil Chem. Soc. 37:44. ‘9'<=>lker, L., and Amich-Galli, J. 1967. The comparative values for metabolizable energy and net energy for production resulting from addition of tallow and other fats to broiler rations. N.R.A. European Office, Rome, Italy. CHAPTER I I Lipid peroxidation in poultry meat. II. The influence <31 dietary oils and alpha-tocopherol supplementation on membranal lipid stability. 88 89 ABSTRACT The effects of different dietary oils and alpha- ‘t: ocopherol on the fatty acid composition of mitochondrial and microsomal lipids of broiler muscle, and their lability t c NADPH-induced peroxidation was investigated. Fatty acid ‘3 onposition of both neutral lipids and phospholipids of :11 itochondria and microsomes was influenced by dietary oil 2 anosition. Supplementation of the diet with alpha- : Qcopherol only appeared to increase the alpha-tocopherol : thentration in the microsomal membranes in the dark meat. rhe incorporation of alpha-tocopherol into the membranes by a 5— etary means also retarded the peroxidative changes. The rate of NADPH-induced lipid peroxidation in microsomes and mitochondria was dependent primarily on the faftty acid composition of the membrane lipids, and, to a leser extent, on the alpha-tocopherol content. Results of th :is study lend support to the hypothesis that lipid E>eJroxidation in uncooked meat is initiated in the membrane- l::’<=D11nd lipids and that the rate of oxidation is influenced I>1’~":‘Lmarily by the fatty acid composition of the membrane 1 ipids. 90 INTRODUCTION Lipid oxidation is a major deteriorative reaction occurring in muscle foods during storage (Pearson et al., 1 9 83) . It was initially believed that adipose tissue was the major focus for the occurrence of lipid oxidation in meat (Watts, 1954). However, later studies indicated that membrane-bound phospholipids are the sites where oxidative changes are initiated in meat and meat products (Gokalp et a l -, 1983; Willemont et al., 1985; Gray and Pearson, 1987) . These changes can be initiated by nonenzymic (Igene et al. , l S '79; Rhee et al., 1987) and/or enzymic reactions (Lin and H12. ltin, 1976; Rhee et al., 1984; German and Kinsella, 1985; MC Donald and Hultin, 1987) . The various mechanisms of eh zymic and non-enzymic initiation of lipid peroxidation have recently been reviewed (Kanner et al. , 1987; Asghar et a1 ., 1988). Biological membranes contain phospholipids which are I:‘Iiech in polyunsaturated fatty acids. They are very sKlsceptible to peroxidation (Buege and Aust, 1978) as the subcellular membranes (mitochondrial and microsomal) are bathed in a fluid containing prooxidants such as oxygen, tll‘ansition metals and peroxidase enzyme (Vladimirov et al. , 1980) . In addition, hydrogen peroxide and the superoxide anion radicals are normally produced in muscle cells (Harel influ£ 91 and Kanner, 1985a,b) . Low concentrations of hydrogen peroxide can activate metmyoglobin to form a porphyrin cation radical which is believed to initiate membranal :l. jfpid peroxidation (Kanner and Harel, 1985) . There is some evidence that the fatty acid composition 6 f the mitochondrial and microsomal lipids in rats can be a ltered by dietary oils (Tahin et al., 1981; Innis and C: l andinin, 1981a,b) . Other reports have indicated little 3 II: no change in the proportion of saturated to unsaturated E atty acids in the phospholipids as a function of dietary 3’ i Js (Gibson et al., 1984; McMurchie et al., 1986). Most a. 33 studies have centered on the membrane-bound lipids of Q bgans such as liver, heart and kidney of rats, and little in :formation is available pertaining to the influence of a i etary oil on the composition of subcellular membranes from skeletal muscles of meat animals. It is well established that alpha-tocopherol s"~lIJplementation of poultry diets results in an increased a~3~1>ha-tocopherol level in the tissue and a concomitant il'1<:rease in the stability of the meat (Marusich et al. , 19'75; Sklan et al., 1983; Sheldon, 1984). However, in formation is lacking as to how the oxidative stability of meInbranal lipids in the dark and white meat of broilers is influencedl by dietary alpha-tocopherol supplementation and 11°" the stability of these membranes influences the overall 92 stability of the meat itself. The major objectives of this study were: ( 1) to determine the effect of dietary oils varying in degree of unsaturation and alpha-tocopherol supplementation on the fatty acid composition of membranal lipids of broiler muscles; and (2) to determine the oxidative s tability of the membranal lipids of broiler dark and white meat and its relationship to the stability of broiler meat an :ring storage . MATERIALS AND METHODS M Feeding Regimens The source of broilers and the dietary treatments were QQscribed in Chapter 1. W0 o_f Sarcoplasm from Meat a_n__d Isolation o_f Wa _114 __J.____M crosomes One hundred grams of meat (dark or white) were a:Lspersed in 600 m1 of chilled sarcoplasmic protein- QXtracting buffer (0.1 M KCl in 10 mM Tris-HCl, pH 7.8-7.9 for white meat: pH 7.4-7.5 for dark meat, and 0.1 mM glycerol), and homogenized in a Waring blender (Dynamic Co. of America, New Hartford, Conn.) for 1.5 min at top speed. The resulting slurry was centrifuged at 600 x G (Sorvall RC ..K hf 93 2 —B, Ivan Sorvall Inc. Norwack, Conn.) for 10 min to precipitate the myofibrillar and connective tissue fractions. The supernatant was collected, and the pellet was redispersed in 400 ml of the above-mentioned buffer, blended for 1 min, and then centrifuged at 600 x G for 10 min. The sarcoplasmic extracts were pooled together a hd subjected to the differential centrifugation procedure c f Schenkman and Cinti (1978) to separate the mitochondrial a hd microsomal fractions. The sarcoplasmic extracts were c emtrifuged at 10,300 x G (8,000 rpm) for 15 min to The supernatant p recipitate the mitochondrial fraction. was saved and calcium chloride was added to a final Q ancentration of 8 mM. After thoroughly mixing, the m inture was centrifuged at 13,200 x G (9,000 rpm) for 30 The subcellular In. in to separate the microsomal fraction. f ractions were not further purified to remove contaminating n‘Efofibrillar proteins. W W Lipids were extracted from the membranes utilizing the dry column method of Marmer and Maxwell (1981) , as described previously (Chapter 1) . my Acid Analysis The method of Morrison and Smith (1964) was used for 94 the preparation of fatty acid methyl esters of the membrane lipids. Quantitation of Alpha-Iogopherol in Membranes Quantitation of alpha-tocopherol in the mitochondria and microsomes was achieved through a combination of the methods of Itoh et al. (1973) and Slover et al. (1983). Detail of the procedures are described in Chapter 1. Measurement of Subcellular Membrane Protein The protein content in the mitochondria and microsomes was measured by the Biuret method of Gornall (1949 ), as modified by Asghar and Yeates (1974). Subcellular material (0.2 g) was sonicated in 20 ml buffer solution (0.1 M KCl in 0.05 mM tris-HCl buffer, pH 7.2) for 25 min. One ml of the dispersion was transferred to a test tube to which was added 4 ml Biuret reagent (1.5 g copper sulfate, 0.112 g sodium potassium tartrate and 40 g NaOH in 1000 ml distilled water). The color was allowed to develop for 30 min. Thereafter, dichloromethane (2 ml) was added, and after thoroughly mixing for 30 sec, samples were centrifuged at 2000 x G for 10 min. The absorbance of the upper aqueous layer was determined at 540 nm using a Spectronic 2000 spectrophotometer (Bausch and Lomb Co., Rochester, NY). The protein concentration was computed 95 from a standard curve established using bovine serum albumin. Wefw-MMWEW ans Thiobarbituric acid-reactive substances (TBARS) were determined by a modification of the procedure described by Buege and Aust (1978). The final mixtures for the peroxidation assay contained microsomal or mitochondrial membranes (0.62 mg protein/ml in 0.05 M tris-HCl buffer, pH 7.2), 5 mM adenosine diphosphate (ADP) and 0.1 mM ferric chloride. NADPH (reduced nicotinamide adenine dinucleotide phosphate) was added at a level of 0.1 mM (final concentration) to initiate the peroxidation reaction. The reaction was carried out in triplicate for 180 min in a 100 m1 Erlenmeyer flask incubated at 37°C in a shaking water bath. At appropriate time intervals (5, 10 15, 20, 30, 60, 90, 120, 150 and 180 min), 2 ml aliquots of the reaction mixture were removed from the Erlenmeyer flask and added to a test tube containing 2 ml of the trichloroacetic acid (TCA)/thiobarbituric acid (TBA) stock solution and mixed thoroughly with a Vortex mixer. A freshly prepared TCA/TBA stock solution consisting of 10% w/v TCA, 0.4% TBA and 0.25N HCl was used. The test tube and contents were heated in a boiling water bath for 15 min and then cooled. The tubes were centrifuged in an IEC clinical centrifuge 96 (Damon/IEC Division. Needham Hts, Mass) at 1000 x G (2,000 rpm) for 10 min to precipitate the proteins. The absorbance of the supernatant was measured at 532 nm and the results reported as nmole malonaldehyde per mg protein -1 using a molar extinction coeficient of 1.56 x 105 M'1 cm (Buege and Aust, 1978). Statisnigal Analyaia 9; Experimental 2424 Statistical analyses were performed using the SAS SERIES package developed by the SAS Institute Inc., Cary, North Carolina (1987). The experimental design for the study of alpha-tocopherol content of the subcellular membranes was a completely randomized block design (Steel and Torrie, 1980). Analyses of variance (ANOVA) of the TBARS data were conducted using a split plot with 5 x 2 x 2 factorials as the whole plot. Duncan’s multiple range test was used to estimate significant difference between means at the 5% significance level (Duncan, 1955). The least square difference (LSD) test was used to determine the significant difference of unsaturated fatty acid content in neutral and phoapholipids. 97 Table 1 Lipid composition of the subcellular membranes isolated from the dark (D) and white (W) meat of broilers fed different dietary oils and alpha- tocopherol Treatments Total1 Neutral2 Phospholipids2 lipid lipid lipid % % % D Mitochondria 6.1 45.7 54.3 Coconut oil D Microsomes 5.6 44.6 55.4 W Mitochondria 5.3 48.7 51.3 W Microsomes 2.9 30.7 69.3 D Mitochondria 6.9 31.0 69.1 Olive oil D Microsomes 4.7 37.7 62.3 W Mitochondria 5.1 37.6 62.4 W Microsomes 3.6 37.5 62.5 D Mitochondria 6.2 33.4 66.6 Linseed oil D Microsomes 2.8 37.9 62.1 W Mitochondria 5.5 36.5 63.5 W Microsomes 2.7 26.6 73.4 D Mitochondria 4.6 36.2 63.8 HSBo3+ alpha- D Microsomes 4.3 37.1 62.9 tocopherol W Mitochondria 4.1 31.4 68.5 W Microsomes 2.8 30.2 69.8 D Mitochondria 6.9 35.1 64.9 H3503 D Microsomes 5.3 35.5 64.5 W Mitochondria 4.9 30.2 69.8 W Microsomes 2.8 32.0 68.0 1 Expressed as a percent of the subcellular membrane on a wet weight basis 2 Expressed as a percent of total lipid 3 Partially hydrogenated soybean oil 98 RESULTS AND DISCUSSIONS Magnum—a1 £4214 ___R_§1_L_BC°m 0 't'0 Total lipid, neutral lipid and phospholipid contents of mitochondria and microsomes isolated from dark and white meat of broilers fed different dietary fats and alpha- tocopherol are summarized in Table 1. In general, the total lipid content of the mitochondrial fraction from both the dark and white meats was higher than that of the microsomal fraction. The relative proportions of the neutral lipids and phospholipids in the mitochondrial and microsomal membranes did not change to any measurable extent in response to dietary oil, except in the case of the coconut oil-fed group. In general, the mitochondria from the skeletal muscles contained 30-49% neutral lipid and 51-70% phospholipid, whereas the corresponding concentrations in the microsomes were 27-45% and 55-73%, respectively. These values are somewhat different from those cited for the mitochondria and microsomes from rats and rabbits. Fiehn and Peter (1971) reported that phospholipids represented 82.2% of the mitochondrial lipids of rats, while the phospholipid content of the microsomal lipids was 78.0%. Other studies have indicated that the mitochondrial lipids from other mammalian species such as rabbits contain as much as 88.8% phospholipid (Waku and Uda, 1971). Thus, there is 99 considerable variation in the phospholipid contents of muscles among species as described by some early workers (Kaucher et al., 1944; Bloor, 1943; Acosta et al., 1966). They concluded that the more active muscles contain a greater quantity of phospholipids. Fatty Acid Com ' 'on The fatty acid data for the neutral lipids and phospholipids of the mitochondria and microsomes are presented in Tables 2-5. The nature of the dietary oils is reflected in the fatty acid composition of the neutral lipids of the membranes and, to a lesser degree, in the fatty acid composition of the phospholipids. Special notice should be directed to the high contents of the diene (C22:2), pentaene (C22:5w3) and hexaene (C22:6) fatty acids, and to the relatively low amounts of C20:4 (arachidonic acid), C22:4 and C22:5w6 in the phospholipids of mitochondria and microsomes isolated from the dark and white meat of broilers fed linseed oil. Another noticeable feature is the higher content of the short chain saturated fatty acids (C12:0 and C14:0) in the mitochondrial and microsomal lipids of broilers fed coconut oil. The oleic acid (C18:1) content was higher and the linoleic acid (C18:2) content lower in the mitochondrial and microsomal lipids isolated from the dark 100 Table 2 Percent fatty acid composition of the neutral lipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D W D W D W D W D W C12:0 2.5 1.0 - - - - - - - - C14:O 2.5 1.8 0.1 0.1 0.1 0.2 0.2 0.1 0.3 0.1 C16 DMAa 4.7 5.2 3.5 5.0 2.6 6.2 3.0 4.6 4.2 5.0 C16:0 13.7 15.8 15.9 14.8 14.9 13.3 14.0 14.9 13.2 15.8 C16:1 2.6 1.4 1.3 0.7 1.9 0.7 1.3 1.0 1.1 0.9 C17:O 1.4 1.2 1.0 1.3 0.6 2.6 1.7 1.5 1.8 1.7 C18 DMAa 1.5 1.3 0.9 2.2 2.0 1.9, 1.0 1.4 1.6 1.6 C18:0 11.6 11.6 9.1 10.3 9.5 9.2 12.0 10.7 11.4 11.7 C18:1 18.7 18.0 21.6 22.1 18.8 12.9 18.8 18.4 18.0 17.0 C18:2 24.3 24.1 25.9 22.2 19.7 18.2 28.2 26.5 28.2 27.2 C18:3 3.5 3.0 3.3 3.8 15.0 15.7 3.1 3.3 4.0 2.9 C20:2 0.6 0.6 0.9 0.8 0.4 0.5 1.7 1.1 1.1 0.9 C20:3 0.9 0.6 0.8 0.7 - - - - - - C20:4 4.4 5.0 4.8 5.4 3.0 3.6 5.1 5.2 5.6 5.0 C22:0 1.4 2.3 2.0 2.1 1.1 2.4 1.1 1.1 1.2 1.5 C22:2 0.3 1.0 0.2 0.5 1.0 1.9 0.2 0.5 0.3 0.5 C22:3 0.3 0.6 0.2 0.5 1.1 1.2 0.4 2.1 0.5 0.9 C22:4 2.3 2.3 2.4 3.0 1.5 1.6 2.7 3.2 2.8 3.0 C22:5w6 0.9 0.9 0.9 1.3 1.3 1.3 1.9 1.3 1.1 1.1 C22:5w3 0.9 1.2 1.2 1.8 2.5 3.0 2.0 1.5 2.1 1.7 C22:6 1.0 1.1 1.0 1.4 3.0 3.6 1.6 1.5 1.5 1.6 Unsat. 60.7 59.8 64.5 64.2 69.2 64.0 67.0 65.7 66.3 62.5 Sat. 33,; 33.7 28.1 28.6 26.2 27.7 29.0 28.3 27.9 30.9 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acids. Fatty acid data underlined are significantly (P < 0.05) from other fatty acid data. 101 Table 3 Percent fatty acid composition of phospholipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D W D W D W D W D W C12:0 0.2 2.0 - - - - - - - - C14:0 1.9 2.7 0.1 0.4 0.1 0.5 0.2 0.1 0.1 0.2 C16 DMAa 3.5 4.9 4.7 4.2 5.3 4.8 4.0 5.1 4.4 4.4 C16:0 19.0 21.9 18.9 23.8 18.8 27.3 20.0 25.4 24.7 24.3 C16:1 1.4 1.4 0.4 0.9 0.5 0.5 1.1 0.4 0.5 1.0 C17:0 1.2 0.7 1.0 0.8 1.1 0.9 0.6 1.0 1.0 0.7 C18 DMAa 0.3 0.5 1.0 1.0 0.5 0.6 0.4 0.8 0.7 0.7 C18:0 15.6 11.0 13.1 10.9 16.2 11.4 15.5 11.9 12.3 11.9 C18:1 18.9 18.1 21.8 23.6 14.9 18.3 15.5 17.4 15.9 17.1 C18:2 17.0 19.5 15.5 14.7 19.1 17.7 20.7 16.4 19.4 17.2 C18:3 3.7 2.5 4.4 4.0 4.4 2.8 2.7 3.5 2.1 3.6 C20:2 0.8 0.9 0.5 0.6 0.4 0.5 0.6 0.8 0.8 0.7 C20:3 0.4 0.9 0.4 0.6 - - - - - - C20:4 10.7 8.0 10.6 8.6 6.0 4.1 11.7 10.9 11.5 11.3 C22:0 1.4 1.9 1.2 1.7 1.1 1.4 1.0 1.2 1.4 1.4 C22:2 0.2 2.0 0.1 0.1 1.8 1.5 0.2 0.5 0.2 0.3 C22:3 0.3 0.4 1.6 0.9 1.0 1.2 0.5 0.4 0.3 0.6 C22:4 1.8 1.0 2.4 1.5 0.5 0.3 2.4 2.0 2.0 2.0 C22:5w6 0.6 0.4 0.8 0.5 0.2 0.2 0.8 0.5 0.7 0.7 C22:5w3 0.7 0.6 1.0 0.7 3.3 2.4 1.3 0.9 1.1 0.9 C22:6 0.7 0.5 0.9 0.6 4.8 3.7 1.1 0.9 0.9 1.0 Unsat. 57.1 56.3 60.2 57.1 56.7 53.2 58.3 54.6 55.3 56.3 Sat. 39.3 38.3 34.2 37.6 37.4 41.4 37.3 39.7 39.6 38.6 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acids. 102 Table 4 Percent fatty acid composition of neutral lipids isolated from microsomal membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D w D w 0 w D w D w 012:0 7.2 4.6 - - - - - - - - 014:0 4.8 3.3 0.4 0.3 0.4 0.5 0.4 0.5 0.3 0.3 016 DMAa 1.4 3.6 1.8 3.5 0.7 0.7 0.5 0.4 2.3 2.9 C16:0 16.9 15.0 15.2 12.7 17.2 17.4 17.2 16.1 13.7 14.9 C16:1 4.9 2.8 3.0 1.8 3.3 2.1 3.0 2.8 1.9 2.4 017:0 0.9 1.1 0.6 1.0 0.2 0.1 0.1 0.1 1.0 1.2 C18 DMAa 1.3 1.3 0.5 1.9 0.2 0.4 0.2 0.2 1.0 1.3 018:0 6.0 7.9 6.8 6.7 6.6 8.7 6.9 7.2 8.2 8.5 C18:1 25.9 21.9 36.4 32.2 21.2 20.8 30.9 29.7 27.3 25.6 018:2 15.3 15.9 17.0 14.7 18.4 17.0 30.3 28.0 28.9 27.3 018:3 5.6 7.2 7.3 7.3 21.3 19.5 4.6 5.7 4.9 4.1 020:0 - - - - - - 0.3 0.3 0.3 0.2 020:2 0.9 1.2 0.7 1.3 0.3 0.3 0.3 0.4 0.8 0.7 020:4 4.1 6.4 6.3 8.7 1.9 3.0 3.0 3.8 3.4 4.3 022:0 1.0 1.3 0.5 1.5 0.5 1.0 0.3 0.9 0.9 1.0 022:2 0.2 0.7 0.1 0.1 0.4 1.0 0.1 0.7 0.1 0.4 022:3 1.0 0.2 0.3 0.3 1.1 1.0 0.6 0.2 1.0 0.6 022:4 1.6 2.6 1.4 2.5 0.4 0.4 0.7 0.3 1.5 2.3 C22:5w6 0.6 0.9 0.5 1.0 0.1 0.2 0.2 0.3 0.5 0.8 C22:5w3 0.4 1.1 0.6 1.4 2.7 2.8 0.3 0.4 1.0 1.2 022:6 0.5 1.2 0.6 1.3 3.2 3.3 0.3 0.3 1.9 1.2 Unsat. 60.6 62.0 74.1 71.4 74.4 71.3 74.2 72.7 72.3 69.7 Sat. 36.7 33;; 23.6 22.3 24.9 27.6 25.2 25.1 24.5 26.2 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acid. Fatty acid data underline are significantly (P < 0.05) from other fatty acid data. 103 Table 5 Percent fatty acid composition of phospholipids isolated form microsomal membranes of dark (D) and white (W) meat of broilers fed different dietary oils and alpha-tocopherol Coconut Olive Linseed HSBO + HSBO oil oil oil alpha- tocopherol D W D W D W D W D W C12:O 0.3 0.2 - - - - - - C14:0 2.0 2.0 0.2 0.3 0.1 0.3 0.2 0.2 0.3 0.1 C16 DMAa 4.1 5.2 5.0 4.7 6.3 5.6 4.5 6.1 4.8 4.8 C16:0 19.4 21.1 23.5 23.7 19.1 21.8 16.8 20.6 23.5 23.8 C16:1 1.3 1.3 1.0 1.0 0.5 0.5 0.5 0.2 0.5 0.4 C17:0 0.9 1.0 0.9 0.9 1.4 1.3 1.6 1.6 1.1 0.9 C18 DMAa 0.4 0.9 0.7 1.4 0.7 1.5 0.9 1.7 1.1 1.0 C18:0 14.6 10.1 9.8 10.1 13.8 11.6 14.9 9.8 11.4 11.5 C18:1 19.6 18.4 21.6 23.5 15.3 15.8 13.9 17.4 16.3 17.4 C18:2 18.3 21.4 14.3 15.8 19.2 16.4 19.0 16.7 20.0 19.9 C18:3 2.5 3.0 4.6 3.6 4.1 3.9 3.7 3.6 2.8 1.8 C20:2 0.3 0.4 0.5 0.6 0.4 0.5 0.5 0.8 0.9 0.7 C20:3 0.6 1.4 0.4 0.7 - - - - - - C20:4 9.3 6.3 9.6 7.7 4.8 5.0 13.7 11.7 10.1 10.1 C22:0 1.6 2.4 1.7 2.0 0.9 1.4 1.2 1.2 1.5 1.6 C22:2 0.3 1.4 1.0 1.0 3.0 3.9 1.1 0.5 0.3 0.4 C22:3 0.6 0.7 0.5 0.5 0.7 0.6 0.7 0.3 0.7 0.3 022:4 2.0 1.2 1.5 1.2 0.4 0.5 2.6 3.1 2.0 2.1 C22:5w6 0.7 0.5 0.9 0.7 0.2 0.2 0.8 0.9 0.7 0.7 C22:5w3 0.6 0.4 1.4 0.9 3.7 3.9 1.9 1.9 1.2 1.2 C22:6 0.7 0.7 1.2 0.8 5.5 5.5 1.5 1.8 1.1 1.3 Unsat. 56.6 57.2 58.4 57.0 57.8 56.6 61.0 58.9 56.4 56.4 Sat. 38.9 37.8 36.0 37.0 35.3 36.3 33.7 33.3 37.8 37.8 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acid. 104 and white meat of broilers fed olive oil, as compared to those from the other groups. The membrane lipids from the HSBO-fed groups had higher C18:2 and C20:4 contents than those from the other groups. These results are not surprising in view of the fact that linolenic acid which is the predominant fatty acid in linseed oil, serves as the precursor for the synthesis of longer chain fatty acids of the omega-3 family such as C22:5w3 and C22:6 by delta-6 and delta-5 desaturases (Cook, 1985; Gurr, 1984: Hadley, 1985). In contrast, linoleic acid (C18:2) which is the principal fatty acid in HSBO, is the precursor of the longer chain fatty acids of the omega- 6 family, including C20:4 (arachidonic acid), C22:4 (docosatetraenoic acid) and C22:5w6 (docosapentaenoic acid) (Cook, 1985). The neutral lipids in the mitochondrial and microsomal fractions from dark meat contained more C16:1 and less arachidonic acid (20:4) and docosadienoic acid (C22:2) fatty acids than did the neutral lipids of white meat, but the reverse was true for the phospholipid fractions. This may be due to the difference in the activities of certain desaturases in dark and white meat. Tahin et al. (1981) reported that the fatty acid composition of mitochondrial or microsomal lipids in rat liver, heart, and brain was influenced by dietary oils, but 105 to different degrees in different organelles. There are less data available concerning diet-induced modification of fatty acid composition of mitochondrial and microsomal lipids isolated from broiler dark and white meat. Despite the large differences in the type of lipid present in the experimental diets, the ratio of saturated fatty acids to unsaturated fatty acids did not change markedly (Tables 2-5). This observation supports the concept that a homeostatic mechanism is in some way responsible for buffering the membranes from undergoing significant changes (McMurchie et al., 1986). The possible explanation of the homeoviscous phenomenon in membrane lipids is to preserve a relatively constant level of membrane fluidity (McMurchie et al., 1986). Concentgation a; Alpha-Toconherol in Subcelluiar Mennranes The alpha-tocopherol concentrations in the mitochondrial membranes of dark and white meat from broilers receiving a basal diet were 2.3-3.6 ug and 1.8-2.8 ug of alpha-tocopherol/g membrane (wet weight basis), respectively (Table 6). Dietary oil had no apparent influence on the deposition of alpha-tocopherol in the mitochondrial membranes in both dark and white meat. The same was true for the microsomal fraction from broiler white meat which contained the least amount of alpha- 106 Table 6 Levels of alpha-tocopherol in dark (D) and white (W) meat from broilers fed different dietary oils and alpha—tocopherol (one day post slaughter) Treatments Alpha-tocopherol (ug/g membrane, wet basis)e Mitochondria Microsomes D W D W Coconut oil 2.3 1.9 31.0C 0.6 (group 1) Olive oil 3.0 2.8 47.6b 0.7 (group 2) Linseed oil 2.8 1.8 20.9d 0.8 (group 3) HSBO + alpha- 3.6 2.6 126.0a 2.2 tocopherol (group 4) HSBO 2.9 1.9 37.2c 0.9 (group 5) a,b,c,d Alpha-tocopherol contents bearing different superscript letters on the same column are significantly different at P < 0.01. e Mean of three analyses. 107 tocopherol (0.6-0.9 ug/g) deposited. In contrast, the microsomes from dark meat contained substantial amounts of alpha-tocopherol and the concentrations were largely influenced by the level of alpha-tocopherol in diet, and to some extent by the nature of the dietary oil. For example, the microsomes isolated from the dark meat of the linseed oil group had the lowest concentration of alpha-tocopherol (21 ug/g) and those from the olive oil group contained the highest level (47.6 ug/g) when the concentrations were based on the basal diet. The microsomes from the dark meat of the HSBO group contained 37.2 ug alpha-tocopherol/g membrane. Supplementation of the diet with alpha-tocopherol resulted in the deposition of 126 ug of this antioxidant per gram of microsomal membrane isolated from dark meat, while microsomes from white meat contained only 2.2 ug/g wet membrane. The different amounts of alpha-tocopherol in the microsomal membranes may directly affect the tissue concentration of this antioxidant in broiler dark and white meat. Sheldon (1984) reported that dietary supplementation with alpha-tocopherol resulted in a 100 to 600% increase in this antioxidant in the dark meat of turkey as compared to the white meat. 108 —o— Coconut Oil 8 - ——.—— Olive Oil —+—— HSBO + Alpha-Tocophcrol —-l— HSBO 6 d nmole Malonaldehydc/mg Microsomes Protein & 2" ' ’7‘- -;/ I 0 I I I I I I I 0 30 60 90 120 150 180 Time (Minutes) Figure 1 NADPH-initiated lipid peroxidation in microsomes from white meat of broilers fed different dietary oils varying in degree of unsaturation and alpha-tocopherol supplementation 109 --1r—-— (Coconut Cfil 10 7 -—0— Olive Oil -—1r——- Lhnwcd (X1 —9— HSBO + Alpha-Tocopherol —.— HSBO 8 -I .5 ii a: 8 8 6~ .2 :5 DD 8 73 '9. “5'3 "U 4 .- '8 O '3 . :2 .2 E 2 'l . ?-:b=”""I———_—W . . 0 I I I I T I I 0 30 60 90 120 l50 180 'Fkne (Nfinumm) Figure 2 NADPH-initiated lipid peroxidation in microsomes from dark meat of broilers fed different dietary oils varying in degree of unsaturation and alpha-tocopherol nmole Malonaldehydc/mg Mitochondria Protein 110 —o— Coconut Oil ——o-—- Olive Oil 8 ‘ _._ Linmd Oil - —-.— HSBO + Alpha-Toccpherol _._ HSBO 2 " ./ 0 ' I I r I I I 0 30 60 90 120 150 180 Time (Minutes) Figure 3 NADPH-initiated lipid peroxidation in mitochondria from white meat of broilers fed different dietary oils varying in degree of unsaturation and alpha- tocopherol 111 —o— Coconut Oil 201 ——.—- Olive on —e— Linseed Oil —0— HSBO + Alpha-Tocopherol nmole Malonaldehydc/mg Mitochondria Protein 0. 8 ‘ _l I _ _/ I ‘f :S—y" J V v V v 0 I I I I I I I 0 30 60 90 120 150 180 Time (Minutes) Figure 4 NADPH-initiated lipid peroxidation in mitochondria from dark meat of broilers fed different dietary oils varying in degree of unsaturation and alpha-tocopherol O N 51; L1: pero isol ADP- III 112 931.063.1372 51941211111 of 11.02:.“ aha-m4 Haida Figures 1-4 show the rate of NADPH-induced lipid peroxidation in the mitochondrial and microsomal membranes isolated from the five groups of broilers. When the NADPH- ADP-ferric chloride initiator was added to the isolated microsomal fractions, the microsomes from the linseed oil- fed group were more rapidly oxidized than those from the other groups (Figures 1 and 2). Microsomes isolated from the alpha-tocopherol group had lower TBARS numbers than those from the HSBO control group. Similar trends were observed when the mitochondria were used as the lipid substrates (Figures 3 and 4). Analysis of variance of the NADPH-induced peroxidation data for the subcellular membranes of the dark and the white meat from broilers is presented in Table 7. The results of the Duncan multiple range test for TBARS variables of subcellular membranes are listed in Table 8. They indicate that the effects of interaction among treatments, type of meats, membranes, and sampling time were significantly different (P < 0.001). Different membranes isolated from dark or white meat of broilers fed different dietary oils and/or alpha-tocopherol, as well as different sampling times, all affected the oxidative stability of membranal lipids. The rapid rate of peroxidation in microsomes from broilers fed linseed oil can be, in part, explained by the 113 Table 7 Analysis of variance of thiobarbituric acid- reactive substances (TBARS numbers nmole malonaldehyde/mg membrane protein) in subcellular membranes of dark and white meat from broilers fed different dieuary oils and alpha-tocopherol Source DF SS MES F values1 Treatments (TRT) 4 1894.2 473.6 135.3 Error TRT 715 2490.6 3.5 Meats, dark (D), white (W) 1 133.1 133.1 45.9 DW x TRT 8 2160.9 270.1 93.1 Error DW x TRT 710 2090.9 2.9 Membrane (MEM) 1 186.5 186.5 71.7 TRT x MEN 8 1969.6 246.2 94.7 DW x MEN 2 137.2 68.6 26.4 0W x TRT x MEM 8 273.3 34.2 13.2 Error DW x TRT x MEM 700 1818.2 2.6 Sampling Time (Time) 11 798.6 72.6 728.0 TRT x TIME 48 2443.5 50.9 509.0 DW x TIME 12 172.5 14.4 144.0 MEM x TIME 12 227.4 19.0 190.0 DW x TRT x TIME 48 364.0 7.6 76.0 TRT x MEM x TIME 48 173.4 3.6 36.0 DW x MEM x TIME 12 48.5 4.0 40.0 DW x TRT x MEM x TIME 48 154.7 3.2 32.0 Error DW x TRT x MEM x TIME 719 4284.8 0.1 1 F values indicate significance at P < 0.001. The effects of interaction among treatments, type of meats, membranes, and sampling time were significantly different (P < 0.001). T802 0 114 Table 8 Oxidative stability of subcellular membranes isolated form dark and white meat of broilers fed different dietary oils and alpha-tocopherol as expressed by TBARS numbers (nmole malonaldehyde/mg membrane protein) Treatments Coconut Olive Linseed HSBO + HSBO (N=144) oil oil oil alpha- tocopherol 1.39b 1.55b 5.50a 1.25C 1.64b Meats Dark meat White meat (N=360) 2.70a 1.83b Membranes Mitochondria Microsomes (N=360) 2.78a 1.76b a,b,c Means bearing different superscript letters on the same row are significantly different at P < 0.05. diff part mic: is a the mic: alw mic grc H ff ("f 115 differences in fatty acid composition of the lipids and, in part, by the lower alpha-tocopherol content in the microsomes as compared to those from the other groups. It is also apparent from the results in Figures 1 and 2 that the rate of NADPH-induced lipid peroxidation in the microsomal fractions from the different groups did not always relate to the alpha-tocopherol concentrations in the microsomes. For instance, microsomes from the coconut oil group contained 31.0 ug/g alpha-tocopherol and exhibited a lower rate of peroxidation than did the microsomes from the olive oil and HSBO groups, even though the latter microsomes contained higher concentrations of alpha- tocopherol (47.6 ug/g and 37.2 ug/g, respectively). This indicated that the fatty acid composition of the microsomes was probably the more important factor determining the rate of peroxidation. The same trends appeared to hold for the mitochondria which only marginally varied in alpha- tocopherol concentration between groups, but differed markedly in peroxidation rate (Figures 3 and 4). Even though mitochondria from the alpha-tocopherol-supplemented group did not accumulate any significant amount of the antioxidant, it is possible that alpha-tocopherol supplementation might have helped in preserving the high level of thiols or thiol-associated enzymes (glutathione reductase, glutathione peroxidase) which did not permit the acc 116 accumulation of hydroperoxides in the systems. The same may hold for microsomes from the white meat of broilers fed the alpha-tocopherol supplement. A study by Franco and Jenkinson (1986) who demonstrated that glutathione was effective in decreasing peroxidation in microsomes from rats fed balanced diets but not from vitamin E-deficient rats, lends support to this proposition. Microsomal and mitochondrial membranes are particularly susceptible to lipid peroxidation owing to the presence of high concentrations of polyunsaturated fatty acids and their close proximity to the myoglobin pigment in meat (Lin and Hultin, 1976). Kanner and Harel (1985) indicated that microsomal lipid peroxidation initiated by hydrogen peroxide-activated metmyoglobin (Meth) could be inhibited by the addition of 1-10 umoles of alpha- tocopherol. Results of the present study indicate that the incorporation of alpha-tocopherol though dietary means into the membranes can increase the stability of membranal lipids to peroxidative changes. It has long been recognized that alpha-tocopherol can function as an antioxidant in tissues (Olcott and Matill, 1941; Dam, 1957). The commonly accepted theory is that alpha-tocopherol inhibits the oxidative reactions of membrane-bound lipids caused by free radical attack. The phenolic group of alpha-tocopherol acts as an electron 117 donor, deactivating the catalyst, as well as suppressing the formation of lipid peroxy radicals and thus preventing propagation of the chain reaction. In the subscellular membranes, alpha-tocopherol is believed to be present adjacent to membrane-bound enzymes such as NADPH oxidase that generate free radicals (Machlin, 1984). Therefore, the incorporation of this antioxidant into the subcellular membranes should be more effective in controlling membrane- bound lipid peroxidation than the addition of the antioxidant in vitro which may not necessarily get imbedded in the membranal structure. The patterns of oxidative stability of meat during storage (Chapter 1) generally followed the same trends as established for the subcellular membranes. SUMMARY AND CONCLUSION Results of this study lead to the conclusion that the alteration of the fatty acid composition of membrane-bound lipids and/or incorporation of alpha-tocopherol into the membranes through feeding will stabilize the membrane-bound lipids. This, in turn, will stabilize the meat during refrigerated and frozen storage. a: Ff D4 Ge; REFERENCES Ascota, S. 0., Marion, W. 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Fatty acids of neutral and phospholipids, rancidity scores and TBA values as influenced by packaging and storage. J. Food Sci. 48:829. Gornall, A. G. Bardawill, C. J. and David, M. M. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751. Gray, J. I. and Pearson, A. M. 1987. Rancidity and warmed- over flavor. In" Advances in Meat Research." A. M. Pearson and T. R. Dutson, eds. Vol. 3 p. 222. Nostrand- Remhold Co., New York, NY. Gurr, M. I. 1984. Biosynthesis of fats. In "Role of Fats in Food and Nutrition." p 88 Elsevier Applied Science Publishers New York. Harel, S. and Kanner, J. 1985a. Hydrogen peroxide generation in ground muscle tissue. J. Agric. Food Chem. 33:1186. Harel, S. and Kanner, J. 1985b. Muscle membranal lipid peroxidation initiated by hydrogen peroxide-activated metmyoglobin. J. Agric. Food Chem. 33:1188. Hadley, N. F. 1985. The Adaptive Role of Lipids in Biological System. p 31. Wiley Interscience. New York. Igene, J. 0., King, J. A,. Pearson, A. M. and Gray, J. I. 1979. Influence of heme pigments, nitrite and non- heme iron on development of warmed-over flavor (WOF) in cooked meat. J. Agric. Food Chem. 27:838. Innis, S. M. and Clandinin, M. T. 1981a. Dynamic modulation of mitochondria inner-membrane lipids in rat heart by dietary fat. Biochem. J. 193:155. Innis, S. M. and Clandinin, M. T. 1981b. Mitochondrial- membrane polar-head—group composition as influenced by diet fat. Biochem. J. 198:231. Ha: MC D MCML 120 Itoh, T., Tamura, T. and Matsumoto, T. 1973. Methylsterol composition of 19 vegetable oils. J. Am. Oil Chem. Soc. 50:300. ' Kanner, J. and Harel, S. 1985. Initiation of membranal lipid peroxidation by activated metmyoglobin and methemoglobin. Arch. Biochem. Bioph. 237:314. Kanner, J. German, J. B. and Kinsella, J. E. 1987. Initiation of lipid peroxidation in biological systems. CRC Crit. Rev. Food Sci. Nutr. 25:317. Kaucher, M., Galbraith, H., Button, V. and Williams, H. H. 1944. The distribution of lipids in animal tissues. Arch. Biochem. 3:203. Lin, T. S. and Hultin, H. O. 1976. Enzymatic lipid peroxidation in microsomes of chicken skeletal muscle. J. Food Sci. 41:1488. Machlin, L. J. 1984. Chapter 3: Vitamin E. In "Handbook of Vitamins. Nutritional, Biochemical, and Clinical Aspects." p 115. Marcel Dekker, Inc. New York. Marmer, W. N., and Maxwell, R. J. 1981. Dry column method for the quantitative extraction and simultaneous class separation of lipids from muscle tissue. Lipids 16:365. Marusich, W. L., Ritter, E. De., Ogrinz, E. F., Keating J. M. Mitrovic, M. and Bunnell, R. H. 1975. Effect of supplemental vitamin E in control of rancidity in poultry meat. Poultry Sci. 54:831 McDonald, R. E. and Hultin, H. O. 1987. Some characteristics of the enzymic lipid peroxidation system in the microsomal fraction of flounder skeletal muscle. J. Food Sci. 52:15. McMurchie, E. J., Gibson, R. A., Charnock, J. S. and Mclntosh, G. H. 1986. Mitochondrial membrane fatty acid composition in the marmoset monkey following dietary lipid supplementation. Lipids 5:315. Morrison, W. R. and Smith, L. M. 1964. Preparation of fatty acid methyl esters and dimethylacetals form lipids with boron trifluoride-methanol. J. Lipid Res. 5:600. Slc Vlad; Waku, 121 Olcott, H. S. and Mattill, H. A. 1941. Constituents of fats and oils affecting the development of rancidity. Chem. Rev. 29:257. Rhee, K. S., Dutson, T. S. and Smith, G. C. 1984. Enzymic lipid peroxidation in microsomal fractions from beef skeletal muscle. J. Food Sci. 49:675. Rhee, K. S., Ziprin, Y. A. and Ordonez, G. 1987. Catalysis of lipid oxidation in raw and cooked beef by metmyoglobin-hydrogen peroxide, nonheme iron, and enzyme systems. J. Agric. Food. Chem. 35:1013. SAS SERIES package. 1987. SAS Institute Inc., Cary, North Carolina. Schenkman, J. B. and Cinti, D. L. 1978.. Preparation of microsomes with calcium. In" Methods in Enzymology." S. Fleischer and L. Packer, eds. Vol. 52. p. 83. Academic Press, New York. Sheldon, B. W. 1984. Effect of dietary tocopherol on the oxidative stability of turkey meat. Poultry Sci. 63:673. Sklan, D., Tenne, E. and Budowski, P. 1983. The effect of dietary fat and tocopherol on lipolysis and oxidation in turkey meat stored at different temperatures. Poultry Sci. 62:2017. Slover, H. T., Thompson, R. H. and Merola, G. V. 1983. Determination of tocopherols and sterols by capillary gas chromatography. J. Am. Oil Chem. Soc. 60:1524. Steel, R. G. D. and Torrie, J. H. 1980. "Principles and Procedures of Statistics." A Biological Approach. 2nd ed. McGaw-Hill Book Co., New York. Tahin, Q. S., Blum, M. and Carafoli, E. 1981. The fatty acid composition of subcellular membranes of rat liver, heart, and brain: Diet-induced modifications. Eur. J. Biochem. 122:5. Vladimirov, Y. A., Olenew, v. I., Suslova, T. B. and Cheremisina, I. P. 1980. Lipid peroxidation in mitochondrial membrane. Adv. Lipid Res. 17:173. Waku, K. and Uda, U. 1971. Lipid composition in rabbit sarcoplasmic reticulum and occurrence of alkyl ether phospholipids. J. Biochem. 69:483. WE W1 122 Watts, B. M. 1954. Oxidative rancidity and discoloration in meat. Adv. Food Res. 5:1. Willemont, C., Poste, L. M., Salvador, J. and Wood, D. F. 1985. Lipid degradation in pork during warmed-over flavor development. Can. Inst. Food Sci. Technol. J. 18:316. CHAPTER I I I Lipid peroxidation in poultry meat. III. Effects of oxidized dietary oil and antioxidant supplementation on broiler growth and meat stability 123 st BE in OX St 124 ABSTRACT Broilers were fed diets containing oxidized sunflower oil, alpha-tocopherol and BHA/BHT supplements. Oxidized oil caused a significant reduction in broiler body and carcass weights, whereas alpha-tocopherol and BHA/BHT supplementation improved growth performance. Meat from these broilers were stored at 4°C and -20°C and evaluated for their oxidative stability. Feeding oxidized oil to broilers resulted in meat that underwent rapid oxidative changes during refrigerated and frozen storage. On the other hand, dietary alpha-tocopherol and BHA/BHT resulted in residual alpha-tocopherol and BHA/BHT in meat, which significantly (P < 0.05) improved the oxidative stability of meat during refrigerated and frozen storage. 125 INTRODUCTION In the broiler feed industry, oxidation of selected feed ingredients is not uncommon and can be costly to the poultry grower (Shermer and Calabotta, 1985). Severe feed oxidation causes a readily detected illness in broilers called encephalomalacia. A low level of oxidation in feed (peroxide value 2 meq/kg feed) may also adversely affect the growth performance of the broilers (Shermer and Calabotta, 1985). In addition, few studies have addressed the effect of oxidized dietary oil on the oxidative stability of broiler meat and meat products during storage. In order to prevent the oxidative deterioration of feed components, synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), are usually added to the diet (Gordon and Machlin, 1959; Williams et al., 1960; Bartov and Bornstein, 1972; Scott et al., 1982). Although the antioxidants are known to effectively control lipid oxidation in feed, little is known about their ability to stabilize lipids in the meat when introduced into the muscles through diet. On the other hand, the natural antioxidant, alpha-tocopherol, has been shown to effectively delay some of the symptoms associated with the presence of oxidized oils in the diet (Shermer and Calabotta, 1985). Dietary supplementation also increases the tocopherol content of the tissues and weft ranc on s The 1 126 subsequently extends the storage life of muscle tissues (Marusich et al., 1975; Uebersax et al., 1978; Combs and Regenstein, 1980; Bartov and Bornstein, 1981, 1983; Sheldon, 1984). However, the extent to which alpha- tocopherol accumulates in the dark and white meat of broilers and its subsequent effect on the oxidative stability of the meat during storage has not been extensively studied. The major objectives of the present study were: (1) to determine the effects of oxidized oil on the fatty acid composition of muscle lipids and on the oxidative stability of broiler meat; and (2) to investigate the influence of dietary BHA/BHT and alpha-tocopherol (short- and long-term feeding) supplementation on the oxidative stability of dark and white meat of broilers. MATERIALS AND METHODS Bagiia; Feeding Begimens Eighty one-day old White Mountain chicks (all male) were obtained from Fairview Farms Inc., Indiana, and randomly divided into five groups. Each group was raised on standard all-mash starter and finisher broiler diets. The first group was fed a diet containing 5.5% oxidized 127 sunflower oil, the final peroxide value being 22 meq/kg feed. The second group was fed a diet containing 5.5% sunflower oil (unoxidized),which was supplemented with alpha-tocopherol (200 mg/kg feed) in the form of alpha- tocopheryl acetate for the last 10 days of the dietary regimen (short-term supplementation). The third group was fed the alpha-tocopherol supplement for six weeks (long- term supplementation). The fourth group received the same level of unoxidized sunflower oil supplemented with BHA and BHT. The consumption of BHA was regulated at the rate of 12.5 mg/bird/day starting from the third week of the feeding trial, with the daily amount fed doubling every successive week. Broilers were fed BHT for the last 5 days before slaughter. On the fifth day before slaughter, broilers received BHT at the rate of 12.5 mg/bird/day and the daily amount fed was doubled every successive day. The fifth group was fed a diet containing 5.5% unoxidized sunflower oil and served as the control group. The broilers were raised and slaughtered under the conditions described previously in Chapter 1. Onidanion g; Sunflowe; Oi; Sunflower oil, purchased from a local store, was oxidized by bubbling air through the oil at 80°C until the peroxide value of the oil reached 400 meg/kg oil 128 (approximately 70 hours). The peroxide value of the oxidized dietary oil was determined using the official iodometric method (AOAC 1970). MWMM The broiler meat was manually deboned and ground by passing twice through a food grinder with a 7mm plate (Kitchen Aid Stand Mixer KS-A, Hobart Corp, Troy, OH). The dark meat and white meat were separately packaged in Nasco Whirl-Pak bags (VWI Scientific Co., Bapavia, IL). Packages of meat from each group were randomized and stored at 4°C and -20°C. Samples were withdrawn at appropriate time intervals for different analyses. Lipid Extragtion and Fatty Acid Determination Lipids were extracted from the meat utilizing the dry column method of Marmer and Maxwell (1981). Fatty acid methyl esters were prepared by the method of Morrison and Smith (1964) and separated by gas chromatography using a SP-2330 packed column. Details of these analyses were described in Chapter 1. WQWS 0' 'ds Phospholipid components were separated by the thin layer chromatographic (TLC) method of Parker and Peterson (196E Quan and (U 129 (1965), as described in Chapter 1. QuentitatienefAIRhe-flgsefltemlinm lie—s' sue Quantitation of the alpha-tocopherol levels in dark and white meat from broilers was achieved through a combination of the methods of Itoh et al., 1973 and Slover et al., 1983. Details of these analyses are presented in Chapter 1. Anaiysis g; BEA ang an: in Muscie Tissues Quantitation of BHA and BHT in broiler meat was carried out by a combination of the methods of Wyatt and Sherwin (1979) and Ito et al. (1980). A stock solution of the antioxidants was prepared by dissolving 1.5 mg BHA or BHT (Eastman Chemical Products, Inc. Rochester, N.Y ) in 10 ml sunflower oil. One ml of the stock solution was transferred to a test tube containing 2 ml aqueous potassium hydroxide (1N) and allowed to sit overnight at room temperature. The antioxidant (BHA or BHT) was extracted from the saponified solution with 5 ml di- isopropyl ether. The BHA and BHT was derivatized by the addition of 50 ul bis-trimethylsilyl trifluoroacetamide and 100 ul silylation grade pyridine (Pierce Chemical Co., Rockford, IL). This mixture was allowed to sit for 30 min at room temperature. The silylated BHA and BHT were used 130 as standard solutions containing 1 ug BHA or BHT per ul of solution. Gas chromatographic analysis of the silylated BHA and BHT derivatives was performed using a Hewlett Packard 5890 gas chromatograph (GC) equipped with a flame ionization detector and a 30m x 0.2 mm o.d. methyl silicon fluid capillary column (Hewlett Packard). The GC was operated in the temperature program mode, with an initial temperature of 180°C for 10 min, followed by a 5°C/min increase to a final temperature of 200°C. The injection port temperature and the flame ionization detector temperature were 200°C and 300°C respectively. The split ratio was 20:1 and helium was used as a carrier gas at a flow rate of 15 ml/min. Quantitation of the BHA and BHT contents in the neutral lipids of broiler meat was accomplished using the same procedure. Results were reported as mg BHA or BHT/kg meat. Lipid Qxidation in naap §ys§ems Representative portions of the meat samples were stored under refrigerated conditions (4°C) and oxidative changes monitored after 1, 3, 5 and 9 days. Similar meat samples were stored in a freezer (-20°C) and withdrawn after 2 and 6 month intervals for the determination of the: (I) (1' D) and ran Dun Sig (DI; 131 their oxidative stability during frozen storage. Measurement of 2-thiobarbic acid-reactive substances (TBARS) in the meat samples was achieved using the distillation method of Tarladgis (1960), as described in Chapter 1. mm The feed consumption, carcass weight, fat content and meat weight data were analyzed using a complete randomized design, while the lipid components and TBA data for dark and white meat from broilers were analyzed by a complete randomized block design (Steel and Torrie, 1980). The Duncan’s multiple range test was used for testing the significance of the differences between mean values (Duncan, 1955). RESULTS AND DISCUSSION Qapcass gnapagtapistics The body and carcass weights of broilers fed the diet containing oxidized oil were 4.2% and 7.3% lower than the corresponding weights of the control group (Table 1). These results suggest that oxidation impaired the nutritional value of sunflower oil and, in turn, the efficiency of feed utilization. These data substantiate 132 Table 1 Effects of oxidized oil and antioxidant supplementation on the feed connsumption, feed conversion, body weight and dressing percent of broilers Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol Total feed consumption 91.8 94.7 92.4 88.2 93.5 (kg) Total body weight (kg) 43.7 46.4 46.7 46.9 45.6 Feed conversion 2.10 2.04 1.98 1.88 2.05 ratio Average body 2.73 2.90 2.92 2.93 2.85 weight (kg) Dressing1 64.8 66.9 66.8 67.6 67.0 percent Average b carcass (kg) 1.77C 1.94a 1.95a 1.98a 1.91 weight a,b,c Average carcass weight within rows bearing different superscript letters differ significantly at P < 0.05. 1 Dressing percent = (carcass weight/body weight) x 100 133 earlier observations made by several researchers (Crampton et al., 1951; Waldroup et al., 1960; Hussein and Kratzer, 1982) that feeding of heat-abused fats/oils depressed animal growth. The poor growth rate of broilers fed oxidized oil may be due to a decrease in the biological value of the heated oil. Aldehydes, ketones, acids, esters, and polymerized oils are direct products of the oxidation process and can result in reduced energy values of broiler feed (Shermer and Calabotta, 1985). Products of oxidation may also have promoted the destruction of the fat-soluble vitamins (A, D, E and K) or other susceptible feed constituents such as carotenoids. Moreover, these oxidative products may react with proteins and amino acids in the feed and impair the biological values of these nutrients. Broilers fed the BHA/BHT-supplemented diet had significantly higher carcass weights (P < 0.05), respectively, than those of the control group (Table 1). Similarly, short-term and long-term alpha-tocopherol supplementation also significantly (P < 0.05) increased carcass weights of the broilers. Short-term supplementation produced increases of 1.8% and 2.5% in body and carcass weights, while long-term supplementation resulted in increases of 1.6% and 3.7%, respectively, when compared to the corresponding weights of the control 134 (Table 1) . ‘ Supplementation of the diet with alpha-tocopherol and BHA/BHT tended to improve the feed utilization efficiency of broilers, as revealed by the low feed conversion ratios (Table 1). The results are consistent with the data in the previous feeding trail (Chapter 1) which showed that alpha-tocopherol supplementation of broiler diets influenced the growth performance of broilers. There is a paucity of data in the literature reflecting the effect of BHA/BHT on the growth performance of broilers. Waldroup et al. (1960) reported that birds receiving diets containing the synthetic antioxidant, ethoxyquin, were significantly heavier than birds receiving diets containing no antioxidant. More studies are needed to substantiate the observation that BHA or BHT can improve growth when added to broiler diets. Lipig Qomponents in Bgoile; Maap The lipid composition of dark and white meat is presented in Table 2. The total lipid content of the dark meat from all broiler groups were higher than that of the white meat. Although the total lipid contents of dark and white meat from broilers fed the oxidized dietary oil and the BHA/BHT supplement were slightly higher, and those from the alpha-tocopherol-fed broilers were somewhat lower than 135 Table 2 Lipid composition of dark (0) and white (W) meat from broilers fed oxidized oil and antioxidants Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol Total1 7.8a 7.3a 5.8b 6.5a 6.2a lipid w 2.5a 2.2a 2.3a 3.1a 2.4a Neutra12 D 87.6a 86.9a 86.4a 86.8a 87.2a lipid w 73.6a 70.8a 70.0a 71.4a 71.9a Phospho-2 D 12.4a 13.1a 13.6a 13.2a 12.8a lipid w 27.4a 29.2a 30.4a 28.6a 28.1a Lyso-PC3 0 7.13 6.3a 6.0a 5.6a 5.5a w 6.9a 6.6a 6.3a 6.6a 7.4a Lyso-PE3 D 8.4a 5.5b 6.3b 6.6b 7.4a w 7.1a 5.8a 5.8a 6.8a 5.2a Sphingo-3 D 8.6a 8.1a 7.2a 9.7a 5.4a lipid w 11.5a 12.4a 10.0a 9.7a 10.1a P03 0 46.5C 49.4b 49.4b 53.1a 52.6a w 48.6a 50.5a 51.5a 53.5a 51.9a PE3 D 23.3b 22.0b 22.1b 22.8b 26.3a w 18.9a 17.9a 17.9a 19.7a 17.8a a,b,c The data within row bearing different superscript letters differ significantyly at P < 0.05. 1,2,3 and phospholipids (3). Expressed as percent of meat (1), total lipid (2) 136 the total lipid from the control group, the differences between treatments were not significant. The amounts of neutral lipids and phospholipids also did not differ significantly in meat from the different treatments. The amounts of lyso-PC, lyso-PE, PC and PE in phospholipids from dark or white meat also were not affected by dietary treatments (Table 2). The fact that oxidized dietary oil did not significantly alter the phospholipid components suggests that a level of rancidity was adequately reached to induce the synthesis of enzymes such as catalase, glutathione peroxidase, and superoxide dismutase (Younathan and McWilliams, 1985) that may decrease free radical effects on fatty acid synthesis. KELLY 8214 Composition The fatty acid profiles of the neutral lipids and phospholipids of dark and white meat are shown in Tables 3 and 4. Sixteen fatty acids were identified in the neutral lipids and phospholipids of dark and white meats. In the neutral lipid fractions, the fatty acids C16:0, C18:0, C18:1 and C18:2 constituted approximately 80% of the total fatty acids, whereas in the phospholipid fractions, C16:0, C18:0, C18:1 C18:2, C18:3 and C20:4 accounted for about 70- 75% of the total fatty acids. The fatty acid composition of the neutral lipids and phospholipids was not significantly oil alpha- tocopherol tocopherol 137 Percent fatty acid composition of neutral lipids alpha- isolated from dark (0) and white (W) meat of Oxidized Short-term Long-term BHA/BHT Sunflower broilers fed oxidized oil and antioxidants oil Table 3 36319338908471322 0M205662121310000 35544..0.20.1183724..11 042056632213100.nwnu. 1 23 30535159095152£11u 0M205662212310m0nw 34644892nw44933522 0M205653212210000 36nw11826767132522 0.”..20.657.2112310000 13 JANA—31169486682422 0.520.6661112210000 1 23 313338201n~4971311 04206463122310nw0.nu. 1 23 39146339873572311 0420546211331000nw 1 23 34724.].4661385 eooeo cocootttt 0620656211221 1 23 no... oeooetttt 06205567m11321 1 23 63 an 00100112302334.4556 CCCCCCCCCCCCCCCCC 75.7 75.1 77.0 76.9 76.6 77.2 77.5 77.9 77.3 77.2 24.3 24.9 23.0 23.1 23.4 22.8 22.5 22.1 22.7 22.8 t = trace amount fatty acid (less than 0.1%) Sat. Unsat. 138 Percent fatty acid composition of phospholipids isolated from dark (D) and white (W) meat of broilers fed oxidized oil and antioxidants Table 4 Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol D W D W D W D W D W C14:0 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.2 0.1 0.2 C16 DMAa 4.5 5.4 5.6 5.9 5.5 6.0 5.7 5.7 5.6 6.1 016:0 15.8 18.6 17.3 18.5 15.5 18.2 16.1 18.5 16.0 18.9 C16:1 0.5 0.5 0.7 0.6 0.9 0.6 0.6 0.7 0.8 0.7 C17:0 1.6 1.9 2.0 1.9 2.1 1.9 2.1 1.8 2.2 1.8 C18 DMAa 1.0 1.8 1.1 1.9 1.1 1.9 0.9 1.6 1.0 1.5 C18:0 12.9 10.9 14.5 11.7 14.7 11.1 14.7 10.7 14.0 11.3 C18:1 14.3 14.0 13.4 14.4 12.5 14.2 12.3 14.6 13.1 14.4 C18:2 18.6 17.3 18.1 17.2 18.8 18.0 17.8 17.9 17.7 18.0 C18:3 0.5 0.1 0.2 0.2 0.2 0.2 0.4 0.2 0.3 0.1 C20:0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 C20:2 1.2 1.2 1.2 1.2 1.2 1.3 2.0 1.3 1.0 1.2 C20:3 2.5 2.4 2.2 2.4 2.2 2.5 2.8 3.3 2.2 2.6 C20:4 10.6 11.0 10.1 12.4 11.0 12.3 10.8 11.3 10.8 12.3 022:4 6.9 6.6 6.7 5.5 6.8 5.9 6.8 6.5 7.3 5.7 C22:5w6 5.6 6.2 4.9 4.4 5.2 4.5 5.0 4.4 5.7 4.0 C22:5w3 1.3 1.0 0.8 0.5 0.9 0.5 0.9 0.6 1.0 0.5 C22:6 0.9 0.8 0.9 1.0 1.1 0.7 0.9 0.6 1.1 0.5 Unsat. 63.0 61.1 59.2 59.8 60.8 60.7 60.3 61.4 61.0 60.0 Sat. 31.5 31.7 34.1 32.6 31.4 33.1 31.3 32.4 32.4 32.4 aDMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plalmalogens. DMAS are not counted as fatty acids. 139 affected by either the oxidized oil or alpha-tocopherol and BHA/BHT supplementations. Although Sklan (1983) indicated that vitamin E enhanced enzymes responsible for delta-9 desaturation and influenced fatty acid metabolism in liver, data obtained in this study did not show any apparent difference in the fatty acid composition of muscle lipids from broilers fed a daily dietary supplement of 200 mg alpha-tocopherol. anggntgations g; Antioxidants in gngilg; Tiggngg The data in Figure 1 indicate that oxidized dietary oil, alpha-tocopherol, and BHA/BHT, significantly (P < 0.01) influenced the amount of alpha-tocopherol deposited in the dark and white meat of broilers. Dark and white meat from the control group of broilers fed sunflower oil (group 5) contained 2.9 mg and 2.4 mg alpha-tocopherol/kg, respectively, and originated from the minimal amounts of alpha-tocopherol in the basal diet. Short-term alpha-tocopherol supplementation (group 2) resulted in the deposition of significantly higher levels (P < 0.05) of alpha-tocopherol in both dark (8.9 mg/kg) and white (5.8 mg/kg) meat as compared to meat from the control group. Overall, these values represented 1.01% of the total alpha-tocopherol consumed by the broilers, 0.62% in the dark and 0.39% in the white meat. 140 d1 mm mm. mm. m. 1mm 1 1.5. m 0m r Em .w. Z.Tm0 .lt.Bl m“... ,m men s 123/45 09 ......okonu céqodqodqodmoddfl 4 vuenouowonouoo o o oouonououeo '0}.}.}.}Q}Q}O}QDO}O}Q>O}. 900». O 9060009 fi§§%00 O O O ponovnononono 600000 Vno».«o«ouo«oo '60.... ’000000004 12‘ :82 max—828852.12 we Dietary Treatments Figure 1 [Elgha-tocopherol concentrations (mg/kg meat) in the dark D and white (W) meat of broilers fed oxidized oil and antioxidants Cit Mar tOcc 141 Meat from broilers subjected to long-term alpha- tocopherol supplementation (group 3) had the highest alpha- tocopherol contents among the five treatments. Dark meat contained 10.6 mg/kg, while white meat contained 6.6 mg/kg. These broilers accumulated 0.68% of the total amount of alpha-tocopherol consumed, 0.40% being deposited in the dark meat and 0.28% in the white meat. Data from both the short-term and long-term groups showed that dark meats accumulated approximately 50% more alpha-tocopherol than did the white meat. Sheldon (1984) explained this observation on the basis that there were physiological variations in the vascular networks of dark and white muscle tissues. The more highly developed vascular system, combined with the higher lipid content, allows alpha- tocopherol to be incorporated to a greater degree in the dark meat than in the white meat. The tissue alpha-tocopherol values agree with those cited by Marusich et a1. (1975) and Sheldon (1984). Marusich et a1. (1975) reported broiler tissue alpha- tocopherol concentrations of 2.7 mg, 3.9 mg, 5.0 mg, and 6.2 mg/kg in white meat when broilers were fed alpha- tocopherol continuously for 8 weeks at levels of 20, 30, 40, and 60 mg/kg feed, respectively. When broilers were fed diets containing 40, 80, and 160 mg alpha-tocopherol/kg feed for the last 4 days before slaughter, the breast meat had 31:1 4.2 mg/ tocophe slaugh1 mg, an 1975 ) . diets aceta‘ breas and 4 142 had alpha-tocopherol concentrations of 1.8 mg, 3.2 mg, and 4.2 mg/kg meat, respectively. Broilers fed alpha- tocopherol at the same levels for the last 5 days before slaughter developed breast muscle containing 2.1 mg, 3.8 mg, and 5.9 mg alpha-tocopherol/kg meat (Marusich et al., 1975). These investigators also found that broilers fed diets containing alpha-tocopherol or alpha-tocopheryl acetate at 160 mg/kg feed for the last 4 days, developed breast meats with alpha-tocopherol concentrations of 5.3 mg and 4.7 mg/kg meat, respectively. Data in Figure 1 also indicate a negative effect of oxidized dietary oil in the broiler diet on tissue alpha- tocopherol concentrations. Broilers fed the oxidized sunflower oil (group 1) retained 2.2 mg/kg and 1.8 mg/kg alpha-tocopherol in the dark and white meat. These values were 24.1% (dark mean) and 25.0% (white meat) smaller than the respective concentrations in the meat from the control group. The lower alpha-tocopherol concentration may be due, in part, to the destruction of a portion of the alpha- tocopherol in the diet by the oxidized dietary oil before it was fed to the broilers. In addition, some of the alpha-tocopherol deposited in the tissue may have been used up in protecting tissue lipids from oxidized oil-induced free radical attack. Century and Horwitt (1959) observed a vitamin E deficiency disease, encephalomalacia, when 143 broilers were fed high levels of oxidized diet. However, information is lacking concerning the relationship between percent oxidized dietary oil in the feed and the vitamin E requirements of the broilers. More studies are required to clearly define the relationship between oxidized dietary oil and alpha-tocopherol concentrations in the meat. The alpha-tocopherol content of white meat from broilers fed BHA/BHT (group 4) was slightly lower, while that of the dark meat was slightly higher than comparative data from the control group (Figure 1). These differences were not significant (P > 0.05). Bartov and Bornstein (1981) demonstrated that supplementation of broiler diets with BHT increased alpha-tocopherol levels in carcass fat. In the present study, higher concentrations of alpha- tocopherol were not observed in the dark and white meat from broilers fed BHA/BHT. Most of the alpha-tocopherol originating from the basal diet might have been deposited in the depot fat, instead of in the muscle tissues. It may be concluded that the presence of BHA or BHT in the broiler diet does not affect the alpha-tocopherol requirement in the muscle tissues. Small amounts of BHA and BHT were found in the broiler muscle. BHA (2.1 mg/kg) was present in only the white meat, while BHT was detected in both dark and white meat at concentrations of 6.5 mg/kg and 3.8 mg/kg, resp BHT lite beta and I)” 144 respectively. These values represented 8.8% of the total BHT and 0.8% of the total BHA consumed. Very little literature information is available on the relationship between the concentrations of BHA or BHT in broiler tissues and in the broiler diet. mmxmmmm The TBA test was used to determine the oxidative stability of dark and white meat from the different groups of broilers (Tables 5 and 6). Oxidized oil-supplementation (group 1) significantly (P < 0.01) decreased the stability of broiler meat. The TBARS numbers of dark meat from broilers fed oxidized sunflower oil increased from 0.48 (1 day) to 2.52 mg/kg after 3 days of storage at 4°C, while TBARS numbers for white meat increased from 0.70 (1 day) to 2.01 mg/kg over the same peroid. These values were significantly higher (P < 0.01) than those of the dark and white meat from the control group (0.27 to 0.70 mg; 0.17 to 0.64 mg/kg), respectively. The oxidized dietary oil in the feed thus adversely affects the shelf life of refrigerated broiler meat, probably through the transfer of free radicals from the feed to the broiler meat. Schermer and Calabotta (1985) reported that oxidized oil, even at peroxide levels as low as 2 meq/kg of feed, can cause serious levels of oxidation in feed and/or feed 145 Table 5 Thiobarbituric acid-reactive substances (TBARS) numbers (mg malonaldehyde/kg meat) of dark and white meat from broilers fed oxidized oil and antioxidants Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol Dark meat stored at 4°C Average 1 day 0.48 0.23 0.21 0.17 0.27 0.27x 3 day 2.52 0.67 0.27 0.26 0.70 0.883Y 5 day 2.98 0.89 0.53 0.52 1.67 1.37y 9 day 4.10 0.92 0.62 0.57 1.89 1.62y Mean 2.52a 0.68c 0.41c 0.38C 1.16b White meat stored at 4°C Average 1 day 0.70 0.17 0.02 0.03 0.17 0.22x 3 day 2.01 0.42 0.26 0.53 0.64 0.77XY 5 day 3.36 0.46 0.40 0.56 0.65 1.09Y 9 day 3.72 0.51 0.43 0.59 1.12 1.27Y Mean 2.45a 0.39c 0.28c 0.43c 0.65b a,b,c TBARS numbers within rows bearing different superscripts differ significantly at P < 0.05. le TBARS numbers within columns bearing different superscripts differ significantly at P < 0.05. 146 Table 6 Thiobarbituric aicd-reactive substnaces (TBARS) numbers (mg malonaldehyde/kg meat) of dark and white meat from broilers fed oxidized oil and antioxidants Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol Dark meat stored at -20°C Average 1 day 0.48 0.23 0.21 0.17 0.27 0.27x 2 months 4.40 0.48 0.42 0.38 0.78 1.29XY 6 months 5.05 0.90 0.60 0.65 1.75 1.79Y Mean 3.31a 0.54b 0.41b 0.40b 0.93b White meat stored at -20°C Average 1 day 0.70 0.17 0.02 0.03 0.17 0.22x 2 months 2.53 0.37 0.34 0.65 0.58 0.89Xy 6 months 4.32 0.56 0.44 0.76 1.10 1.44y Mean 2.52a 0.37b 0.27b 0.48b 0.62b a,b TBARS numbers within rows bearing different superscripts differ significantly at P < 0.01. x,y TBARS numbers within columns bearing different superscripts differ significantly at P <0.10. 147 ingredients before feeding. However, there is little information in the literature pertaining to meat quality of broilers fed meal containing oxidized dietary oil. Meat from broilers fed alpha-tocopherol (groups 2 and 3) had significantly (P < 0.05) lower TBARS numbers than meat from the control broilers (Table 5). White meat from group 4 (long-term alpha-tocopherol supplementation) had the highest alpha-tocopherol content (Figure 1) and the lowest TBARS number (0.28 mg/kg meat) among all dietary treatments. As expected, meat from broilers given a short- term alpha-tocopherol supplement (group 2) had slightly higher TBARS numbers than broiler meat from group 3 (long- term alpha-tocopherol), but these values were significantly (P < 0.05) lower than those for the control group. Meat from the broilers fed the BHA/BHT-supplemented diet was more stable towards oxidation (P < 0.05) than was the control. Webb et al. (1972) demonstrated that feeding BHT as 0.01, 0.02 or 0.04% of the broiler diet did not significantly reduce rancidity development. A diet containing a combination of 0.02% ethoxyquin plus 0.02% BHT significantly (P < 0.05) reduced TBA numbers from 1.99 to 1.50 mg/kg meat. In the present study, a higher level (0.08%) of BHT was fed to the broilers during the last 5 days before slaughter, and succesfully reduced oxidative deterioration in broiler meat during refrigerated storage. 148 It can be concluded that the efficiency of BHA/BHT in preventing rancidity development in meat is dependent on the levels of BHA/BHT in the broiler diet. More studies are required to substantiate the observation that synthetic antioxidants such as BHA or BHT can influence the meat stability of broilers by feeding. Similar trends were observed in raw meat stored at -20°C for 6 months (Table 6). Dark and white meat from broilers fed oxidized dietary oil had the highest TBARS numbers and these were significantly (P < 0.05) different from those for meat from the control group of broilers. The data obtained from this study generally confirm the findings of Sklan et al. (1983) who reported that, when dietary tocopherol levels were increased from 5 to 60 mg/kg feed, tissue tocopherol concentrations increased and minimized the oxidative changes of meat during storage. Bartov et al. (1983) also found that tissue tocopherol levels significantly influenced the oxidative stability of poultry meat. Sheldon (1984) concluded that tocopherol- supplemented diets were effective in reducing lipid oxidation of poultry meat during storage. 149 CONCLUSIONS Results of this study confirm previous findings that the alpha-tocopherol tissue content depends not only on dietary levels, but also on the duration of time the supplemented diets are fed to broilers. BHA/BHT concentrations in the dark and white meat of broilers also clearly reflected dietary levels. Both BHA/BHT and alpha- tocopherol supplementation significantly delayed the oxidative deterioration in dark and white meat during refrigerated and frozen storage. Dark and white meat from broilers fed oxidized sunflower oil underwent rapid oxidative deterioration during refrigerated and frozen storage. This was due, in part, to the low levels of tissue alpha-tocopherol and, in part, to the incorporation of oxidized dietary oil components in the muscle lipid system. REFERENCES Association of Official Analytical Chemists. "Official Methods of Analysis," 11 th ed. 1970. p. 445. No. 28.023 and 28.024. Bartov, J. and Bornstein, S. 1972. Comparisons of BHT and ethoxyquin as antioxidants in the nutrition of broilers. Poultry Sci. 51:859. Bartov, I. and Bornstein, S. 1981. Stability of abdominal fat and meat of broilers: Combined effect of dietary vitamin E and synthetic antoxidants. Poultry Sci. 60:1840. Bartov, I., Basker, D. and Angel, S. 1983. Effect of dietary vitamin E on the stability and sensory quality of turkey meat. Poultry Sci. 62:1224. Century, B. and Horwitt, M. K. 1959. Effect of fatty acids on chick encephalomalacia. Proc. Soc. Exp. Biol. Med. 102: 375. Combs, G. F. Jr., and Regenstein, J. M. 1980. Influence of selenium, vitamin E, and ethoxyquin on lipid peroxidation in muscle tissues from fowl during low temperature storage. Poultry Sci. 59:347. Crampton, E. W., Farmer, F. A., Berryhill, F. M. 1951. The effect of heat treatment on the nutritional value of some vegetable oils. J. Nutrition 43:431. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics 11:1. Gordon, R. S., and Machlin, L. J. 1959. A method of evaluation of antioxidants based on vitamin A protection. Poultry Sci. 38:1463. Hussein, A. S. and Kratzer, F. H. 1982. Effect of rancidity on the feeding value of rice bran for chickens. Poultry Sci. 61:2450. Ito, Y., Toyoda, M., Suzuki, H., Ogawa, S. and Iwaida, M. 1980. Gas-liquid chromatographic determination of butylated hydroxytoluene in modified powdered milk. J. Food Protect. 43:832. Itoh, T., Tamura, T. and Matsumoto, T. 1973. Methylsterol composition of 19 vegetable oils. J. Am. Oil Chem. Soc. 50:300. 150 151 Marmer, W. N., and Maxwell, R. J. 1981. Dry column method for the quantitative extraction and simultaneous class separation of lipids from muscle tissue. Lipids 16:365. Marusich, W. L., DeRitter, E., Ogrinz, E. F., Keating, J., Mittovic, M., and Bunnell, R. H. 1975. Effect of supplemental vitamin E in control of rancidity in poultry meat. Poultry Sci. 54:831. Morrison, W. R., and Smith, L. M. 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron trifluoride methnol. J. Lipid Res. 5:600. Parker, F. and Peterson, N. F. 1965. Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J. Lipid Res. 6:455. Scott, M. L., Nesheim, M. C. and Young, R. J. 1982. "Nutrition of The Chicken." M. L. Scott., M. C. Nesheim and R. J. Young eds. M. L. Scott & Associates. Ithaca, New York. Sheldon, B. W. 1984. Effect of dietary tocopherol on the oxidative stability of turkey meat. Poultry Sci. 63:673. Shermer, W. D. and Calabotta, D. F. 1985. Controlling feed oxidation can be rewarding. Feedstuffs. p 24. Nov. 25, 1985. Slover, H. T., Thompson, R. H. and Merola, G. V. 1983. Determination of tocopherols and sterols by capillary gas chromatography. J. Am. Oil Chem. Soc. 60:1524. Sklan, D., Tenne, Z. and Budowski, P. 1983. The effect of dietary fat and tocopherol on lipolysis and oxidation in turkey meat stored at different temperatures. Poultry Sci. 62:2017. Steel, R. G. D., and Torrie, J. H. 1980. "Principles and Procedures of Statistics." A Biological Approach. 2nd ed. McGaw-Hill Book Co., New York, NY. Tarladgis, B. G., Watts, B. M., Younathan, M. T., Dugan, Jr. L. R. 1960. A distillatin method for the quantitative determination of malonaldehyde in rancid foods. J. Am. Oil Chem. Soc. 37:44. 152 Uebersax, M. A., Dawson, L. E., and Uebersax, K. L. 1978. Storage stability (TBA) of meat obtained from turkeys receiving tocopherol supplementation. Poultry Sci. 57:937. Waldroup, P. W., Douglas, C. R., McCall, J. T. and Harms, R. H. 1960. The effects of santoquin on the performance of broilers. Poultry Sci. 39:1313. Webb, J. E., Brunson, C. C. and Yates, J. D. 1972. Effects of feeding antioxidants on rancidity development in pre-cooked, frozen broiler parts. Poultry Sci. 51:1601. Williams, W. P., Jr., Davies, R. E., Ferguson, T. M. and Couch, J. R. 1960. Antioxidants and alfalfa meal carotenoids. Poultry Sci. 39:1307. Wyatt, D. M. and Sherwin E. R. 1979. Analysis of BHA in polythylene by direct-sampling gas chromatography. Food Technol. 33:46. Younathan, M. T. and McWilliams, D. G. 1985. Hematological status of rats fed oxidized feed lipids. J. Food Sci. 50:1396. CHAPTER IV Lipid peroxidation in poultry meat. Iv. The influence of oxidized dietary oil and antioxidant supplementation on membranal lipid stability. 153 ABSTRACT The effects of oxidized oil, dietary alpha-tocopherol and BHA/BHT- supplementation on the fatty acid composition of mitochondrial, microsomal and lipoprotein fractions of broiler muscles, and on their lability to metmyoglobin/ hydrogen peroxide-catalyzed peroxidation were investigated. Oxidized oil in the broiler diets induced rapid membranal lipid oxidation and decreased lipid stability towards metmyoglobin-hydrogen peroxide-catalyzed peroxidation. Supplementation of the broiler diets with alpha-tocopherol increased the alpha-tocopherol concentrations in the microsomal and lipoprotein fractions of the dark meat as well as in the lipoprotein fraction of the white meat. This, in turn, stabilized the membrane- bound lipids against metmyoglobin-hydrogen peroxide- initiated peroxidative changes. 154 155 INTRODUCTION Oxidation of membrane-bound lipids in biological membranes causes membrane damage and cellular death (Pryor, 1976)). Biological membranes contain phospholipids which are rich in polyunsaturated fatty acids. In addition, they are in contact with a fluid containing prooxidants such as oxygen, transition metals, peroxidase enzymes (Vladimirov et al., 1980), hydrogen peroxide, and the superoxide anion radical (Harel and Kanner, 1985a,b). These membranes are very susceptible to peroxidation (Buege and Aust, 1978) and are believed to be the sites where peroxidative changes are initiated in raw meat (Gray and Pearson, 1987). Oxidative changes in meat lipids may be initiated by nonenzymic (Pearson et al., 1977; Rhee et al., 1987) and/or enzymic reactions (German and Kinsella, 1985; McDonald and Hultin, 1987). The various mechanisms of enzymic and non- enzymic initiation of lipid peroxidation in meats have recently been reviewed by Kanner et al. (1987) and Asghar et al. (1988). Although it is generally accepted that oxidative changes in meat are initiated at the membrane level, little information is available on the levels of alpha-tocopherol deposited in the subcellular membranes (mitochondria, 156 microsomes and lipoproteins) of dark and white muscles as a result of feeding broilers with alpha-tocopherol- supplemented diets. Feeding oxidized dietary oil to experimental animals has been reported to cause various types and degrees of abnormalities (Rasheed et al., 1963), particularly those associated with a vitamin E deficiency in the diet (Dam and Granados, 1945). In addition, damage to the membranes of functional subcellular particles such as mitochondria and microsomes by lipid peroxidation may give rise to a wide array of secondary effects (Tappel, 1962). Due to a lack of published data concerning the effects of oxidized dietary oil on membranal lipid stability, this study was conducted to provide such information. The principal objectives of this study were: 1) to investigate the effects of oxidized oil and dietary antioxidant supplementation on the antioxidant concentrations in the subcellular membranes of broiler meat; and 2) to determine the influence of these dietary treatments on the peroxidative stability of membranal lipids. 157 MATERIALS AND METHODS Broiler med Regimens The source of the broilers and the dietary treatments utilized in this study are described in Chapter 3. Wfimlfimaflmolt n2: WWMWW Subcellular components were separated using the method of Schenkman and Cinti (1978), as described in Chapter 2. After the precipitation of the microsomal fraction, the supernatant was saved and lead acetate was added to produce a solution which was 4 mM with respect to lead acetate. This mixture was then centrifuged at 10,300 x G (8,000 rpm) for 30 min. The resulting percipitative was subsequently designated the lipoprotein fraction. The subcellular fractions were not further purified to remove any contaminating myofibrillar proteins due to trace lipid and no alpha-tocopherol content in this protein which possessed no effect on the results of this study. Lipid Ex act'on The dry column method of Marmer and Maxwell (1981) was utilized to extract the lipids from the membranes. Fntty Acid Analysis The boron trifluoride-methanol procedure of Morrison 158 and Smith (1964) was used for the preparation of fatty acid methyl esters of the membrane lipids. The procedures used for sample preparation and analysis are described in Chapter 1. Quantitntion g: nlnna-Tgconhenoi in Membnanes Identification and quantitation of alpha-tocopherol in the membranes were achieved through a combination of the methods of Itoh et al. (1973) and Slover et al.(1983), as described in Chapter 1. Identification, derivatization and quantitation of BHA and BHT in the subcellular membranes were achieved through a combination of the methods of Wyatt and Sherwin (1979) and Ito et a1. (1980), as described in Chapter 3. Measurement 9; §ubgellular Membrane Protein The protein content of the mitochondria, microsomes and lipoproteins was estimated using the Biuret method (Gornall et al., 1949), as modified by Asghar and Yeates (1974). Details of this procedure are described in Chapter 2. 159 Measurgment p; MetmyoglobinzHydrogen Peroxide-induced Lipid Penoxidatipn in Menpnanes The peroxidative stability of the isolated membranes was determined by a modification of the procedure described by Kanner and Harel (1985). The final mixtures for the peroxidation assay contained subcellular membrane (0.62 mg _protein/ml), metmyoglobin (30 uM) and hydrogen peroxide (30 uM) in a low pH buffer. This buffer was composed of 0.2 M KCl, 0.05 M NaOH and 0.13 M lactic acid, pH 5.4-5.5 and was designed to represent the low acidic condition of meat after rigor mortis. The peroxidation reaction was carried out in 100 ml beakers held in a 37°C water bath. When the temperature of the model system reached 37°C, hydrogen peroxide and metmyoglobin were added to initiate the peroxidation reaction. The reaction was monitored over a 3 hr period, and the extent of membranal lipid peroxidation was determined as described in Chapter 2. §tnti§picai Analysis 9; Experimental pnpn Statistical analyses were performed using the SAS SERIES package developed by the SAS Institute Inc., Cary, North Carolina (1987). The statistical design for the analysis of membranal alpha-tocopherol data was described in Chapter 2. 160 RESULTS AND DISCUSSIONS Mgnppnnni Lipig Composition Total lipid, neutral lipid and phospholipid contents of mitochondria, microsomes and lipoproteins isolated from the dark and white meat of broilers fed the various dietary regimens are summarized in Table 1. In general, the mitochondrial fractions contained the highest amount of total lipid, while the lipoprotein fractions had the smallest total lipid content. The relative proportion of neutral lipids and phospholipids in the mitochondrial, microsomal and lipoprotein fractions did not change to any noticeable extent in response to dietary alpha-tocopherol or BHA/BHT supplementations. The diet containing the oxidized oil also had no effect on the proportion of neutral and phospholipids in the subcellular membranes of broilers. The neutral lipids isolated from the mitochondrial, microsomal and lipoprotein fractions comprised 35-49%, 34-43% and 28-34%, respectively, of the total lipids, while the phospholipids from the corresponding membranes contributed 51-65%, 57-66% and 66- 71%, respectively. The phospholipid percentages obtained in this study for broilers were somewhat lower than those cited in the literature for rats and rabbits. Higher phospholipid contents in the mitochondria (82.2%) and 161 Table 1 Lipid composition of the subcellular membranes isolated from dark (D) and white (W) meat of braoilers fed oxidized.oil and antioxidants Treatments Total1 Neutral2 Phospho~---2 lipid lipid lipid % % % Oxidized oil D Mitochondria 6.0 37.8 62.2 D Microsomes 4.1 39.8 60.2 D Lipoproteins 2.5 31.1 68.9 W Mitochondria 3.9 34.8 65.2 W Microsomes 3.0 38.7 61.3 W Lipoproteins 3.1 29.2 70.8 Short-term D Mitochondria 4.1 49.3 50.7 alpha-tocopherol D Microsomes 3.8 33.9 66.1 D Lipoproteins 2.0 41.5 58.5 W Mitochondria 3.3 36.6 63.3 W Microsomes 3.4 42.9 57.1 W Lipoproteins 1.9 33.5 66.5 Long-term D Mitochondria 4.9 39.6 60.4 alpha-tocopherol D Microsomes 3.8 34.8 65.2 D Lipoproteins 2.0 33.0 67.0 W Mitochondria 4.8 37.7 62.3 W Microsomes 3.5 36.3 63.7 W Lipoproteins 2.3 30.0 70.0 BHA/BHT D Mitochondria 4.8 37.1 62.9 D Microsomes 3.9 33.6 66.4 D Lipoproteins 2.1 29.9 70.1 W Mitochondria 3.5 37.6 62.4 W Microsomes 3.4 37.8 62.2 W Lipoproteins 1.9 29.7 70.3 Sunflower oil D Mitochondria 6.5 37.9 62.1 D Microsomes 4.9 43.0 57.0 D Lipoproteins 2.2 36.9 63.1 W Mitochondria 5.6 35.9 64.1 W Microsomes 3.6 37.1 62.9 W Lipoproteins 2.9 31.4 68.6 1 Expressed as a percent of the subcellular membrane on a wet weight basis Expressed as a percent of total lipid 162 microsomes (78.0%) of rats, and in the mitochondria (88.8%) of rabbits have been reported (Fiehn and Perter, 1971: Waku and Uda, 1971). Other researchers have reported that more active muscles contain greater quantities of phospholipids (Kaucher et al., 1944; Bloor, 1943: Acosta et al., 1966). The lower phospholipid contents of membranes from broilers could be due to the nature of the species and to the lower activity of the muscles. The variation in lipid components may be also due, in part, to the crudeness of the subcellular membrane preparation. Fatty Apig Composition pf Membranal Neutrai Lipids Fatty acid composition data for the neutral lipids of the mitochondria and microsomes are presented in Tables 2 and 3. Seventeen fatty acids were identified in the neutral lipids of both the mitochondrial and microsomal membranes isolated from dark and white meat. In general, C16 fatty acids (palmitic C16:0 and palmitoleic C16:1) and C18 fatty acids (stearic C18:0, oleic C18:1, and linoleic C18:2) were the dominant fatty acids. The presence of oxidizd sunflower oil in the diet of broilers selectively affected the longer chain unsaturated fatty acids (e.g., C20:4) in the neutral lipids. The lower C20:4 (arachidonic acid) content in the oxidized oil group may be due to the conversion of this fatty acid to prostaglandins for the 163 Table 2 Percent fatty acid composition of the neutral lipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broilers Fatty Oxidized Short-term Long-term BHA/BHT Sunflower acid oil alpha- alpha- oil tocopherol tocopherol D W D W D W D W D W C12:0 - - 0.4 0.4 0.2 0.2 - - - - Cl4:0 0.5 0.6 0.3 0.8 0.6 0.3 1.0 0.4 0.3 0.4 C16 DMAa 1.5 0.8 2.7 1.4 1.3 2.7 3.4 3.0 3.5 2.9 C16:1 15.6 17.7 12.5 17.0 14.7 12.8 12.1 14.9 14.4 13.5 C17:0 2.5 2.8 3.1 2.3 2.9 2.1 1.7 1.8 1.8 1.6 C18 DMAa 0.2 0.2 0.7 1.2 1.0 2.1 0.9 1.4 0.9 1.4 C18:0 10.5 10.2 10.5 8.7 9.0 10.6 12.1 12.6 12.7 11.1 C18:1 21.3 22.7 19.4 22.8 22.2 18.7 15.7 17.7 16.2 18.6 C18:2 33.9 32.7 28.8 28.1 28.5 29.7 29.5 27.7 28.7 29.7 C18:3 - - 0.4 0.6 0.8 1.0 - - - - C20:0 - - 0.8 0.9 0.5 0.5 - - - - C20:2 1.6 1.5 1.0 2.0 0.8 1.5 1.3 2.3 2.8 2.3 C20:3 0.7 0.5 0.8 0.6 0.5 0.8 0.8 0.9 0.9 1.2 C20:4 6.4 5.5 11.6 9.0 11.8 9.2 11.1 8.7 10.1 8.4 C22:4 2.3 1.9 3.1 1.2 1.9 3.1 3.3 3.0 2.9 3.9 C22:5w6 2.1 2.4 1.2 1.3 0.6 1.4 2,3 1.3 1.0 1.5 C22:5w3 - - 0.7 1.0 0.6 0.9 1.9 2.2 1.0 1.5 C22:6 - - 0.5 0.3 1.4 0.9 1.2 1.2 1.0 1.1 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acids. 164 physiological defense system to cope with the oxidative stress in the broilers. Carpenter (1981) stated that lipid peroxides are produced during the enzymic conversion of arachidonic acid (C20:4) to prostaglandins, thromboxane, prostacyclin, and leukotrienes. In addition, the oxidized lipids in the broiler diet may be a source of free fatty acid radicals which may propagate peroxidative reactions in the membranal-bound lipids, consequently increasing the suceptibility of longer chain unsaturated fatty acids to oxidation. There is a paucity of data in the literature on the effect of oxidized oil on membranal lipid composition or stability. More studies are required to substantiate the observations that oxidized oil in the diet would influence the susceptibility of membranal lipids toward free radical attack. The fatty acid composition data for the lipoprotein fractions followed the same pattern as for the mitochondria and microsomes. These data are presented in Appendix A. There were measureable quantities of two dimethyl- acetal (DMA) derivatives of hexadecanal and octadecanal in the fatty acid profiles of the neutral lipids isolated from the mitochondrial and microsomal fractions. These results would indicate that the neutral lipids were contaminated by small amounts of phospholipids including plasmalogens, which would explain, in part, why the phospholipid 165 Table 3 Percent fatty acid composition of the neutral lipids isolated from microsomal membranes of dark (D) and white (W) meat of broilers Fatty Oxidized Short-term Long-term BHA/BHT Sunflower acid oil alpha- alpha- oil tocopherol tocopherol D W D W D W D W D W C14:0 0.4 0.5 0.9 0.3 0.3 0.3 0.4 0.3 1.1 0.5 C16 DMAa 2.9 5.7 4.1 4.3 2.7 2.6 2.4 2.9 1.9 2.3 C16:0 13.6 13.6 11.7 12.5 12.7 12.1 12.7 11.9 13.4 14.1 C16:1 1.6 2.6 2.0 1.5 1.9 1.6 1.8 1.4 1.7 1.2 C17:0 1.5 1.3 1.8 1.5 1.6 1.3 1.6 1.7 1.8 1.8 C18 DMAa 0.9 1.4 0.9 1.3 0.8 1.1 0.9 1.5 0.7 1.0 C18:0 13.7 14.5 13.6 13.3 13.1 12.9 12.7 12.9 12.9 13.8 C18:1 17.9 20.2 16.8 17.0 17.7 18.1 17.7 17.7 16.9 16.5 C18:2 29.3 30.3 27.7 26.5 29.8 29.5 29.8 28.6 30.1 29.5 C20:2 2.0 1.6 2.4 2.3 2.2 1.8 1.8 2.4 1.7 1.6 C20:3 2.9 3.3 1.0 1.5 0.8 1.2 1.2 1.4 1.3 1.3 C20:4 9.5 8.8 12.8 13.0 12.0 12.2 13.6 12.4 11.3 11.6 C22:4 2.9 1.1 2.2 3.1 2.7 2.8 2.9 3.4 3.0 2.0 C22:5w6 0.9 0.8 1.3 1.2 1.6 2.4 1.2 1.3 1.3 1.4 C22:5w3 t t 0.5 0.5 0.5 0.1 0.2 0.2 0.7 0.6 C22:6 t t 0.5 0.4 0.6 0.1 0.4 0.1 0.8 0.8 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acids. 166 percentages of the total lipids were lower than reported values for other animal species. MEM osi 'ongfmgnslglmflI—oliem The fatty acid data in Tables 4 and 5 indicate that the predominant fatty acids in the membranal phospholipids of dark and white meat of broilers were palmitic (C16:0), stearic (C18:0), oleic (C18:0), linoleic (C18:2), linolenic (C18:3 and arachidonic acids (C20:4). These fatty acids accounted for approximately 85% of the total fatty acids. The presence of oxidized oil, alpha-tocopherol and BHA/BHT in the diets of broilers did not significantly (P > 0.05) affect the fatty acid composition of the membranal phospholipids. In these analyses, dimethylacetal derivatives of hexadecanal and octadecanal in the phospholipids fraction of membranal lipids were again observed. The mechanism of their formation has been discussed in Chapter 1. The fatty acid composition for the lipoprotein fractions was similar to that of the mitochondria and microsomes. These fatty acid data are presented in Appendix B. Concentration pf Aipha-Tocopherol in Subceliular Membranes The data in Table 6 reveal that the alpha-tocopherol 1| [I‘ll] (I11! 167 Table 4 Percent fatty acid composition of the phospholipids isolated from mitochondrial membranes of dark (D) and white (W) meat of broiler Fatty Oxidized Short-term Long—term BHA/BHT Sunflower acid oil alpha- alpha- oil tocopherol tocopherol D w D w D w D w D w 012:0 - - - - 0.2 0.2 0.6 0.4 0.2 0.3 014:0 0.2 0.3 0.3 0.2 0.2 0.3 0.4 0.3 0.2 0.3 016 DMAa 5.3 5.9 4.8 6.0 5.1 5.7 4.4 5.8 4.4 5.6 016:0 16.0 22.7 19.6 20.1 14.6 20.9 18.6 21.3 19.0 20.7 016:1 0.4 2.2 0.1 0.5 0.1 0.4 0.4 0.5 0.5 0.6 017:0 1.8 2.0 1.1 1.9 1.9 1.6 1.4 1.7 1.4 1.7 018 DMAa 1.0 2.7 0.6 1.8 1.1 1.4 0.7 1.5 0.6 1.4 018:0 18.5 9.4 18.1 10.7 17.3 12.4 17.8 11.1 17.5 11.2 018:1 12.1 13.4 13.0 13.9 10.4 14.3 12.6 13.3 12.8 13.6 018:2 22.9 19.6 21.1 19.5 21.7 19.9 23.0 20.0 21.2 19.9 020:2 1.4 1.4 1.3 2.4 2.1 1.7 1.5 2.0 2.1 2.3 020:3 1.2 1.3 1.2 1.7 1.4 1.5 1.0 1.5 1.5 1.9 020:4 11.5 11.3 12.8 12.9 15.2 13.1 12.5 12.9 11.7 12.7 022:4 4.5 4.2 3.6 4.9 5.0 4.0 2.7 4.4 4.1 4.4 C22:5w6 1.7 1.7 1.3 1.7 1.8 1.4 1.1 1,9 1,4 1,6 C22:5w3 0.7 1.1 0.7 1.1 1.1 0.8 0.7 0.8 0.9 1.0 022:6 0.7 0.9 0.5 0.8 0.8 0.7 0.7 0.9 0.6 0.9 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydroglysis of plasmalogens. DMAs are not counted as fatty acids. 168 Table 5 Percent fatty acid composition of the phospholipids isolated from microsomal membranes of dark (D) and white (W) meat of broiler Fatty Oxidized thort-term Long-term BHA/BHT Sunflower acid oil alpha- alpha- oil tocopherol tocopherol D W D W D W D W D W C12:0 0.2 0.2 0.3 0.4 0.2 0.3 0.3 0.3 0.3 0.2 C14:0 0.2 0.3 0.3 0.5 0.2 0.3 0.2 0.4 0.2 0.3 C16 DMAa 5.3 6.5 5.3 5.4 5.3 6.5 5.5 6.0 5.0 5.9 C16:0 19.8 21.6 18.7 21.7 18.8 22.8 19.2 22.1 18.6 20.2 C16:1 0.7 0.7 0.6 0.7 0.5 0.7 0.2 0.5 0.5 0.7 C17:0 1.4 2.0 1.3 1.3 1.7 1.7 1.6 1.6 1.6 1.9 C18 DMAa 0.8 2.1 0.8 1.5 0.9 1.6 0.9 1.3 0.8 1.7 C18:0 16.2 9.5 16.6 9.5 16.3 10.5 16.9 11.1 17.2 9.8 C18:1 13.2 15.6 14.4 14.3 13.1 15.3 12.1 14.6 12.6 14.0 C18:2 22.8 21.8 21.7 19.6 20.9 19.2 21.7 20.1 21.4 19.6 C20:2 1.6 1.7 1.7 1.3 2.2 1.7 1.5 2.1 2.0 3.2 C20:3 1.2 1.2 1.1 1.3 1.1 1.2 1.1 1.4 1.5 2.1 C20:4 9.9 9.3 11.0 11.2 12.0 11.9 11.5 11.2 11.0 11.0 C22:4 3.6 4.5 3.5 3.3 3.7 3.7 4.1 4.2 4.2 5.4 C22:5w6 1.3 1.9 1.2 1.3 1.4 1.4 1.7 1.5 1.5 1.8 C22:5w3 0.7 0.5 0.8 1.1 0.7 0.7 0.8 0.6 0.9 1.2 C22:6 0.9 0.6 0.8 1.1 0.9 1.1 1.0 1.0 0.7 0.9 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acids. 169 concentrations in the mitochondrial, microsomal and lipoprotein fractions of meat from broilers receiving a basal diet were 0.86-2.07, 0.86-48.3, and 0.47-0.97 ug/g (wet weight basis), respectively. Most of the alpha- tocopherol derived from the basal diet was deposited in the microsomes of the dark meat. For example, the microsomes from the dark meat of broilers fed unoxidized sunflower oil contained 48.3 ug/g alpha-tocopherol, which was significantly (P < 0.01) higher than the level found in the oxidized oil-group (21.1 ug/g). The lower alpha-tocopherol content in the latter group can be explained, in part, on the basis that the membrane used the antioxidant to protect it against free radical attack. It is also possible that some of the alpha-tocopherol in the feed was destroyed by the presence of the highly oxidized sunflower oil. However, the broiler diets were prepared weekly and were stored in a cool room until fed to the broilers. Supplementation of the diet with alpha-tocopherol did not significantly (P > 0.05) increase the level of this antioxidant in the mitochondrial membranes isolated from dark and white meat when compared to similar membranes isolated from the control group. Only a small amount of alpha-tocopherol was found in the mitochondria of dark and white meat. The microsomal fraction from the white meat of the 170 Table 6 Concentration of alpha-tocopherol in the subcellular membrane of dark (D) and white (W) meat from broilers fed different dietary regimens Treatments Alpha-tocopherol (ug/g membrane, wet basis) Mitochondria Microsomes Lipoproteins D w D w D w Oxidized oil 1.18 0.43 21.14d 0.21 0.12c 0.01b Short-term 2.11 0.90 91.71b 1.42 1.32c 65.01a alpha-tocopherol Long-term 2.31 1.56 135.67a 4.01 70.41a 66.19a alpha-tocopherol BHA/BHT 0.78 0.68 24.85d 0.85 8.20b 0.26b Sunflower oil 2.07 0.86 48.30c 0.86 0.97c 0.47b a,b,c,d Mean alpha-tocopherol contents within columns bearing different superscripts differ significantly at P < 0.01. 171 long-term alpha-tocopherol-supplemented group contained only slightly higher alpha-tocopherol levels (4.0 ug/g) than microsomes from the control group (0.9 ug/g)(non significant difference). In contrast, the microsomes from dark meat of broilers fed the alpha-tocopherol supplement contained significantly (P < 0.01) higher levels of the antioxdant than the microsomes from the control (unoxidized sunflower oil) group, the concentrations being largely influenced by the duration of supplementation. Microsomes isolated from the dark meat of the short-term alpha- tocopherol-fed group contained 91.7 ug/g, while those from the long-term-fed group contained 135.7 ug/g membrane. The amounts of alpha-tocopherol in the lipoproteins of dark and white meat from the long-term alpha-tocopherol-fed group were 70.4 ug/g and 66.2 ug/g, respectively, which were significantly higher (P < 0.01) than the corresponding membranes from the control group. In the short-term alpha- tocopherol-fed group, only the lipoproteins isolated from the white meat showed significantly (P < 0.05) higher alpha-tocopherol concentrations (65.0 ug/g) (Table 6) than the corresponding fraction from the control group. Mowri et al. (1981) reported that rat liver contains an alpha-tocopherol-binding protein which plays an important role as a carrier for the transportaton of alpha- tocopherol from the lipoprotein to other membranes of the 172 cells. If the mechanism of transportation in muscle is the same as in the liver, the lipoprotein fraction might be serving as a pool for the temporary storage of alpha- tocopherol in the muscle. As alpha-tocopherol levels in microsomes or mitochondria reached their maixmum capacity, the overload of this antioxidant would be stored in the lipoprotein fractions. Supplementation of the broiler diet with BHA/BHT appeared to antagonise the deposition of alpha-tocopherol in the microsomes and mitochondria of dark meat, but had no effect on the subcellular systems from white meat. In contrast, the lipoproteins fraction isolated from dark meat of broilers fed BHA/BHT had 8.2 ug/g alpha-tocopherol, which was significantly (P < 0.01) higher than the control group (0.47 ug/g)(Table 6). The BHA or BHT might have competed with alpha-tocopherol for the structural position in the membrane, or their presence might have reduced the alpha-tocopherol requirement for the physiological defense system. As expected, membranes isolated from dark and white meat of broilers fed oxidized oil had only 25% to 50% of the alpha-tocopherol content of those from the corresponding control group (Table 6). Most of the alpha- tocopherol in the membranes seemed to be utilized in protecting the membranal lipids from free radical attack 173 originating from the ingestion of oxidized oil. Oxidapivg Stapiiity p; ngnpnnn§;§pnnd Lipid The rates of metmyoglobin/hydrogen peroxide-catalyzed lipid peroxidation in the mitochondrial and microsomal fractions isolated from the five groups of broilers are presented in Figures 1-4. Generally, oxidative changes were much more extensive in the subcellular membranes isolated from dark meat as compared to membranes from white meat. These results confirm the findings of Harel and Kanner (1985b) who reported a higher rate of lipid peroxidation in microsomes from dark muscle tissue of broilers compared to white muscle microsomes. The reason for these differences was not explained by these investigators. However, higher total lipid contents (Table 1) may contribute to the higher TBARS numbers for the membranes from the dark meat. When the metmyoglobin/hydrogen peroxide initiator (ratio 1:1) was added to the systems, all subcellular fractions from the oxidized oil-fed group (group 1) were more rapidly oxidized than those from the other groups. This indicated that the membranes from the dark and white meat of broilers fed oxidized oil were the least stable. As expected, membranes from the alpha-tocopherol and BHA/BHT groups had lower TBARS numbers than those from the 30" 25'“ .5 E O“ .5 203 8 .2 g: 1 23 50 g 15" '9. '8 . -o 13 = % 10" 2 l 2 E 5 0 f 0 Figure 1 174 —o— Short-Term Alpha-Tocopherol —-e— Lon g-Term Alpha-Tocopherol —+—— BHA/BHT -—I— Sunflower Oil '_' I I r I T I 30 60 90 120 150 180 Time (Minutes) Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in mitochondrial membranes from dark meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementation 175 10"--u-- (kafileCXl nmole Malonaldehyde/mg Mitochondria Protein -—o—— Short-Term Alpha—Tocopherol —o— Long-Term Alpha-Tocopherol —e— BHA/BHT —-I-— Sunflower Oil “:‘5,_—__—_ ‘ . Figure 2 l l 30 60 90 120 150 180 Time (Minutes) Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in mitochondrial membranes from white meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementation 176 6 “ —._ Short-Term Alpha-Tocopherol ‘ —u— Long-Term Alpha-Tocopherol 5 . —-e—— BHA/BHT —.— Sunflower Oil nmole Malonaldehydc/mg Microsomes Protein DJ - 0 I I I I F I I 30 60 90 120 150 180 Time (Minutes) Figure 3 Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in microsomal membranes from dark meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementation 177 ——-o— Short-Term Alpha-Toccpherol ‘ —-a-— Lon g—Term Alpha-TocOpherol .—o— BHA/BHT —I— Sunflower Oil nmole Malonaldehyde/mg Microsomes Protein 0 ' r l l I l r 30 60 90 120 150 180 Time (Minutes) Figure 4 Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation in microsomal membranes from white meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementation 178 control group (group 5). The oxidized fatty acids in the broiler diet might have been transferred to the membranes, which, combined with the low alpha-tocopherol content in the membranes, influenced the rapid peroxidative changes. The results of the Duncan multiple range test for TBARS values of metmyoglobin/hydrogen peroxide-catalyzed membranal lipid peroxidation of the dark and white meat of broilers are presented in Table 7. These data indicate that membrane-bound lipid peroxidation was significantly (P < 0.001) affected by the oxidized.oil and by alpha- tocopherol and BHA/BHT supplementations, as well as by different types of meat and membranes. The presence of oxidized oil in the broilers' diet significantly (P < 0.001) increased the rate of oxidative changes in membranes, whereas the alpha-tocopherol and BHA/BHT supplements slowed down the rate of oxidation in the membranes. Membranes isolated from dark muscles had significantly higher (P < 0.001) TBARS values than those of the membranes from white muscles. Oxidative trends in the mitochondria from the five broiler groups were similar to trends for the microsomes (Figures 1-4). Even though the mitochondria contained only marginally different alpha-tocopherol concentrations between groups (Table 6), they exhibited significantly (P < 0.01) different rates of peroxidation. The natural 179 Table 7 Oxidative stability of subcellular membranes isolated form dark and white meat of broilers fed different dietary regimens as expressed by mean of TBARS numbers (nmole malonaldehyde/mg protein) Treatments Oxidized Short-term Long-term BHA/BHT Sunflower (N=288) oil alpha- alpha- oil tocopherol tocopherol 5.16a 0.69C 0.54c 0.71C 1.37b Meat samples Dark meat White meat (N=720) 2.31a 1.07b Membranes mitochondria Microsomes (N=720) 3.29a 1.32b a,b.c Means with different letters are significantly at P < 0.001. 180 antioxidant, alpha-tocopherol, when incorporated into the subcellular membranes may have combined with thiol containing enzymes (glutathione reductase, glutathione peroxidase) to destroy the formation of hydroperoxides in the systems (Machlin, 1984). Franco and Jenkinson (1986) demonstrated that glutathione was effective in decreasing the rate of lipid peroxidation in microsomes from rats fed normal diets, while microsomes from rats fed vitamin E- deficient diets were more rapidly oxidized under the same peroxidative conditions. These results suggest that alpha- tocopherol and thiol-containing enzymes have a synergistic effect in preventing free radical attack on the membranes. Harel and Kanner (1985a,b) reported that low concentrations (1-10 uM) of alpha-tocopherol can inhibit microsomal lipid peroxidation in vitro when initiated by hydrogen peroxide-activated metmyoglobin. Similar levels of alpha-tocopherol can be incorporated into membranes by dietary means (Table 6) and this, in turn, can effectively depress membranal lipid peroxidation in yiyp. The mitochondria and microsomes isolated from dark meat and the lipoprotein fraction isolated from the white meat of broilers fed BHA/BHT underwent peroxidation at a significantly (P < 0.01) lower rate than corresponding membranes from the control group. Thus, BHA/BHT might have exerted their antioxidant function by being accumulated in 181 the membranes, or by preserving thiols or thiol-containing enzymes (glutathione reductase, glutathione peroxidase). Lipoprotein fractions isolated from the five groups of broilers exhibited similar peroxidative trends. These data are presented in the Appendix C (dark meat) and D (white meat). Generally, the trends in membranal lipid oxidation reflected the pattern of oxidative changes in meat during storage (Chapter 3). SUMMARY AND CONCLUSION It may be concluded that the incorporation of alpha- tocopherol or BHA/BHT into the membranes through dietary means would be held to stabilize the membrane-bound lipids. This, in turn, would effectively reduce lipid oxidation in whole meat during refrigerated and frozen storage. REFERENCES Acosta, S. 0., Marion, W. W. and Forsythe, R. H. 1966. Total lipids and phospholipids in turkey tissues. Poultry Sci. 45:169. Asghar, A. and Yeates, N. T. M. 1974. Systematic procedure for the fractionation of muscle protein, with particular reference to biochemical evaluation of meat quality. Agr. Biol. Chem. 38:1851. Asghar, A., Gray, J. I., Buckley, D. J., Pearson, A. M. and Booren, A. A. 1988. Perspectives in warmed-over flavor. Food Technol. 42:(in press) Bloor, W. R. 1943. "Biochemistry of The Fatty Acids." Van Nostrand-Reinhold. Princeton, New Jersey. Buege, J. A. and Aust, S D. 1978. Microsomal lipid peroxidation. In "Methods in Enzymology." S. Fleischer and L. Packer, eds. Vol. 52. p. 302. Academic Press, New York. Century, B. and Horwitt, M. K. 1959. Effect of fatty acids on chick encephalomalacia. Proc. Soc. Exp. Biol. 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In "Vitamins and Hormones." Vol. 20 P. 493. R. S. Harris and J. G. Wool, eds. Vladimirov, Y. A., Olenew, v. I., Suslova, T. B. and Cheremisina, I. P. 1980. Lipid peroxidation in mitochondrial membrane. Adv. Lipid Res. 17:173. Waku, K. and Uda, U. 1971. Lipid composition in rabbit sarcoplasmic reticulum and occurrence of alkyl ether phospholipids. J. Biochem. 69:483. Wyatt, D. M. and Sherwin, E. R. 1979. Analysis of BHA in polyethylene by direct-sampling gas chromatography. Food Technol. 33:46. Chapter V Lipid peroxidation in poultry meat. V. The influence of dietary oils and antioxidant supplementation on the oxidative stability of cooked meat during storage 185 186 ABSTRACT Experiments were designed to investigate the effects of different dietary oils and antioxidant supplementation on the oxidative stability of cooked meat during refrigerated storage. The rate of oxidation in cooked meat was significantly influenced by the fatty acid composition of the raw meat. Fatty acids of the omega-6 family influenced the formation of hexanal, while fatty acids of the omega-3 family were highly correlated with TBARS numbers and sensory panel scores in the cooked meat during storage. Inclusion of oxidized oil in the broiler diet caused extensive lipid oxidation and rapid development of warmed- over flavor in cooked meat during storage. Supplementation of the broiler diet with synthetic antioxidants such as BHA or BHT had a limited effect on the oxidative stability of cooked meats. However, alpha-tocopherol appeared to survive cooking and exert a positive influence on the oxidative stability of cooked meat during refrigerated storage. 187 INTRODUCTION Cooking meat brings about a number of changes including the conversion of ferrous iron in the prophyrin ring of the heme pigments to the ferric form (Younathan and Watts, 1959), the denaturation of hemoproteins (Banks, 1961; Eriksson et al., 1971), and the disruption of the muscle membrane system. The latter process results in the exposure of labile lipid components to oxygen and various catalytic species, including heme iron, nonheme iron, and other heavy transition metals (Younathan and Watts, 1959; Sato and Hegarty, 1971; Igene et al., 1979a). Asghar et al. (1988) have summarized the catalytic roles of heme and nonheme iron in cooked meat and how they accelerate the propagation phase of the oxidative process by generating free radicals from preformed lipid hydroperoxides. Homolytic decomposition of the hydroperoxides ultimately leads to the formation of short chain aldehydes, ketones, free fatty acids, and some polymers (Lundberg, 1962), all of which are believed to contribute to the oxidized flavor in cooked meat during storage (Bailey et al., 1980; Wu and Sheldon, 1988). As an example, hexanal is produced by the autoxidation of linoleic acid and can be used as a index of the degree of oxidation in cooked meat systems (Shahidi et al., 1987: Wu and Sheldon, 1988). 188 Since low thresholds of activation energy are involved in the development of oxidized flavors in foods (Gray, 1978), refrigeration or freezing of cooked meat is not particularly helpful in preventing oxidation. Although application of synthetic antioxidants in controlling warmed-over flavor (WOF) is a feasible alternative, these compounds can only be added to a limited number of meat products (Rhee, 1987). Thus, the meat industry is looking for alternatives which can effectively control WOF in cooked meat and simultaneously fullfil the safety requirement. Previous studies have shown that stabilization of the membrane-bound lipids in raw broiler meat through dietary means has a positive influence on the oxidative stability of meat during storage (Chapters 1-4). However, little information is available in the literature as to how these dietary treatments affect the oxidative stability of cooked meat. Therefore, the objective of the present study was to evaluate the effects of different dietary oils (including oxidized oil) and dietary antioxidant supplementation on the oxidative stability of cooked broiler meat during refrigerated storage. 189 MATERIALS AND METHODS Bnoiier Feeding Regimens The source of the broilers and the dietary treatments utilized in this study are described in Chapters 1 (feeding experiment 1) and Chapter 3 (feeding experiment 2). Cooking Progegune Three packages of raw dark and white meat from each dietary treatment were withdrawn from a freezer (-20°C) after 6 months storage and thawed overnight in a walk-in cooler (4°C). One hundred and fifty grams of the dark or white meat from each group of broilers were placed in Nasco Whirl-Pak bags (VWI Scientific Co., Bapavia, IL) and cooked in a thermostatically controlled water bath set at 75°C until the internal temperature of the meat reached 70-71°C. The meat was held at this final temperature for 5 min. Lipid Extraction Fnom Cooked Meat Lipids were extracted from the cooked meat samples utilizing the dry column method of Marmer and Maxwell (1981). "'3‘ m m (I) m (I) 190 memmmmm Both objective and subjective methods were used to assess the oxidative changes in cooked meat during storage. The objective methods included the determination of the hexanal content and 2-thiobarbituric acid-reactive substances (TBARS) numbers, and subjective evaluation was made by an untrained sensory panel. The samples were examined immediately after cooking (0 day) and after 1, 2, 'and 3 days of refrigerated (4°C) storage for sensory panel scores, and after 1 and 3 days for hexanal and TBARS determinations. Hexanal Leterminapion One hundred grams of cooked meat were weighed into a 2 L boiling flask, 750 ml of distilled water were added, and the flask was attached to a Likens-Nickerson extraction apparatus. Isopentane (25 ml) was used as the extracting solvent. The system was allowed to reflux for 6 hr. The solvent extract was dried by passing through anhydrous sodium sulfate (20 g), then concentrated under a nitrogen stream to a final volume of 1 ml. Extracts were stored in screw—cap vials (2 dram) at -20°C if not immediately analyzed. Hexanal in the extracts was analyzed using a Hewlett Packard 5890A gas chromatograph equipped with a 30 m x 0.25 191 mm o.d. silicon column (heliflex bonded fsot Suprerox) (Alltech Associates, Inc. Deerfield, IL). The temperature for the gas chromatographic analysis was programmed from 40°C (initial hold for 10 min) to 170°C at a rate of 10°C per min. Injection and detector temperatures were 200°C and 300°C, respectively. The split ratio was 20:1 and helium was used as the carrier gas at a flow rate of 15 ml/min. Hexanal was identified and quantitated by comparing the relative retention time and peak,area of a known amount of standard (Sigma Chemical Co., St Louis, MO) and reported as mg/kg meat. Thiobngpitunic Acid (TBA) Procedure The TBARS numbers in cooked meat were determined using the distillation procedure of Tarladgis et al. (1960), as described in Chapter 1. Sensony Lyaiuation pf Qooked Meat Six sensory panelists were recruited from staff personnel and graduate students. The panelists had previous experience in the sensory evaluation of poultry meat, with emphasis on warmed-over flavor (WOF). Training sessions were held to familiarize the panelists with the 192 evaluation procedures and scoresheet (Appendix E). The scoring system was adapted from the "Guidelines for Cookery and Sensory Evaluation of Meat” (American Meat Science Association, 1978). Anslxsisefzxperimentelw Experimental data were analyzed using a complete randomized block design (Steel and Torrie, 1980). Analysis of the data was conducted using group means. The correlation coefficients for the relationship between the hexanal content, sensory scores, and TBARS numbers, were also calculated using the FX-4000P CASIO program (Casio Computer Co., Ltd. Tokyo, Japan). Duncan’s multiple range test was used to estimate significant differences at the 5% probability level (Duncan, 1955). RESULTS AND DISCUSSIONS Lipid Composition 9; Cooked Meat Lipid composition data for the cooked meat are summarized in Table 1. The level of total, neutral and phospholipids did vary to some extent between groups. However, the neutral lipid/phospholipid ratio (N/P ratio) in cooked meat for all treatments decreased as compared to 193 Table 1 Lipid composition of cooked dark (D) and white (W) meat from broilers fed different dietary oil and antioxidants Feeding experiment #1 (Chapter 1) Coconut Olive Linseed HSBO + HSBOa oil oil oil alpha- tocopherol Total D 8.8 8.7 8.4 7.3 8.0b lipid w 2.9 2.9 3.0 2.7 2.5 Neutral D 79.4 82.7 84.4 82.4 83.4 lipid W 56.0 56.3 58.2 56.6 56.4 Phospho- D 20.6 17.3 15.6 17.6 16.4 lipid W 44.0 43.7 41.8 43.4 43.6 Feeding experiment #2 (Chapter 3) Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol Total D 7.2 8.0 7.3 7.2 7.1b lipid W 2.7 2.9 2.9 3.1 2.9 Neutral D 86.8 86.2 85.5 85.4 86.4 lipid W 63.0 62.8 62.9 62.6 63.5 Phospho- D 13.7 13.8 14.5 14.6 13.6 lipid W 37.0 37.2 37.1 37.4 36.5 a Partially hydrogenated soybean oil b Expressed as a percent of meat, means of three replicated analyses. 194 that in raw meat (Chapters 1 and 3). For example, the N/P ratio of cooked dark and white meat from broilers fed coconut oil was 3.85 and 1.27, while the N/P ratio in raw meat were 4.83 and 1.56, respectively (Chapter 1). This suggests that loss of neutral lipids in the form of drippings occurred during cooking whereas the phospholipid content was affected to a lesser degree. These results agree with the observation of Igene et al. (1979b) who reported that drippings from cooked broiler meat contained mostly water and neutral lipids. Qxidatixe Stability 21 922520 Dark and finite Meet During Refpigepnted Sporage The TBARS values of cooked meat from all groups generally increased over the storage period (Tables 2 and 3). This trend agrees with the findings of previous investigators (Younathan and Watts, 1959; Keller and Kinsella, 1973; Igene et al., 1979a). The rapid oxidation of lipids in cooked meat has been attributed to the exposure of membrane-bound lipids to oxygen as well as to the catalytic effects of non-heme and heme iron (Pearson et al., 1977). The TBARS numbers for the linseed oil-fed group increased from 1.65 mg/kg in the dark meat at 0 day to 5.02 mg/kg after 3 days of refrigerated storage (Table 2). These values were significantly higher (P < 0.05) than 195 Table 2 Effect of storage at 4°C on thiobarbituric acid- reactive substances (TBARS) numbers of cooked dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol (Feeding experiment #1) Days of Coconut Olive Linseed HSBO + HSBO storage oil oil oil alpha- tocopherol Mean 0 D 0.90 0.84 1.65 0.29 0.531 0.84x 1 D 0.93 0.97 3.41 0.39 0.71 1.27y 3 D 1.11 1.13 5.02 0.51 0.85 1.72y Mean 1.01b 0.98b 3.36a 0.400 0.70b Mean 0 w 0.48 0.31 1.20 0.19 0.301 0.50x 1 w 0.75 0.42 2.25 0.23 0.42 0.81Y 3 w 0.80 0.61 3.84 0.30 0.59 1.23y Mean 0.68b 0.45b 2.43a 0.24c 0.44b 1 Expressed as mg malonaldehyde/kg meat a,b,c TBARS numbers within rows bearing different superscripts differ significantly at P < 0.05. X'Y TBARS numbers within columns bearing different superscripts differ significantly at P < 0.05. 196 Table 3 Effect of sotrage at 4°C on the thiobarbituric acid-reactive substances (TBARS) numbers of cooked dark (D) and white (W) meat from broilers fed oxidized dietary oils and antioxidants (Feeding experiment #2) Oxidized Short-term Long-term BHA/BHT Sunflower alpha- alpha- oil tocopherol tocopherol Days of storage oil Mean 0 D 1.29 0.45 0.19 0.80 0.901 0.73X 1 D 6.19 1.89 0.54 2.07 2.54 2.65Y 3 D 7.96 2.47 0.97 3.71 5.75 4.172 Mean 5.17a 1.60c 0.57C 2.19bc 3.07c Mean 0 w 1.27 0.31 0.19 0.40 0.911 0.62x 1 w 4.73 0.81 0.60 1.03 2.91 2.02Y 3 w 5.18 1.23 0.70 1.74 4.57 2.68Y Mean 3.73a 0.78c 0.50C 1.06bc 2.80b 1 Expressed as mg malonaldehyde/kg meat. a,b,c TBARS numbers within rows bearing different superscripts differ significantly at P < 0.05. x,y,z TBARS numbers within columns bearing different superscripts differ significantly at P < 0.05. 197 corresponding values for the control group fed hydrogenated soybean oil (HSBO). On the other hand, TBARS numbers for dark meat from the alpha-tocopherol-fed group (0.40 mg/kg) were significantly lower (P < 0.05) than those for meat from the control group (0.70 mg/kg meat). Similar trends were observed for the cooked white meat, although the overall TBARS numbers were about 50% lower than the corresponding values for dark meat. The data in Table 2 also indicate that the cooked dark and white meat from the coconut-and olive oil-fed groups had slightly higher TBARS numbers, during storage than the meat from the control HSBO group. However, the differences were not significant. The higher total lipid content in the raw meat from these two groups (Table 1) may be responsible for the higher TBARS numbers. Cooked dark meat from broilers fed oxidized oil had a TBARS number of 5.17 mg/kg, whereas values for the cooked dark meat from the short-term and long-term alpha-tocopherol group and the control group were 1.60, 0.57 and 3.07 mg malonaldehyde/kg meat, respectively. TBARS numbers obtained for the oxidized oil-fed group were significantly higher (P < 0.05), and those from the antioxidant-supplemented groups were significantly lower (P < 0.05) than the corresponding values for the control (sunflower oil) group. Similar 198 trends were observed for the cooked white meat. Oxidized fatty acids in the broiler diet might be the source of the initial hydroperoxides and free radicals in meat, leading to an increase the susceptibility of cooked meat to oxidation during refrigerated storage. In contrast, supplementation of the broiler diets with alpha-tocopherol and BHA/BHT resulted in the deposition of these antioxidants in meat, thus decreasing the oxidative deterioration of cooked meat during storage. Two factors may account for the greater stability of meat from the alpha-tocopherol groups compared to the meat from the BHA/BHT fed group. Firstly, higher amounts of alpha-tocopherol were incorporated in the muscle than BHA/BHT (Chapters 1 and 3). Secondly, alpha-tocopherol is shown to survive cooking temperatures (Bunnell et al., 1965; Machlin, 1984). However, the cooked meat was not analyzed for its alpha-tocopherol content in this study. Hexanai Contents i Coo e Meat Hexanal concentrations in cooked meat from different groups are presented in Tables 4 and 5. Hexanal was present in significantly (P < 0.05) higher quantities in cooked meat from the coconut oil, olive oil and HSBO-fed groups as compared to meat from the alpha-tocopherol- supplemented group (Table 4). Cooked dark and white meat 199 Table 4 Hexanal concentrations (mg /kg meat) in cooked dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol Days of Coconut Olive Linseed HSBO + HSBO storage oil oil oil alpha- at 4°C tocopherol Mean 0 D 4.6 5.5 3.2 4.0 5.7 4.6x 1 D 8.4 7.8 5.1 5.1 9.0 7.2y 3 D 12.6 13.1 5.2 6.0 15.2 10.4z Mean 8.6a 8.8a 4.5b 5.6b 10.0a Mean 0 w 5.2 6.6 3.1 2.9 6.8 4.9x 1 w 9.0 8.3 3.2 3.4 7.0 6.2Y 3 w 10.0 10.8 4.9 4.9 10.0 12.5z Mean 8.1a 8.5a 3.7c 3.7C 8.8b a,b,c Hexanal numbers within rows bearing different superscripts differ significantly at P < 0.05. x,y,z Hexanal numbers within columns bearing different superscripts significantly at P < 0.05. 20) from the control (HSBO) broilers had the highest hexanal content (9.97 mg/kg and 8.75 mg/kg, respectively), while the linseed oil-fed group contained the lowest amount of hexanal (4.49 mg/kg and 3.37 mg/kg, respectively). Generally, hexanal concentrations in cooked dark and white meat increased during storage. Hexanal concentrations in the cooked dark meat were higher than those in the corresponding white meat samples. These '. results confirmed those of Love and Pearson (1976) who observed that the hexanal concentrations in the headspace over oxidizing cooked meat samples increased as the extend of oxidation increased. In feeding experiment #2 (Table 5), cooked meat from broilers fed long-term alpha-tocopherol supplements had significantly lower (P < 0.05) hexanal contents in both dark (5.2 mg/kg) and white meat (3.1 mg/kg) compared to meat from the control group, which were 10.8 and 9.0 mg/kg, respectively. The cooked meat from the BHA/BHT- supplemented group also had a lower hexanal content immediately after cooking. Hexanal production increased rapidly when the meat was held at refrigerated temperature for 1 day, presumably due, in part, to the low levels of BHA/BHT initially present and, in part, to presumed loss of these antioxidant during cooking. In contrast, cooked meat from the broilers fed oxidized oil contained the highest 201 Table 5 Hexanal concentrations (mg/kg meat) in cooked dark (D) and white (W) meat from broilers fed oxidized dietary oil and antioxidants Days of Oxidized Short-term Long-term BHA/BHT Sunflower storage oil alpha- alpha- oil at 4°C tocopherol tocopherol Mean 0 9.3 3.7 3.6 3.8 8.2 5.7x 1 15.4 9.1 5.8 12.7 10.4 10.7Y 3 15.8 10.9 6.1 13.2 13.7 11.9y Mean 13.5a 7.9C 5.2d 9.9b 10.8b Mean 0 7.4 2.2 1.2 2.4 7.8 4.2x 1 9.4 5.5 3.7 8.6 9.1 7.3yz 3 12.3 6.2 4.3 9.5 10.2 8.52 Mean 9.7a 4.6bc 3.1c 6.9b 9.0a a,b,c,d Hexanal numbers within rows bearing different superscripts differ significantly at P < 0.05. x,y,z Hexanal numbers within colimns bearing different superscripts differ significantly at P < 0.05. t 202 hexanal contents among samples examined, 13.5 mg/kg for dark meat and 9.7 mg/kg for white meat. In food systems containing substantial amounts of linoleic acid, the hexanal content is a good indicator of lipid oxidation (Frankel et al., 1981; Min and Kim, 1985; Shahidi et al., 1987). However, the hexanal content did not appear to be a good indicator of the extent of oxidation in meat from the linseed oil-fed group. Low hexanal contents were observed, even though high TBARS numbers were recorded for both raw and cooked meat. Since other omega-6 fatty acids such as C20:4, C20:5 and C22:5w6 are also precusors of hexanal, the low content of these fatty acids in meat from the broilers fed linseed oil (Chapter 1) would result in lower hexanal formation. The hexanal concentration in cooked meat and the total percentage of omega-6 fatty acids in raw meat were found to be positively correlated (r = 0.569). In contrast, the percentage of omega-3 fatty acids, i.e., C18:3, C22:5w3 and C22:6w3 (Chapter 1) in raw meat exhibited a negative correlation with hexanal concentration in cooked meat samples (r= -0.944, P < 0.05). Results from this experiment indicated that hexanal production and TBARS numbers of cooked meat from feeding experiment #1, with the exception of the linseed oil group (Tables 2 and 4), were highly interrelated (r = 0.978 in 203 the dark meat and r = 0.971 in the white meat, P < 0.05). For cooked meat from broilers from feeding experiment #2 (Tables 3 and 5), the correlation coefficients between hexanal contents and TBARS numbers were 0.953 for dark meat and 0.943 for white meat (P < 0.05). Shahidi et al. (1987) reported that hexanal contents were linearly correlated with TBARS values (r = 0.995, P < 0.05) for cooked pork loin stored at 4°C for 35 days. Therefore, it may be concluded that the hexanal content of cooked meat can reflect the oxidative state of meat in the early stages of storage. Sensogy Panel Sgopes fig; Cooked Meat Sensory panel scores for overall off-flavor in dark and white meats are presented in Tables 6 and 7. In the first feeding experiment (Table 6), panelists sniffed increasingly pronounced off-flavors in cooked meats with duration of storage at 4°C. The most significant increases in off-flavor in cooked meats were observed between 1 and 3 days in all samples. Although meat from the linseed oil- fed broilers had relatively low hexanal contents, these samples were rated as having more significant (P < 0.05) off-flavors (4.3 for dark meat and 4.0 for white meat) than control meat samples (3.8 for dark and 3.4 for white meat). Therefore, other volatile compounds such as 2- ./— 204 TAble 6 Sensory panel socres of cooked dark (D) and white (W) meat from broilers fed different dietary oils and alpha-tocopherol Days of Coconut Olive Linseed HSBO + HSBO storage oil oil oil alpha- at 4°C tocopherol Mean 0 D 2.60 2.50 4.00 1.65 2.25 2.60x 1 D 2.80 3.30 4.50 1.70 3.30 3.14y 3 D 4.70 4.55 6.00 2.80 4.50 4.502 Mean 3.37b 3.45b 4.83a 2.08c 3.35b Mean 0 w 2.85 2.75 3.75 1.50 2.40 2.56x 1 w 3.95 3.75 3.85 1.65 3.00 3.24x 3 w 4.85 4.25 5.00 3.00 4.00 4.22y Mean 3.88ab 3.56b 4.20a 2.05C 3.13b a,b,c Sensory panels scores within rows bearing different superscripts differ significantly at P < 0.05. X'Y Sensory panels scores within columns bearing different superscripts differ significantly at P < 0.05. Sensory panel scores: 1 = Undetectable off-flavor Trace off-flavor Slightly off-flavor Moderately off-flavor Pronounced off-flavor Very pronounced off-flavor mmbum II II II II II 205 pentylfuran (Smouse and Chang, 1967: Badings, 1970: Bailey et al, 1980), 2-enals (MacNeil and Dimick, 1970a,b), acetaldehyde, and dimethyl trisulfide (Wu and Sheldon, 1988) must also contribute to the development of off-flavor in cooked meat. In feeding experiment #2 (Table 7), cooked dark and white meat from broilers fed the oxidized sunflower oil had the highest off-flavor scores among the five treatments, whereas meat from broilers fed long-term with alpha- tocopherol had the lowest off-flavor scores. A significant difference (P < 0.05) between scores for meat from the short-term and long-term alpha-tocopherol supplementations was also observed, suggesting that tissue alpha-tocopherol concentrations (Chapter 3) also influenced the oxidative stability of cooked meats. Bartov et al. (1983) found no difference in sensory quality of breast and thigh meat from turkey fed with and without low level of vitamin E (40 mg/kg feed). These observations may be due, in part, to the low level of vitamin E in the tissue which reflected the low dietary level. Dark and white meat from broilers fed BHA/BHT had very low off-flavor scores immediately after cooking. However, off-flavor scores increased rapidly within 24 hr of refrigerated storage. This increase in off-flavor scores was highly correlated with the increase in hexanal 206 Table 7 Sensory panels scores of cooked dark (D) and white (W) meat from broilers fed oxidized dietary oil and antioxidants Days of Oxidized Short-term Long-term BHA/BHT Sunflower storage oil alpha- alpha- oil at 4°C tocopherol tocopherol Mean 0 D 3.75 1.90 1.85 1.95 3.40 2,57x 1 D 5.80 3.70 2.60 4.90 5.15 4.43Y 3 0 5.95 4.30 2.75 5.05 5.25 4.66Y Mean 5.17a 3.30c 2.40d 3.97b 4.60a Mean 0 w 3.10 1.40 1.05 1.45 3.25 2.05x 1 w 3.80 2.50 1.90 3.50 3.70 3.08Y 3 w 4.80 2.75 2.10 3.85 4.10 3.52Y Mean 3.90a 2.22b 1.68C 2.93b 3.68a a,b,c,d Sensory panels scores within rows bearing different superscripts differ significantly at P < 0.05. X'Y Sensory panels scores within column bearing different superscripts differ significantly at P < 0.05. Sensory panel scores 1 = Undetectable off-flavor Trace off-flavor Slightly off-flavor Moderately off-flavor Pronounced off-flavor Very pronounced off-flavor mtnwsuaw II II II II II 207 concentrations in the meat samples (r = 0.999 for both dark and white meat, P < 0.01). Webb et al. (1972) stated that 0.04% BHT in the diet did not significantly reduce rancidity development in precooked, frozen broiler parts and that the sensory panel could not distinguish the differences. Higher level of BHA or BHT in broiler diets may be required to inhibit the development of off-flavor in cooked meat. There is very little information in the literature regarding the effect of BHA or BHT supplementation on oxidative changes in cooked meat during storage. In the present study, the percentage of omega-3 fatty acids such as C18:3, C22:5w3, and C22:6 in raw meat (Chapters 1 and 3) were found to be highly correlated with sensory scores for all cooked meat samples (r = 0.952 for dark meat and 0.745 for white meat, P < 0.05). Results of this study indicate that the hexanal contents and sensory scores of cooked meats were highly correlated in the cooked dark and white meat of broilers from feeding experiment #1 (r = 0.930 and 0.973, respectively)(P < 0.05) and from experiment #2 (0.999 for both dark and white meat) (P < 0.01). The correlation coefficients of TBARS numbers and sensory scores for cooked meat from feeding experiment #1 were 0.954 (dark meat) and 0.929 (white meat), while cooked meat from feeding 208 experiment #2 were 0.906 and 0.947 (P < 0.01), respectively. These results indicate that hexanal contents, sensory scores, and TBARS numbers of cooked meat were interrelated. 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Relationship of meat pigments to lipid oxidation. J. Food Sci. 24: 734. SUMMARY AND CONCLUSIONS Two feeding trials were conducted to ascertain the effect of different dietary oils and antioxidant supplements on (1) the oxidative stability of dark and white meat (raw and cooked) of broilers during refrigerated (4°C) and frozen storage (-20°C), and (2) the oxidative stability of subcellular membranes (mitochondria, microsomes and lipoproteins) in dark and white muscle of broilers. The systems of NADPH/ADP/ ferric chloride and metmyoglobin/hydrogen peroxide were used to initiate membranal lipid peroxidation in these studies. Results indicated that dietary oils varying in their degree of unsaturation significantly (P < 0.01) affected the fatty acid composition of the neutral lipids and, to a lesser extent, the fatty acid composition of the phospholipids in dark and white meat. Antioxidants such as alpha-tocopherol, and BHA/BHT supplementation had no influence on the fatty acid composition of the neutral lipids or phospholipids, either in the total muscle or in the subcellular membranes. Supplementation of the broiler diets with alpha- tocopherol resulted in higher tissue alpha-tocopherol concentrations. Concentrations of alpha-tocopherol in the dark and white meat from broilers fed 200 mg/kg feed 212 213 supplements for 10 day (short-term), and 40 days (long- term) supplments were 8.9 mg and 5.8 mg/kg, and 10.6 mg and 6.6 mg/kg, respectively. These values represented an overall deposition of 0.68 to 1.01% of the total alpha- tocopherol consumed, of which the dark meat accounted for 0.39 to 0.62% and the white meat accounted for 0.28 to 0.40%, respectively. Similar trends were observed in the subcellular membranes, showing that tissue alpha-tocopherol concentrations reflected dietary levels. Microsomes isolated from the dark meat of broiler fed short-term alpha-tocopherol contained 91.7 ug/g, while those from the the group fed the long-term supplement contained 135.7 ug/g membrane. The levels of alpha-tocopherol in the lipoproteins were 70.4 ug/g (dark meat) and 66.2 ug/g (white meat) for the long-term alpha-tocopherol group, while lipoproteins from the white meat from broilers fed the short-term with alpha-tocopherol contained 65.2 ug/g. These values were significantly higher (P < 0.05) than the corresponding membranes from the control group. Results showed that dietary oils, BHA/BHT, and alpha- tocopherol supplementation significantly (P < 0.05) influenced the oxidative stability of dark and white meat, and the subcellular membranes isolated from the meat. It was primarily the fatty acid composition and, to a lesser 214 extent, the alpha-tocopherol concentration in broiler muscles which influenced the rates of peroxidation of the muscle lipids. However, alpha-tocopherol supplements appeared to be the more cost-effective way of retarding lipid peroxidation in broiler meat compared to feeding the more expensive oils (coconut oil and olive oil) to broilers. Oxidized sunflower oil in the broiler diet increased the susceptibility of membranes to metmyoglobin/hydrogen peroxide-initiated lipid peroxidation. The oxidized fatty acids in the broiler diet might have been transferred to the membranes, which, combined with the low levels of alpha-tocopherol in the membranes, influenced the rapid peroxidative changes. The rates of membrane lipid peroxidation were parallel to the rates of lipid peroxidation in the corresponding raw and cooked meat during storage. The amounts of polyunsaturated fatty acids, particularly those of the omega-6 (C18:2, C20:2, C20:3, C20:4, and C22:5w6) and omega-3 (C18:3, C22:5w3, and C22:6) families were significantly altered by different dietary oils. The total amount of omega-6 fatty acids in raw meat was correlated to the hexanal concentrations in the cooked meat, while the omega-3 fatty acid content in raw meat was directly related to the sensory panel scores and TBARS 215 numbers (r = 0.769 and 0.758, respectively, P < 0.05). Results of these studies provided evidence that lipid peroxidation in raw meat is initiated in the membrane-bound lipids. Alteration of the fatty acid composition of the membranal lipids as well as the introduction of an antioxidant such as alpha-tocopherol into them through dietary means, has a positive influence on the rates of membranal lipid peroxidation and this, in turn, was reflected in the oxidative stability of raw and cooked meat during storage. FUTURE RESEARCH NEEDS In spite of the numerous publications on the subject, many questions relative to the mechanism of lipid peroxidation in meat systems remain unanswered. For example, further knowledge for various forms of iron present in muscle is needed to more fully elucidate their contribution to lipid peroxidation. The term nonheme iron has been used most frequently in meat science to distinguish it from heme iron on the basis of solubility and precipitation by trichloroacetic acid, which can hardly be regarded as specific for the separation of heme and nonheme iron in meats. To follow the changes in "dialyzable iron" content during cooking of meat seems to be more meaningful than designating it nonheme iron. Clearly, more studies are needed to establish the relative significance of the different sources of iron in lipid peroxidation in meat systems. Recent data also indicate that peroxidation of endogenous lipids in biological tissues seems to involve both enzymic and nonenzymic reactions. However, studies are needed to further evaluate their relative roles in the peroxidation process. Similarly, more information is needed to clearly define the nature of the initiator of lipid peroxidation in raw muscle tissues. 216 APPENDICES 217 Appendix A Percent fatty acid composition of the nertral lipids isolated from lipoprotein fractions of dark (D) and white (W) meat of broilers fed oxidized oil and antioxidants Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha oil tocopherol tocopherol D W D W D W D W D W C14:0 0.4 0.7 0.4 1.1 0.4 0.4 1.1 0.7 0.5 0.4 C16 DMAa 1.8 1.5 1.6 1.4 1.5 1.1 1.5 1.2 0.7 0.8 C16:0 15.9 16.0 15.6 14.1 14.2 13.8 16.0 15.1 15.5 15.1 C16:1 2.9 3.0 2.7 2.2 2.2 1.9 2.7 3.5 2.9 2.6 C17:0 1.0 0.9 0.9 0.7 1.1 0.8 0.9 0.7 0.8 0.7 C18:0 13.7 14.5 13.6 13.3 13.1 12.9 12.7 12.9 12.9 13.8 C18:1 10.8 11.2 11.6 12.4 11.2 11.9 12.2 12.5 12.8 12.7 C18:2 31.4 32.8 33.7 34.0 32.4 32.5 32.7 31.1 32.8 33.7 C20:2 1.6 1.7 0.8 1.7 1.3 2.7 0.5 0.9 0.8 1.4 C20:3 0.7 1.1 1.0 1.0 0.6 1.5 0.3 0.1 1.8 1.0 C20:4 8.7 6.4 8.3 7.4 8.4 8.4 8.3 7.4 7.9 7.7 C22:4 2.5 1.0 1.1 0.9 2.2 1.9 0.5 2.9 1.1 2.0 C22:5w6 t t t t 1.8 1.5 0.8 1.2 t t C22:6 t t t t 0.5 0.5 1.1 1.3 t t a DMA = Dimethylacetal derivative of hexadecanal derived from acid hydrolysis of plasmalogens. DMA is not counted as a fatty acid. t = trace amound of fatty acid. 218 Appendix B Percent fatty acid composition of the phospholipids isolated from lipoprotein fractions of dark (D) and white (W) meat of broilers fed oxidized oil and antioxidants Oxidized Short-term Long-term BHA/BHT Sunflower oil alpha- alpha- oil tocopherol tocopherol D W D W D W D W D W C12:0 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.3 0.2 0.2 C14:0 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.2 0.4 0.3 C16 DMA 5.5 6.4 4.9 6.0 5.5 6.5 5.4 6.2 6.1 6.6 C16:0 18.9 21.1 16.9 20.2 16.6 21.0 17.0 19.9 17.1 22.2 C16:1 0.6 0.6 0.8 0.7 0.7 0.6 0.7 0.6 0.9 0.6 C17:0 1.6 1.8 1.8 2.0 1.9 2.0 2.1 2.0 2.0 2.0 018 DMAa 1.0 1.3 1.0 1.8 1.1 1.9 1.1 1.7 1.1 1.7 C18:0 16.1 11.0 14.8 10.4 16.0 11.3 15.4 10.9 16.6 11.7 C18:1 13.6 15.1 13.7 14.7 12.7 15.0 12.6 14.0 12.4 14.9 C18:2 23.2 20.7 22.1 18.8 22.0 19.0 22.1 18.9 21.4 19.0 C20:2 1.0 1.6 2.7 2.7 1.9 2.0 1.8 2.8 1.0 1.4 C20:3 0.9 1.4 1.5 1.9 1.3 1.5 1.2 1.9 1.2 1.5 C22:4 10.7 10.8 11.2 11.6 12.1 11.4 12.5 11.8 12.2 11.0 C22:5w6 1.4 1.6 1.6 1.7 1.6 1.5 1.8 1.8 1.7 1.5 C22:5w3 1.6 1.7 1.1 1.0 1.0 0.9 0.9 1.0 0.9 0.8 C22:6 t t 0.8 0.8 0.7 0.7 0.7 0.9 0.7 0.7 a DMA = Dimethylacetal derivatives of hexadecanal and octadecanal derived from acid hydrolysis of plasmalogens. DMAs are not counted as fatty acids. t = trace amount of fatty acid. 219 Appendix C Metmyoglobin/hydrogen peroxide-initiated lipid peroxidation nmole Malonaldehyde/mg Lipoprotein Protein in lipoprotein fractions from dark meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementations O .1. 1 Oil ——-0— Short-Term Alpha-Tocopherol . -—t— Long-Term Alpha-Tocopherol ——o—- BHA/BHT _n— Sunflower Oil 3 .. 2 d l - - . ...______—- v / 0 I I I I I I I 0 30 60 90 120 150 180 Time (Minutes) 220 Appendix D Metmyoglobin/hydrogen peroxide-initaiated lipid peroxida- nmole Malonaldehyde/mg Lipoprotein Protein tion in lipoprotein fractions from white meat of broilers fed oxidized dietary oil, alpha-tocopherol and BHA/BHT supplementations O .1. 1 Oil —*—— Short-Tenn Alpha-Tocopherol . ——a— Long-Term Alpha-Tocopherol ——e— BHA/BHT 4' —o— Sunflower Oil 3 4 2'1 1 1 cl . __ ..e_.._.__ Afi 0 I I I I I I I 0 30 60 90 120 150 180 Time (Minutes) 221 Appendix E Sensory panel score form for the evaluation of cooked broiler meat for off-flavor (warmed-over and oxidized flavor) NAME DATE Be certain you record the code numbers. Then, sniffing the sample, taste it if necessary, when you open the lid of sample package. Place a circle on the scale at the point you feel accurately describes the flavor of the sample. CODE Sensory panel score Undectable off-flavor Trace off-flavor Slightly off-flavor Moderately off-flavor Pronounced off-flavor Very pronounced off-flavor Chm-FLAME“ II II II II II II