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FINES wiII be charged if book is returned after the date stamped beIow. 1 MAY 3 Q 5123 I u‘ 163 OCT 182003 , 982593 . _..___,, ._ V Y '7§1.1.:r; T5315} 10 4 s _..___ l'llfl .I, ROLE OF MEAT LIPIDS AND MEAT PIGMENTS IN DEVELOPMENT OF RANCIDITY AND WARMED-OVER FLAVOR IN FROZEN AND COOKED MEAT By John Oamen Igene 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 1978 ABSTRACT ROLE OF MEAT LIPIDS AND MEAT PIGMENTS IN DEVELOPMENT OF RANCIDITY AND WARMED—OVER FLAVOR IN FROZEN AND COOKED MEAT By John Oamen Igene Experiments were designed to study the influence of total lipids, total phospholipids, individual phospho- lipids (PC and PE) and triglycerides on lipid oxidation and off-flavor development in frozen meat and cooked meat model systems. Experiments were also undertaken to explore the effect of length of frozen storage of whole meat on the stability of its constituent lipids and on development of TBA values. The effect of meat pigments, nitrite and non- heme iron on development of warmed-over flavor (WOF) in cooked meat was also studied. The 2-thiobarbituric acid (TBA) test and or taste panel scores were used to assess the extent of lipid oxidation in frozen stored and cooked meat samples. The compositional changes in the lipid content of the samples were evaluated using GLC and TLC techniques. The levels of heme and non-heme iron in extracted meat pigments were determined using atomic absorption spectrophotometry. John Oamen Igene Studies with frozen model meat systems showed that both triglycerides and phospholipids contribute to development of rancidity, although phospholipids make the greatest contribution. The influence of triglycerides in the develop- ment of rancidity in frozen meat model systems was shown to depend upon the degree of unsaturation and the length of frozen storage. The relationship between rancidity and oxidation of the polyunsaturated fatty acids (PUFAs) was confirmed, particularly in the phospholipids. Evidence is presented showing that both total phos— pholipids and PE are major contributors to development of WOF in cooked meat model systems. The triglycerides en- hanced development of WOF in the model meat system only when combined with the phospholipids. Phosphatidyl choline (PC) did not influence WOF in the model system. Changes in the PUFAs of the phospholipids were shown to be directly related to development of WOF, especially in PE. Results revealed that changes in total lipids during frozen storage of raw meat were largely due to losses in the triglyceride fraction. The phospholipid content of raw meat was relatively constant, irrespective of the length of freezer storage. Cooking significantly (P<0.0l) elevated the percentage of phospholipids in relation to total lipids and accounted for a significant (P<0.00l) in- crease in the rate of lipid oxidation. Cooked meat held at A°C for “8 hrs after cooking was more susceptible to develOpment of WOF than similar samples held at -18°C John Oamen Igene for A8 hrs. PE, PC, total phospholipids and their PUFAs were significantly less stable in cooked than in raw frozen meat. Thus, involvement of PUFAs in the develop- ment of WOF was verified. The stability of the different types of meat, either raw frozen or cooked was in the order of: beef > chicken white meat > chicken dark meat. Both removal of meat pigments and addition of 156 ppm of nitrite significantly (P<0.001) inhibited the development of TBA values in cooked meat. Taste panel evaluation confirmed the beneficial effects of removal of heme pigments and addition of nitrite to meat for con- trolling the development of WOF. Thus, results suggested that heme pigments may catalyze lipid oxidation. The percentage of bound heme iron in fresh meat pig- ment extract was slightly over 90% while the level of free non-heme iron was less than 10%. Cooking, however, released a significant amount of non-heme iron from bound heme pigments, which accelerated lipid oxidation in cooked meat. Thus, the rate of lipid oxidation in cooked meat is due in part to release of non-heme iron during cooking which then catalyzes lipid oxidation. Although earlier studies have suggested that myoglobin may catalyze lipid oxidation, this study showed that pigments pg£_§g_do not greatly accelerate the development of WOF, but serve as a source of non-heme iron in cooked meat. Thus, results showed that non-heme iron was the major pro-oxidant in the development of WOF in cooked meat. Addition of 2.0% EDTA John Oamen Igene effectively chelated the non-heme iron, and thus, sig- nificantly reduced lipid oxidation. To my elder brother, Benedy 0. Igene, his wife, Mrs. D. Igene and their children, Obayando Victor Ideyenmi Ebosereme Oiyimhebedan ("Mom") 11 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation and thanks to his major professor, Dr. A. M. Pearson, for his guidance and involvement in this study and for his assistance and patience in preparing the Dissertation. Sincere appreciation is expressed to Dr. L. L. Bieber, Dr. L. R. Dugan, Jr., Dr. P. Markakis and Dr. J. F. Price for serving on the guidance committee. Special thanks are expressed to Dr. R. A. Merkel, Dr. T. H. Coleman and Mr. J. R. Anstead for their cooperation and assistance in obtain- ing the meat and poultry used in this study; to Dr. J. L. Gill and Dr. R. R. Neitzel for their assistance in statisti- cal design and analysis of data. Thanks are also expressed to Continental Diversified Industries, Chicago, IL for donat- ing the retortable pouches used for packaging the samples in this study. The author is especially grateful to his entire family for their continuous understanding and encouragement through- out his course of studies and lastly to the Bendel State Government of Nigeria for sponsoring his studies at Michigan State University. 111 TABLE OF CONTENTS Chapter Page LIST OF TABLES. . . . . . . . . . . . . . . . . . 1x LIST OF FIGURES . . . . . . . . . . . . . . . . . xiv INTRODUCTION. 1 LITERATURE REVIEW . . . . . . . . . . . . . . . A Introduction. . . . . . . . . . . . . . . . A Distribution of Animal Fats A Fatty Acid Composition. . 5 Phospholipids in Animal Tiesues 7 Fatty Acid Composition of the Phospholipids . . . . . . . . . . . . . . . . . 11 The Role of Lipids in the Develop- ment of WOF . . . . . . . . . . . . . . . . . 13 Role of Lipids on Storage Stability of Frozen Meats . . . . . . . . . . . lA Mechanism of Lipid Oxidation. . . . . . . . . . 15 Products of Lipid Oxidation . . . . . . . . . . l7 Catalysts of Lipid Oxidation. . . . . . . . . . 19 Influence of Grinding and Cooking . . . . . . . . . . . . . . . . . . 21 Control of Lipid Oxidation. . . . . . . . . . . 23 EXPERIMENTAL. . . . . . . . . . . . . . . . . . . . 25 Solvents and Chemicals. . . . . . . . . . . . . 25 Methyl Ester Mixtures . . . . . . . . . . . . . 25 TLC Standard Mixture. . . . . . . . . . . . . . 25 Thin-Layer Plates . . . . . . . . . . . . . . . 26 Gas- -liquid Chromatography Column Packings . . . . . . . . . . . . . . . 26 Source of Meat. . . . . . . . . . . . . . . . . 26 iv Chapter Page Methods . . . . . . . . . . . . . . . . . . . . . 27 Meat for Evaluating the Effect of Frozen Storage on Lipids and Their Role in the Development of Oxidized Flavor. . . . . . . . . . . . . . . 27 Preparation of Meat for Rancidity of WOF Evaluation . . . . . . . . . . . . . . . 27 Methods of Analyses . . . . . . . . . . . . . . . . 28 TBA Test. . . . . . . . . . . . . . . . . . . . 28 Taste Panel Evaluation of WOF in Cooked Meat. . . . . . . . . . . . . . . 29 Extraction of Lipid from Muscle Tissue. . . . . . . . . . . . . . . . . . . . . 29 Drippings from Cooked Meat. . . . . . . . . . . 30 Thin-Layer Chromatography (TLC) o o o o o o c o o o o o o o o o o o o 0 3O Separation of Phospholipid Components. . . . . . . . . . . . . . . . . 30 Quantitation of Phospholipids (Phosphorus Determination). . . . . . . . . 31 Purification of PC and PE . . . . . . . . . 32 Preparation of Methyl Esters. . . . . . . . 32 Gas-Liquid Chromatography (GLC) . . . . . . . . 32 Statistical Treatment . . . . . . . . . . . . . 33 Experimental Systems. . . . . . . . . . . . . . . . 3“ Experiment A. . . . . . . . . . . . . . . . . . 3A Experiment B. . . . . . . . . . . . . . . . . . 3A Experiment C. . . . . . . . . . . . . . . . . . 3“ Experiment D. . . . . . . . . . . . . . . . . . 35 Preparation of Model Meat System. . . . . . . . . . . . . . . . . . . . . 35 Design of Experimental Treatments Involving Model Systems . . . . . . . . . . . . 37 Experiment A. . . . . . . . . . . . . . . . . . 37 Experiment B. . . . . . . . . . . . . . . . . . 39 Chapter Page Experiment C. . . . . . . . . . . . . . . . . . A0 Experiment D. . . . . . . . . . . . . . . . . . Al RESULTS AND DISCUSSION. . . . . . . . . . . . . . . A6 Role of Meat Lipids on the Development of Rancidity. . . . . . . . . . . . . . . . . . . . A6 Beef Lipids . . . . . . . . . . . . . . . . . . A6 Chicken Dark Meat Lipids. . . . . . . . . . . . 51 Chicken White Meat Lipids . . . . . . . . . . . 55 Stability of Phospholipids During Frozen Storage. . . . . . . . . . . . . . . . . 58 Phosphatidyl Ethanolamine (PE). . . . . . . . . 58 Phosphatidyl Choline (PC) . . . . . . . . . . . 61 Total Phospholipids (TP). . . . . . . . . . . . 61 Changes in Fatty Acid Composi- tion Due to Storage . . . . . . . . . . . 62 Beef Triglycerides. . . . . . . . . . . . . . . 62 Chicken Dark Meat and White Meat Triglycerides . . . . . . . . . . . . . . . . 6A Stability of Fatty Acids in Total Phospholipids . . . . . . . . . . . . . . . 66 Changes in the Fatty Acid Profiles of PC and PE . . . . . . . . . . . . . . . . . . 69 Summary of Results. . . . . . . . . . . . . . . . . 73 Experiment B. . . . . . . . . . . . . . . . . . . . 75 Role of Triglycerides, Total Lipids and Total Phospholipids on Development of WOF in Cooked Meat Model Systems . . . . . . 75 Changes in Phospholipid Components Due to Cooking and Storage at A°C . . . . . . . 79 Changes in Fatty Acid Composition as a Result of Cooking . . . . . . . . . . 82 Triglycerides . . . . . . . . . . . . . . . 82 Total Phospholipids . . . . . . . . . . . . 82 Changes in PC and PE in Beef Phospholipids . . . . . . . . . . . . . . . 86 vi Chapter Page Changes in PC and PE in Chicken Dark Meat 0 O O O O O I O O O O O O 0 O O O O 0 88 Changes in PC and PE of Chicken White Meat. 0 O O O O O O O O O 0 O 0 O O O O O 88 The Importance of Phospholipids on Development of WOF. . . . . . . . . . . . . . . 91 Summary of Results. . . . . . . . . . . . . . . . . 97 Experiment C. . . . . . . . . . . . . . . . . . . . 99 Effect of Length of Frozen Storage on the Stability of Meat Lipids and on Production of Off-Flavors. . . . . . . . . . 99 Levels of Extracted Lipids From Muscle Tissues. . . . . . . . . . . . . . . 99 Beef Lipids . . . . . . . . . . . . . . . . .100 Chicken Dark Meat Lipids. . . . . . . . . . . .102 Chicken White Meat Lipids . . . . . . . . . . .106 Effect of Frozen Storage on the Levels of Extractable Lipids . . . . . . . . . . . . .109 Composition of Lipids in the Drippings. . . . . . . . . . . . . . . . . .110 Composition and Stability of Phospholipid Components . . . . . . . . . . . .112 Lysophosphatidyl Choline (LPC). . . . . . . . .112 Phosphatidyl Ethanolamine (PE). . . . . . . . .115 Phosphatidyl Choline (PC) . . . . . . . . . . .116 Total Phospholipids (TP). . . . . . . . . . . .117 Phospholipid Components in the Drippings . . . . . . . . . . . . . . . .120 Changes in Fatty Acid Composition of Lipids During Frozen Storage and Cooking. . . . . . . . . . . . . . . . . 120 Triglycerides. . . . . . . . . . . . . . ... . 120 Changes in the Fatty Acid Composition of Total Phospholipids . . . . . . . 127 Beef Phospholipids . . . . . . . . . . . . 127 Chicken Dark Meat Phospholipids. . . . . . 129 vii Chapter Chicken White Meat Phospholipids. Fatty Acid Composition of Lipids from Drippings. . . . . . . . . . . Changes in the Fatty Acid Profiles of PC and PE During Frozen Storage and Cooking of Meat . . . . . . . . . Beef. . . . . Chicken Dark Meat . . Chicken White Meat. Effect of Frozen Storage of Meat in the Raw State on its TBA Value Following Cooking and Holding at A°C or -18°C for A8 Hrs. . . . . . . . . . . . Beef. . . . Chicken Dark Meat Chicken White Meat. Summary of Results. . Experiment D. . . . . . . . . . . . Influence of Heme Pigments, Non-Heme Iron and Nitrite on Development of WOF in Cooked Meat. Changes in TBA Numbers and Taste Panel Scores. . . . . . . . . . . . . Role of Heme and Non-heme Iron as Pro- oxidants of Lipids Oxidation. Summary of Results. SUMMARY AND CONCLUSIONS . . BIBLIOGRAPHY. . . . . . . . . . . . . viii Page 131 13A 136 136 1A0 1A3 1A7 1A7 151 152 163 167 167 172 173 181 18A 187 Table 10 11 LIST OF TABLES Design of Model Meat Experimental Treatments . . . . . . . . . . . . . . Design and Formulation of Treatments . . . . . . . . . . . . . . . Experimental Treatments to Compare the Effect of Heme and Non—heme Iron on the Development of WOF. . . The effect of adding beef triglycerides total phospholipids and total lipids on TBA values of model beef meat systems stored at -l8°C. . . . . . . . . . Effect of adding chicken dark meat tri— glycerides, total phospholipids and total lipids on TBA values of chicken dark meat model meat systems stored at -18°C . . . . Effect of adding chicken white meat triglycerides, total phospholipids and total lipids on TBA values of chicken white meat model meat systems stored at —18°C Changes in individual and total phospho- lipids during frozen storage (—18°C) of beef and chicken model meat systems. Changes in the fatty acid composition of triglycerides during frozen (-18°C) storage of model meat systems (0-8 months). Changes in the fatty acid composition of total phospholipids during frozen storage (-l8°C) of model meat systems (0-8 months). . . . . . . . . . . . . Changes in the fatty acid composition of PC during frozen storage (-l8°C) of model meat systems (0-8 months). . . Changes in the fatty acid composition of PE during frozen storage (- 18°C) of model meat systems (0- 8 months). . . . . ix Page 38 A3 A5 A7 52 56 59 63 67 71 72 Table Page 12 TBA numbers and sensory scores for cooked beef chicken dark meat and white meat model systems. . . . . . . . . . . . . . . . 76 13 Mean changes in phospholipids due to cooking and storage at A°C . . . . . . . . . 80 1A Mean fatty acid composition of tri- glycerides in model meat systems . . . . . . 83 15 Changes in the fatty acid composition of total phospholipids of meat model systems due to cooking and storage at “0C. 0 I O O O O O O O O O O O O O 0 O O 0 O Bu 16 Changes in fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine of beef model system. . . . . . 87 17 Changes in fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine of dark meat model systems. . . 89 18 Changes in fatty acid composition of phosphatidyl choline and phoSphatidyl ethanolamine of white meat model system . . . . . . . . . . . . . . . . . . . 90 19 The influence of PC, PE, total serum phospholipid and nitrite on develop- ment of WOF using beef model system. . . . . 93 20 TBA numbers and sensory scores for cooked meat model systems containing added PC, PE, serum phospholipids or nitrite. . . . . . . . . . . . . . . . . . . 95 21 Effect of length of frozen storage, cooking and storage temperature upon beef lipids. . . . . . . . . . . . . . . . . 101 22 Effect of length of frozen storage, cooking and storage temperature upon chicken dark meat lipids . . . . . . . . . . 10A Table Page 23 Effect of length of frozen storage, cooking and storage temperature upon chicken white meat lipids. . . . . . . . . . 107 2A Composition of lipids in cooked drippings. . 111 25 Effect of cooking, storage, temperature and length of frozen storage on the stability of the individual phospholipid components in beef, chicken dark and white meat (combined). . . . . . . . . . . . 113 26 Changes in the fatty acid composition of beef triglycerides during frozen storage and cooking. . . . . . . . . . . . . . . . . 123 27 Changes in the fatty acid composition of chicken dark meat triglycerides during frozen storage and cooking . . . . . . . . 125 28 Changes in the fatty acid composition of chicken white meat triglycerides during frozen storage and cooking . . . . . . . . . 126 29 Changes in the fatty acid composition of beef phospholipids during frozen storage and cooking. . . . . . . . . . . . . . . 128 30 Changes in the fatty acid composition of chicken dark meat phospholipids during frozen storage and cooking . . . . . . . . 130 31 Changes in the fatty acid composition of chicken white meat phospholipids during frozen storage and cooking . . . . . . . . . 132 32 Fatty acid composition of the triglycerides and phospholipids obtained in the cooked drippings from beef and chicken dark meat. . 135 33 Changes in the fatty acid composition of phosphatidyl choline (PC) in beef phos- pholipids during frozen storage and cooking. . . . . . . . . . . . . . . . . . 137 3A Changes in the fatty acid composition of phosphatidyl ethanolamine (PE) in beef phospholipids during frozen storage and cooking. . . . . . . . . . . . . . . 139 xi Table 35 36 37 38 39 A0 A1 A2 A3 AA A5 Page Changes in the fatty acid composition of phosphatidyl choline (PC) in chicken dark meat phospholipids during frozen storage and cooking. . . . . . . . . . . . . 1A1 Changes in the fatty acid composition of phosphatidyl ethanolamine (PE) in chicken dark meat phospholipids during frozen storage and cooking . . . . . . . . . 1A2 Changes in the fatty acid composition of phosphatidyl choline (PC) in chicken white meat phospholipids during frozen storage and cooking. . . . . . 1AA Changes in the fatty acid composition of phosphatidyl ethanolamine (PE) in chicken white meat phospholipids during frozen storage and cooking. . . . . . . . . . . . . 1A5 Effect of length of frozen storage at —l8°C and cooking on the level of TBA numbers in beef (LD). 0 O O O O O O I O O O O O O O 0 O 1119 Effect of length of frozen storage at -18°C and cooking on the level of TBA numbers in chicken dark meat . . . . . . . . 152 Effect of length of frozen storage at -l8°C and cooking on the level of TBA numbers in chicken white meat. . . . . . . . 155 Influence of frozen storage of meat in the raw state on TBA values of the cooked meat prior to holding at A°C or -18°C for A8 hrs . . . . . . . . . . . . . . . . 158 Levels of lipids and moisture in meat samples (% fresh tissue) . . . . . . . . . . 169 Summary of fatty acid composition of the triglycerides in beef, chicken dark and white meat . . . . . . . . . . . . . . . . 170 Summary of fatty acid composition of the total phospholipids in beef, chicken dark and white meat . . . . . . . . . . . . . . . 171 xii Table A6 A7 A8 Mean TBA numbers and sensory scores in cooked beef, chicken dark meat and white meat (A replicates for each treatment) Role of heme and non-heme iron on the development of TBA numbers in cooked beef . . . . . . . . . . . . Concentrations of total iron, heme iron and free non-heme iron in treated and un— treated meat pigment extract . . . . . . xiii Page 173 177 180 Figure 10 11 LIST OF FIGURES Structure of the diacylglycerophospho- lipids O O O O O O O I O O O O O O O 0 Preparation of model meat system Preparation and design of experiment to compare the effect of heme and non-heme- iron on the development of WOF Effect of adding beef triglycerides, total phospholipids and total lipids on TBA values of model beef meat systems stored at -18°C. . . . . . . . . Effect of adding chicken dark meat tri- glycerides, total phospholipids and total lipids on TBA values of chicken dark meat model meat systems stored at -l8°C . . Effect of adding chicken white meat tri- glycerides, total phospholipids and total lipids on TBA values of chicken white meat model meat systems stored at -l8°C Influence of storage at -l8°C on the stability of phospholipids in meat model systems. . . . . . . . . . . . . . . . TBA number and sensory scores in cooked beef model meat systems containing purified phospholipids and nitrite. . . . . . . Influence of length of frozen storage and cooking on the levels of beef lipids . . . . . . . . . . . . . . . . Influence of length of frozen storage and cooking on the levels of chicken dark meat lipids . . . . . . . . . . . Influence of length of frozen storage and cooking on the levels of chicken white meat lipids. . . . . . . . . xiv Page 36 AA 50 5A 57 60 9A 103 105 108 Figure Page 12 Influence of frozen storage and cooking on the composition and stability of LPC, SP, PE, PC and total phospholipid phosphorus (TP) in meat. . . . . . . . . . . 11A 13 Effect of cooking on qualitative changes of PC and PE on thin layer plates. A represents total phospholipids from fresh uncooked beef tissue, while B shows the total phospholipids from cooked beef. Note the changes in size of the spots for PC and PE. . . . . . . . . . . . . . . . 119 1A Thin layer plate showing the composition of the phospholipid components in the drippings of cooked meat. A and C represent total phospholipids from cooked beef drippings. B shows a standard mixture containing LPC, PC, PE and cholesterol (CHL).. X indicates position of the missing PE in the cooked drippings. . . . . . . . . . . . . . . . . . 121 15 Influence of the length of storage at -18°C on the TBA number of beef muscle . . . 150 16 Influence of the length of storage at -18°C on the TBA number of chicken dark meat muscle. . . . . . . . . . . . . . . . . 153 17 Influence of the length of storage at -l8°C on the TBA number of chicken white meat muscle. . . . . . . . . . . . . . . . . 156 18 TBA number of cooked meat measured within 1 hour following cooking . . 159 XV INTRODUCTION In recent years, the marketing of precooked or par- tially cooked meat and meat products and frozen raw meats for consumer convenience has become an accepted procedure. An inherent problem associated with precooked or partially cooked meat products is development of oxidized flavors. The development of oxidized flavors associated with cooked, uncured meat and meat products becomes apparent in a matter of hours and is principally due to oxidative degra- dation of lipids. Control of the resulting off—flavors, which have been aptly called warmed-over flavor (WOF) by Tims and Watts (1958), is related to the degree of lipid unsaturation. Even though lipid degradation has been associated with the development of undesirable flavors, a thorough understanding of the role of meat lipids in the production of off-flavors, and particularly for WOF has been lacking. Although phospholipids have been indirectly implicated as r“ being the major contributors to WOF (Wilson, et al., 1976; Younathan and Watts, 1960), the relative contributions of total and individual phospholipids and of total lipids and triglycerides to WOF have not been fully studied. It is well known that raw fresh frozen meats gradually become oxidized during freezer storage, with the slow development of off-flavor being described as rancid (Greene, 1969). The essential difference between rancidity and WOF is that the latter becomes apparent within a few hours following cooking and storage, and especially following reheating, as compared to months or even years for normal rancidity. Keller and Kinsella (1973) have suggested that oxidation of the triglycerides may be the major factor involved in the deterioration of meat during freezer storage. On the other hand, Cadwell et a1. (1960) and Greene (1971) have reported that changes in the phospholipids during frozen storage of raw meats results in rancidity and brown— ing. Thus, the primary objective of this research was to determine the role of meat lipids in the development of rancidity during freezer storage and of WOF in cooked meats. First, it was necessary to develop a model meat system consisting of lipid-free muscle fibers as base material to which each lipid component could be added for testing their role in the development of off-flavors associated with rancidity or WOF in both fresh and cooked meat. Knowledge gained from the model system should pro- vide a good understanding of the behavior of lipids in intact meats. In addition, it was deemed desirable to evaluate how variation in the composition of the triglycerides, total lipids and phospholipids influences oxidative changes in fresh and cooked meats. To this end, beef, chicken dark meat and chicken white meat were stored at -l8°C. At designated time intervals, packages of meat were removed from the freezer and analyzed to determine the role of different lipid components in development of off-flavors. Specifically, the study was designed to determine the role of the following treatments on lipid oxidation in meats: (a) To evaluate the changes that occur in the lipids of intact raw meats stored at -l8°C; (b) To test the stability of raw meats previously stored at -l8°C and then cooked and stored at —18°C or at +A°C for A8 hours; (c) To determine the influence of frozen storage of raw meats held at -18°C in relation to develop- ment of WOF; and (d) Finally, to evaluate the effect of meat pigments and nitrite on the development of WOF. LITERATURE REVIEW Introduction The flavor of meat and meat products is greatly in- fluenced by the lipid constituents (Ory and St. Angelo, 1975). No other characteristic of meat, except possibly tenderness, is so important to consumer acceptance as flavor (Doty, 1961). It is well established that lipid degradation is associated with the development of undesir- able flavors and odors in meat and meat products (Watts, 195A; Hornstein gt 11., 1961). Thus, the present review will discuss the composition of lipids in meat and poultry and their role in flavor problems associated with heat processed meats. In addition, pro-oxidants and antioxi- dants will be reviewed in relationship to development of rancidity and warmed—over flavor (WOF). Distribution of Animal Fats Watts (1962) and Love and Pearson (1971) have reviewed the composition of animal fats. They classified meat lipids as depot or adipose tissue and as intramuscular or tissue lipids. They pointed out that depot fats are largely localized as subcutaneous deposits, although large quanti- ties may be present in the thoracic and abdominal cavities and between the muscles as intermuscular deposits. In addition, the depot fats consist largely of triglycerides and are deposited essentially as fat globules within the individual cells (Watts, 1962). Moreover, the triglycerides vary greatly in amount and composition according to the species, ration, environment, sex and other factors (Watts, 1962). In contrast, tissue lipids (mainly phos- pholipids) are relatively constant in proportion and are an integral part of various cellular structures, such as the cell wall (Kono and Colowick, 1961), the mitochondria (Holman and Widmer, 1969) and the sarcoplasmic reticulum (Newbold et al., 1973). Fatty Acid Composition Natural animal fats are composed principally of the straight chain even numbered carbon fatty acids, typically containing 16 and 18 carbon atoms (Dugan, 1971). According to Hilditch and Williams (196A), the most abundant and commonly occurring fatty acid in animal fat is oleic acid. They also stated that other unsaturated fatty acids that are widely distributed, though not uniformly among animals, include linoleic and palmitic. They also reported that saturated fatty acids constitute about one third of all fatty acids in animal fats, with palmitic acid being the most abundant and seldom being absent. Stearic acid is the next most abundant fatty acid and is also seldom absent (Hilditch and Williams, 196A). In monogastric animals, dietary fatty acids are usually reflected in the composition of the depot fat (Cook 32 al., 1971). In ruminants, on the other hand, the depot fats are not influenced to any great extent by diet (Shorland gt 31., 1957). Most diets of animals usually contain linoleic and linolenic acids (Gunstone, 1967). By elonga— tion and desaturation, these two fatty acids provide the C20 and C22 polyunsaturated fatty acids of animal phos- pholipids (Gunstone, 1967; Poukka and Oksanen, 1972). Sprecher (1977) has pointed out that linoleate, linolen- ate, oleate and palmitoleate each serve as the initial unsaturated fatty acid precursor in biosynthesis of an independent family of polyunsaturated fatty acids. He concluded there is no direct crossover between unsaturated metabolites from one sequence to the other as shown below: 18:2 + 18:3 + 20:3 + 20:A + 22:A + 22:5 18:3 + l8:A + 20:A + 20:5 + 22:5 + 22:6 18:0 + 18:1 + 18:2 + 20:2 + 20:3 16:0 + 16:1 + 16:2 + 18:2 + 18:3 + 20:3 + 20:A Minor amounts of odd numbered fatty acids, especially of saturated C15 and C17 as well as 015:1 and Cl7zl and branched chain fatty acids, have been demonstrated to occur in animal fats, especially in those from ruminants (Shorland, 1962). Phospholipids in Animal Tissues Small quantities of phospholipids are present as proteolipids and are found in animal tissues (Dugan, 1971). Lea (1957) and Younathan and Watts (1960) concluded that phospholipids may play an important role in the flavor and stability of meat and meat products during storage. In spite of the growing interest in the study of meat phos- pholipids, much remains to be known about their contribu— tion to flavor deterioration in muscle tissue. Phospholipids are mixed esters of fatty acids and phos- phoric acid combined with either glycerol or sphingosine (Eibl, 1977). Lehninger (1970) and Gurr and James (1971), have comprehensively reviewed the composition and struc- ture of the phospholipids. They stated that a wide spec- trum of chemical species is made possible, firstly, by considerable variation in the types and combinations of fatty acids, and secondly, by esterification of different organic bases, amino acids and alcohols to the phosphate group. They further pointed out that phospholipids not only derive their lipid properties from long chain fatty acid moieties, but also have polar characteristics donated by ionization of the phosphate and base groups. They reported that biosynthesis of the complete phospholipid molecule (denovo) occurs by two main pathways. One involves the transfer of a phosphate base from a water soluble nucleotide (cytidine diphosphobase) to a diglyceride. The other pathway involves transfer of a phosphatidic acid from a lipid soluble nucleotide (cytidine diphosphatidi— glyceride) to the base. Pearson gt gt. (1977) have further reviewed the struc- ture and composition of meat phospholipids. They pre- sented the structurecfi‘diacyglycerophospholipids as is shown in Figure 1. They further emphasized the fact that most of the phospholipids in muscle are present as phos— phoglycerides with the base esterified to the phosphate usually being choline, ethanolamine or serine. Not all phospholipids have their hydrocarbon residues linked exclusively by an ester to glycerol (Lehninger, 1970). Gurr and James (1971) stated that plasmalogens possess a vinyl ether linkage, which is probably located at the l—position. Dugan (1971) has reviewed a less abun- dant, but nevertheless important group of phospholipids, in which sphingosine is the alcoholic moiety. He pointed out that when sphingosine is esterified to phosphoryl choline it is usually referred to as sphingomyelin. The level of phospholipids (0.5-1.0%) remains nearly constant when expressed as a percentage of muscle, but varies inversely as a function of total lipid (Dugan, 1971). There is, however, considerable variation in the O u CHO 3 CH2 0 C°Rl H — é — H R2-C°O — c — H I ' O I I II CH2OH CH2 O°C°R3 (A) L-Glyceraldehyde (B) Triglyceride O H 0 CH2'O°C°R1 (1) H I R2~C-O — C — H O (2) u H CH2-O-P'OX (3) I OH (c) l,2-Diacyl-Sn-glycerol-3-phosphoryl-X R R 1’ different fatty acid residues. In structure C, Sn stands for stereochemical numbering and X for one of the follow- ing substituents: 2, and R3 represent alkyl groups corresponding to Substituent Phospholipid H Phosphatidic acid HO°CH2-CH2°NH2 Phosphatidyl ethanolamine HO°CH2°CH2N+(CH3)3 Phosphatidyl choline HO'CH2°CH'NH2 Phosphatidyl serine COOH Figure 1. Structure of the diacylglycerophospholipids. 10 phospholipid content between species (Kaucher _t gt., 19A3), and from location to location within the same species (Peng and Dugan, 1965; Acosta gt_gt., 1966). Pearson gt gt, (1977) stated that poultry meat and fish are higher in phospholipids than red meats. When expressed as per- centage of raw muscle tissue, Peng and Dugan (1965) and Acosta gt gt. (1966) have shown that dark meat from poultry contains more phospholipids than white meat. In contrast, Katz gt gt. (1966) have reported that chicken dark meat contains only about half as much phospholipids as white meat. Campbell adeMrkki(l967) and Fooladi (1977) have shown that the phospholipid concentration is higher in cooked meat than in raw meat, regardless of whether it is expressed as percentage of fat or as percentage of total tissue. The phospholipid components that have been isolated and identified from most skeletal tissues are somewhat similar (Peng and Dugan, 1965; Braddock and Dugan, 1972; Keller and Kinsella, 1973; Body and Shorland, 197A). The main components of phospholipids are phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), sphingomyelin (SP), phosphatidyl inositol (PI), lysophosphatidyl choline (LPC) and other minor components (Pearson gt gt,, 1977). Hornstein gt gt, (1961) and Dugan (1971) have reported the composition of beef phospholipids to be 50-60% PC, 30-A0% PE and 10% SP. More recently, Keller and Kinsella 11 (1973) have reported the composition of beef phospholipids as being 53-58% PC, 23-25%, PE, 5-7% SP, 5-7% PI, 1-A% PS and 1-6% others. Peng and Dugan (1965) have reported the quantitative amounts of phospholipids present in chicken dark meat and white meat. In the dark meat, they found 52—58% PC, 2A—30% PE, 7—9% PS and 3-A% SP. On the other hand, the levels of the corresponding components in the white meat were 58-62%, 15-l6%, 9-10% and 2-A%, respectively. The data presented above indicate non-uniformity in the values for phospho- lipid components reported by different workers for the same tissues. Perhaps the reason for this paradox is the fact that phospholipids are labile and difficult to handle (Younathan and Watts, 1960). Fatty Acid Composition of the Phogpholipids Phospholipids from animal sources contain fatty acids mostly with a chain length between 16 and 20, in which palmitic, stearic, oleic, linoleic and arachidonic acids are predominant (Gurr and James, 1971). The most common arrangement for phosphoglycerides is for the saturated fatty acids to be located at the a-position and unsaturated fatty acids at the B-position (Peng and Dugan, 1965). Irrespective of species, the phospholipids are characterized by their high levels of polyunsaturated fatty acids (Hornstein gt gt,, 1961). The phospholipids from.a 12 particular tissue are appreciably more unsaturated than triglycerides from the same source (Lea, 1957). However, Braddock and Dugan (1972) concluded that differences in fatty acid composition of the triglyceride and phospho- lipid fractions from fish are negligible. The components of phospholipids tend to have a characteristic fatty acid composition (Hornstein gt_gt., 1961; Peng and Dugan, 1965; Body and Shorland, 197A). PE has a higher proportion of unsaturated fatty acids than PC (Hornstein gt gt., 1961; Keller and Kinsella, 1973). Body and Shorland (197A) reported a higher content of poly- unsaturated fatty acids (PUFAS) in PE (l7-A3%) as compared to PC (7—25%) and SP (1-A%). However, the proportion of PUFAS reported for PE and PC between and within muscle tissues seems to vary a great deal. According to Body and Shorland (197A), the fatty acid profile of a given phospholipid component varies with the feeding regime of the animal and the conditions of analysis. In spite of the variable results reported for the fatty acid composition of the phospholipids, the levels of stearic and arachidonic acids seem to be markedly higher in PE than in any other component (Hornstein gt gt., 1961; Keller and Kinsella, 1973). Phospholipids from both chicken dark and white meat have a similar degree of un— saturation (Peng and Dugan, 1965; Katz gt gt., 1966). 13 The Role of Lipids in the Development of WOF Hornstein (1967) concluded that fat influences meat flavor through autoxidative degradation of the unsaturated fatty acids. He then stated that the resulting carbonyl compounds may contribute desirable or undesirable flavors, 'depending on their concentration. The development of WOF in cooked meat is generally accepted to be the result of autoxidation of tissue lipids (Younathan and Watts, 1960; Ruenger gt_gt,, 1978). Among tissue lipids, the phos- pholipids have been implicated as the lipid component most readily susceptible to oxidation in cooked meat (Younathan and Watts, 1960) or in freeze-dried beef (E1- Gharbawi and Dugan, 1965; Chipault and Hawkins, 1971). The phospholipids are more complex than the neutral lipids and tend to oxidize very rapidly, at least par- tially due to their high content of PUFAS, which are very labile (Lea, 1957). Oxidation of tissue lipids seems to occur in two stages, that is, the phospholipids are oxidized first and the neutral lipids later (El-Gharbawi and Dugan, 1965; Chipault and Hawkins, 1971). Hornstein gt gt. (1961) concluded that phospholipids do not contribute to desirable meat flavor, especially in lean meat. Corliss and Dugan (1970) and Tsai and Smith (1971) reported that the nature of the nitrogenous components bound in ester linkage to the phosphoric acid moiety may influence the oxidation of the unsaturated fatty acids in 1A the phospholipid molecule. Tsai and Smith (1971) studied the effects of phosphorylated and non—phosphorylated bases, such as ethanolamine, choline and serine, on the oxidation of methyl linoleate in an aqueous system. Only the phos- phorylated and non-phosphorylated bases of ethanolamine exerted a pro-oxidant effect. Corliss and Dugan (1970) also reported that the ethanolamine moiety exerted a greater pro—oxidant effect than the choline portion. Recently, Igene and Pearson (1978) have provided con- vincing evidence that total phospholipids are principally responsible for the development of WOF in cooked beef and poultry. The triglycerides are much less susceptible to oxidation than the phospholipids (Younathan and Watts, 1960; Love and Pearson, 1971). Hence, the triglycerides appear to exert only a minor influence on development of WOF (Igene and Pearson, 1978). Role of Lipids on Storage Stability of Frozen Meats The storage stability of frozen meats, just like cooked meats, depends essentially on the composition of their lipid constituents and more especially on the degree of unsaturation (Watts, 195A; Greene, 1969; Igene gt gt., 1976). Thus, mutton, beef, pork, poultry and fish can be arranged in order of decreasing stability, reflecting their degree of increasing lipid unsaturation (Wilson gt gt., 1976). It is often assumed that tissue lipids are quite 15 stable in frozen storage (Kimoto gt gt., 197A). Some re- search workers (Sulzbacher and Gaddis, 1968; Bratzler gt gt,, 1977) have concluded that autoxidation of the tri- glycerides, principally in the adipose tissue, is respon- sible for the development of rancidity of raw frozen meats. This view is contrary to that held by others (Cadwell gt gt., 1960; Watts, 1962; Greene, 1969), who have con- cluded that oxidative changes in tissue lipids are pri- marily due to autoxidation of the phospholipids. Microbial growth does not occur in meat stored below —9°C (Kimoto gt gt., 197A). However, lipolytic degrada- tion of the phospholipids during freezer storage has been attributed as the cause of rancidity in beef (Awad gt gt., 1968), poultry (Davidkova and Khan, 1967) and fish (Brad- dock and Dugan, 1972). Mechanism of Lipid Oxidation Autoxidation (uptake of oxygen) of food lipids is promoted by heat, light and trace metal catalysts, es- pecially copper and iron (Ingold, 1967; Waters, 1971). The rate and degree of autoxidative degradation of lipids is directly related to the amount of unsaturation (Love and Pearson, 1971). Dugan (1961) and Lundberg (1962) have reviewed the mechanisms involved in autocatalytic oxida- tion of lipids. The free radical chain theory of auto- oxidation (Farmer and Sutton, 19A3; Bolland and Koch, 19A5) 16 has been widely accepted as the mechanism of oxidation in non-conjugated unsaturated fatty acids, which form the great bulk of food fats of both animal and vegetable origin (Lea, 1953). According to the autocatalytic theory as explained by Dugan (1961), oxidation takes place at a reactive methylene group adjacent to a double bond. This results in produc- tion of a hydroperoxide (ROOH) which still retains the original degree of unsaturation. At the same time, a resonance system set up by the free radical leads to the production of conjugated isomers. The hydroperoxides may then decompose to yield more free radicals, which can initiate new reaction chains. The steps involved are illustrated in the modified scheme taken from Sato and Herring (1973) and are shown below: I. Initiation: Initiators . RH + R + H. (unsaturated fat mole) (fatty free radicals) II. Propagation: R' + O + ROO' (peroxide free radical) 2 ROO' + RH + ROOH + R' ROOH + R0' + 'OH RO' + RH + ROH + R' 17 According to Sato and Herring (1973), the free radicals formed can combine with atmospheric oxygen to form peroxide free radicals, which can then react with the substrate to form more free radicals that then propagate the reaction. They theorized that termination occurs as shown below: III. Termination: R' + R' + R—R R' + ROO' + ROOR ROOH + ROO‘ + ROOR + 02 They concluded that these reactions lead to the formation of inactive stable non-reactive end products. Products of Lipid Oxidation The hydroperoxides are the primary products of the re- action of oxygen with unsaturated lipids (Sato and Hegarty, 1973). The secondary products of lipid oxidation (alcohols, ketones, acids, lactones, etc.) are believed to be formed largely through the decomposition of the hydroperoxides (Herz and Chang, 1970). The mechanism of further break- down of the hydroperoxides has been reported to be mono- molecular as well as bimolecular (Mabrouk and Dugan, 1960; Lundberg, 1962) as shown below: l8 ROOH + R0' + 'OH (monomolecular) 2ROOH + 2RO' + H2O (Bimolecular) The catalytic and thermal decomposition of the hydro- peroxides is of great importance to flavor in general and to WOF in particular. The most numerous members of any class of compounds identified in meat flavor concentrates are the carbonyls (Herz and Chang, 1970). Although meat flavor Egg gg resides in the water soluble meat extract (Kramlich and Pearson, 1958), Hornstein and Crowe (1960, 1963) have suggested that the characteristic species flavor is due to the carbonyls. Although there are both water and fat soluble carbonyls, Sanderson gt gt. (1966) reported that those involved in meat flavor are primarily lipid soluble. Hexanal has been reported to be a product of lipid oxidation of linoleate (Gaddis gt gt., 1961). El-Gharbawi and Dugan (1965) found the concentration of hexanal to increase during the storage of freeze-dried beef, while Cross and Ziegler (1965) demonstrated that hexanal occurred in greater quantities in uncured than in cured ham. Love and Pearson (1976) also reported that hexanal is one of the principal products associated with lipid oxidation and is implicated as a component of WOF. Recently, Ruenger gt gt. (1978) reported that heptanal and n-nona-3-6-dienal were related to the development of WOF in turkey meat. The l9 identification of these undesirable flavor compounds, which are typical end products of lipid oxidation, further supports the fact that WOF is due to lipid oxidation. Catalysts of Lipid Oxidation Robinson (192A) first implicated the porphyrins (hemo- globins, myoglobin and cytochromes) as the catalysts of lipid oxidation in meats, and attributed the catalysis to their iron content. It is generally accepted that hemo- globin and other iron porphyrins accelerate lipid oxida- tion, with the hemoproteins being implicated as the major pro-oxidants in meat products (Tappel, 1952; Younathan and Watts, 1958). Watts (195A) indicated that the reaction between lipid and hemoproteins brings about destruction of the pigment as well as oxidation of the fat. Banks (19AA) suggested that the active catalyst results from a combination of a fatty peroxide with an iron porphyrin. Maier and Tappel (1959) proposed that catalytically active hemes form un— stable compounds with fat peroxides, which then decompose to give two free radicals, each of which is capable of initiating an oxidative chain. Although it has been suggested that lipid and myglo— bin oxidation are intimately related (Watts, 195A), it has yet to be conclusively demonstrated whether the oxidation of the lipid occurs first and causes the oxidation of the 20 pigment or vice-versa. Tappel (1955) showed that hematin compounds catalyze the oxidation of unsaturated fatty acids, and that the catalytic activity is dependent on the pres- ence of iron. Younathan and Watts (1959) also reported the catalytic activity of myoglobin in tissue oxidation. They found that uncured cooked meat, containing ferric globin hemochromogen showed greater oxidation shortly after cooking. They concluded that it was the ferric form of the pigment, which is the active catalyst in tissue rancidity. According to Labuza (1971), the rapid rate of oxidation in cooked meat may be due to the denaturation of myoglobin during the cooking process. He suggested that unfolding of the protein allows greater exposure and access of the iron to the previously formed peroxide. Haurowitz gt gt. (19A1) reported that the pro-oxidant effect of hemin or hemoglobin on linoleic or linolenic acid was due to the destruction of the pigment and the subsequent release of inorganic iron. Non—heme iron and other heavy transition metals have been reported to function as catalysts of lipid oxidation in cooked meats (Kwoh, 1970). The mechanism of metal catalysis was reviewed by Ingold (1967) and Waters (1971). Wills (1966) showed that both non-heme iron and hemopro- teins are involved as catalysts in lipid peroxide forma- tion. Sato and Hegarty (1971), Kwoh (1970) and Love and Pearson (197A) have presented data showing that non-heme 21 iron is the major pro-oxidant in cooked meats. They con- cluded that meat pigments Egg gg have no catalytic effects on lipid oxidation in cooked meats. Hirano and Olcott (1971) and Kendrick and Watts (1969) have demonstrated that heme compounds may act as either accelerators or inhibitors of lipid oxidation, their action depending on the ratio of heme to unsaturated fatty acids. There is now strong evidence that ferrous iron is a more active catalyst of lipid oxidation than ferric iron (Smith and Dunkley, 1962; Waters, 1971; Love and Pearson, 197A). Influence of Grinding and Cooking It has been postulated that any process causing dis- ruption of the muscle membrane system, such as grinding or cooking, results in exposure of the labile lipid com- ponents to oxygen and thus accelerates development of oxidative rancidity (Pearson gt gt., 1977). Sato and Hegarty (1971) have reported a very rapid increase in TBA values, and hence of WOF, for raw meats one hour after grinding and exposure to air at room temperature. They suggested that any catalysts of lipid oxidation present in the muscle system are brought into contact with the oxidation-susceptible lipids and contribute to the rapid development of WOF. It is well known that heating accelerates the develop- ment of oxidized flavor (rancidity) in meat and meat 22 products (Younathan and Watts, 1959; 1960; Keller and Kinsella 1973; Wilson gt gt., 1976; Fooladi, 1977). The rapid oxidation of lipids in cooked meat has been attributed to the conversion of ferrous iron of the porphyrin to the ferric form during heating (Younathan and Watts, 1959). According to Yamauchi (1972 a,b) as quoted by Pearson gt gt. (1977), the development of rancidity is most rapid in meat that is heated at 70°C for one hour. He demonstrated that the TBA value of cooked meat decreased as the cook- ing temperature was increased above 80°C. Recently, Huang and Greene (1978), confirmed that meat subjected to high temperatures and or long periods of heating developed lower TBA numbers than similar samples subjected to lower temperatures for shorter periods of time. They postulated that antioxidant substances produced during the browning reaction exert TBA retarding activity; and which pro- gresses as the meat is heated. Similarly, Zipser and Watts (1961) and Sato and Herring (1973) have presented evidence for antioxidant activity in retorted beef. They attributed the decreased amount of oxidation to develop- ment of the browning reaction, which takes place during the heating process. According to Hamm (1966), the Maillard reaction in meats begins at about 90°C and increases with further increases in temperatures and heating times. 23 Control of Lipid Oxidation By using various chemical compounds, such as anti- oxidants and chelating agents as well as by exclusion of oxygen, it is possible to control lipid oxidation in cooked meats with some degree of success (Sato and Herring, 1973). Antioxidants may interfere with or delay the onset of oxidative breakdown in fats and fatty foods (Blanck, 1955). According to Shelton (1959), primary or phenolic antioxidants, which include the tocopherols, butylated hydroxyanisole (BHA)or butylated hydroxytoluene (BHT) in combination with or without chelating or synergistic agents, may break the oxidative reaction chains. Uri (1961) has reviewed the mechanism of antioxidants in foods. It is necessary to ensure that the antioxidants used in meat and meat products are not harmful to the consumer (Sato and Herring, 1973). There is evidence that potent natural antioxidants (the tocopherols) are present in meats (Igene gt gt., 1976), and that antioxidant substances can be produced in meats by high temperature treatment (Pearson gt gt., 1977). Zipser and Watts (1958) and Huang and Greene (1978) have found that production of antioxidant substances by pro- longed heat treatment can stabilize uncured canned meats against oxidative rancidity. The use of extracts of soy- bean and soy products, which contain natural antioxidant substances (flavonoids), has been found to retard oxidation 2A in sliced cooked beef (Watts, 1962; Pratt, 1972). The inhibition of lipid autoxidation by adding nitrite, phos- phate, ascorbate and other curing ingredients has been reviewed by Pearson gt gt. (1977). Sato and Hegarty (1971) showed that nitrite will completely eliminate WOF at 220 ppm and will inhibit development at 50 ppm. Recently, Fooladi (1977) and Igene gt gt. (1978) have demonstrated that 156 ppm of nitrite effectively prevents WOF in cooked meat and poultry. Zipser gt gt. (196A) proposed that nitrite forms a stable complex with iron porphyrins in heat de- natured meat, thereby inhibiting the development of WOF. EXPERIMENTAL Materials Solvents and Chemicals All chemicals and solvents utilized in this investiga— tion were of reagent grade. All solvents were freshly redistilled before use unless otherwise specified. Methyl Ester Mixtures Standard mixtures for the determination of relative retention times by GLC were obtained from Applied Science Lab; (State College, PA) and Supelco, Inc. (Bellefonte, PA). The standard mixtures contained a wide range of fatty acids, starting with C10 through C22:6 and included C2“ 0' TLC Standard Mixture Polar lipid mixtures for TLC containing cholesterol, phosphatidyl ethanolamine, lecithin and lysolecithin were obtained from Supelco Inc. In addition, pure bovine lecithin, phosphatidyl ethanolamine and a serum lipid mixture containing specified amounts of various phos- pholipid components were also obtained from Supelco Inc. 25 26 Thin—Layer Plates Precoated 20 x 20 cm silica gel G thin layer plates (0.5 mm thick) were obtained from Fisher Scientific Co. (St. Louis, MO) and used for all TLC separations. Gas—liquid Chromatography Column Packings Acid—washed and salinized (80/100 mesh) supelcoport coated with 10% diethylene glycol sucinate (DEGS) was ob- tained from Supelco Inc. and used for the GLC separations. Source of Meat Beef, chicken dark meat and chicken white meat were used in these studies. The beef and chicken (old layers) were obtained from the Michigan State University Meat and Poultry Processing Laboratories. Portions of longissimus dorsi (LD) muscle were exercised from beef carcasses at 2A hrs postmortem. Thigh (dark meat) and breast (white meat) meat were removed from the chicken carcasses at 2A hrs postmortem. 27 Methods Meat for Evaluating the Effect of Frozen Storage on Lipids and Their Role in the Development of Oxidized Flavor In order to establish a basis for changes during frozen storage, fresh bone-in samples of beef (LD) muscle were cut into identical thicknesses (2 inch) and sizes (2 lb lots), wrapped with freezer paper, numbered and stored at -l8°C. In the same manner, fresh cuts of un- skinned thighs and breast meat from chicken carcasses were randomly wrapped in packages of A lb each and stored at -18°C. The packaged meat was held at -18°C for 13 months. At designated storage periods, samples were removed from the freezer and held at A°C for 18-2A hrs to allow thawing, after which the meat was tested for both rancidity and WOF. Preparation of Meat for Rancidity or WOF Evaluation External fat was trimmed from the beef while any ad- hering skin was removed from the chicken samples. The meat was then cut into pieces after removing the bones and ground twice through a 3/16 inch plate (except where otherwise specified) using a Hobart meat grinder. For assessment of rancidity, portions of the ground meat were removed for TBA evaluation and lipid analysis. 28 For evaluation of WOF, about Aooégof meat were packed into each of two retortable pouches and heat sealed. The bags were cooked in boiling water to an internal temperature of 70°C. Immediately after cooking, the bags were opened, the drippings collected and the meat thoroughly mixed. Prior to storing the cooked meat at A°C or -18°C, the TBA values of the freshly cooked meat were measured. Then equal amounts of the cooked meat were held at A°C and at —18°C for A8 hrs in unsealed retortable pouches. At the end of A8 hrs, the TBA values as well as comprehensive lipid analyses of the cooked meat were determined. Methods of Analyses TBA Test The distillation method of Tarladgis _t _t. (1960) was utilized to measure the development of oxidative rancidity by the TBA test. However, a modification in the distillation procedure was made for nitrite treated samples. Hougham and Watts (1958), Younathan and Watts (1959) and Zipser and Watts (1962) have shown that nitrite interferes with the distillation step by nitrosation of malonaldehyde. Hence the modified TBA test of Zipser and Watts (1962), in which sulfanilamide is added to the sample, was utilized for all preparations containing nitrite. TBA numbers were expressed as mg malonaldehyde/kg meat. 29 Taste Panel Evaluation of WOF in Cooked Meat Sensory evaluation was carried out by trained panelists after A8 hrs storage of the cooked meat at A°C. At each setting, all panelists were presented with four different coded samples representing different treatments. A control sample consisting of freshly cooked meat was also presented along with the treated samples. All experimental samples were reheated and served while hot. The panel scoring system was as follows: 1 = very pronounced WOF; 2 = pro— nounced WOF; 3 = moderate WOF; A = slight WOF; and 5 = no WOF. Extraction of Lipid from Muscle Tissue Total lipid was extracted from fresh or cooked meat including the experimental treatments involving meat model systems by the procedure of Folch _t _t. (1957). This procedure involves the use of chloroform-methanol (2:1) by homogenizing the tissues several times in a Waring blender followed by filtration. The combined filtrate was transferred to a separatory funnel to allow the chloroform and aqueous layer to separate. The chloroform layer was evaporated to a constant weight of lipid under reduced pressure at 30-A0°C using a Rotavapor-R (Bfichi, Switzerland). Separation of the total lipids into triglycerides and phospholipids was achieved by the method of Choudhury gt gt. 30 (1960). This method involves the separation of total lipids on activated silicic acid in which neutral lipids are preferentially removed by washing with chloroform. The phospholipids combine with the activated silicic acid and are solubilized and extracted with methanol. The ex- tracted total lipids, triglycerides and phospholipids in raw and cooked meat were expressed as the percent of fresh or cooked tissue. Drippings from Cooked Meat Following cooking, the drippings were collected, cooled and the volume determined using a graduated cylinder. Then the lipid present in the drippings was extracted using the procedure of Folch gt gt. (1957). The levels of tri- glycerides and phospholipids were determined by the method of Choudhury gt gt. (1960). The amount of lipids present in the drippings were expressed as percentage of original tissue. Thin—Layer Chromatpgraphy (TLC) Sgparation of Phospholipid Components - The total phos- pholipids extracted from fresh or cooked meat were separated into their components using preparative TLC. Exactly 50 mg/ml of total phospholipids (equivalent to 0.A75 mg phos- pholipid/spot) were spotted on each plate under a stream 31 of nitrogen gas using a Hamilton microsyringe equipped with a Chaney adaptor. A standard mixture of authentic phos- pholipids (Supelco, Inc.) was simultaneously spotted on the left hand side of the plate. The plates were developed in chloroform: methanolzwater (65:25:A, v/v). After the plates were dried in a stream of nitrogen gas, 50 mg/ml of the same sample representing total phospholipids (for determination of total phosphorus) were spotted on the right hand side of the plate for determination of phos- phorus. The spots were identified by spraying the plates with potassium dichromate/sulfuric acid and charring for 10—15 minutes at 175°C using a forced air oven. Quantitation of Phospholipids (Phosphorus Determination) - After the TLC separation of lysolecithin (LPC), sphingo- myelin (SP), phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) from the total phospholipids (including the spots representing total phospholipids), they were scraped from the TLC plates into 30 ml micro-Kjeldahl flasks for digestion with perchloric acid as described by Rouser gt gt. (1966). A standard curve was prepared using aliquots of monobasic potassium phosphate solution containing 1-10 ug of phosphorus. Absorbance of 820 nm was multiplied by a factor of 11.0 (after correcting for the blank), which was calculated from a standard curve to convert the readings to ug of phosphorus. The values for 32 phosphorus in ug were subsequently converted to mg phosphorus/g phospholipid, and expressed as such in this study. Purification of PC and PE - Plates for the determination of fatty acids in PC and PE were prepared as described earlier herein except they were sprayed with iodine vapors. Spots containing PC and PE were immediately recovered and eluted with chloroform-methanol (A:l, v/v), evaporated to dryness under nitrogen gas and redissolved in chloroform. Before converting them to methyl esters, PC and PE were checked for purity by TLC. Preparation of Methyl Esters - Total lipids, triglycer- ides, total phospholipids, PC and PE from all meat samples used for fatty acid analysis were converted to methyl esters by the Boron-trifluoride/methanol procedure as described by Morrison and Smith (196A). Gas-Liquid ChromatographyA(GLQ) GLC analysis of all fatty acid methyl esters was per— formed using a Perkin-Elmer model 900 gas chromatograph equipped with a hydrogen flame ionization detector (FID). The column, 6 ft x 2 mm (i.d.) stainless steel, was packed with 10% (w/w) diethylene glycol succinate (DEGS) on supelcoport (Supelco, Inc.). The column was set at 185°C, 33 the injection port at 220°C and the detector at 250°C. The carrier gas was helium and the flow rate was maintained at 30-A0 ml/min while hydrogen gas and air were adjusted to 30 and 285 ml/min, respectively. Quantitative identification of the emerging peaks was done using retention times of standard mixtures of known fatty acid methyl esters (Applied Science Lab., Inc., State College, PA; Sulpelco, Inc.). Peak areas were cal- culated quantitatively as the product of peak height and width at half height. Results were expressed as percent of the total area. Statistical Treatment Statistical analysis was calculated using the STAT SERIES developed by the Michigan State University Agricul- tural Experimental Station and was raw on control Data Corporation (CDC) 6500 computer. Alternately, some statis- tical analyses were carried out using a Wang programmable computer. Analysis of variance for TBA values, taste panel scores and phosphorus changes were also computed. Standard deviations, correlation and regression coefficients were also calculated. The significance of the computed correlation coefficients was determined by the "r" distribution table from Snedecor and Cochran (1973). The significance between treatments was determined using either Tukey's test for multiple comparisons or the student "t" test for differences 3A between two means. Graphs were plotted using SPSS (Statis- tical Package for the Social Science) version 7.0 (MSU, March 18, 1978). Experimental Systems Four broad groups of experiments were conducted to ascer- tain the role of meat lipids and meat pigments on the development of rancidity and WOF in frozen and in cooked meat, respectively. Experiment A The effects of type of lipids on the development of rancidity in model meat systems during frozen storage at —18°C were determined. Experiment B The effects of type of lipids on the development of warmed—over flavor (WOF) were measured in cooked model meat systems. Experiment C The effect of length of frozen storage in relation to its effect on the stability of meat lipids as well as on the production of off-flavors was determined. 35 Experiment D The role of meat pigments, nitrite, heme and non-heme- iron on development of WOF was assessed. Prgparation of Model Meat System For Experiments A and B, a model meat system was de- veloped to study the relative contributions of triglycerides, total lipids and phospholipids to the development of ran- cidity during freezer storage and of WOF following cooking and storage at A°C. Lipid-free muscle fibers were used as the matrix for the model system. Fresh raw beef, chicken dark meat or white meat were each ground once through a 3/16 inch plate using a Hobart meat grinder. Then total lipids contained in the ground meat were extracted by the method of Folch gt gt. (1957). The phospholipids were separated from total lipids using the procedure of Choudbury gt gt, (1960). The solvent was removed from the residue (protein matrix) by drying under vacuum and later in a stream of nitrogen gas at room temperature. Following removal of the solvent, the model systems were either used immediately or packed in cryovac bags, frozen and stored at -18°C. The preparation of the model system is outlined schematically in Figure 2. 36 Fresh Raw Meat (Beef, chicken dark or white meat) Grinding (3/16 inch plate) I z "I V Ground Meat Extraction of lipid (CH013:MeOH) i 1 Protein Matrix (residue) Total Lipids Vacuum dried , Separation in I I silicic acid i I ‘ 7 Muscle fibers Chloroform Methanol elution elution l l Triglycerides Phospholipids Figure 2. Preparation of model meat system. 37 Design of Experimental Treatments Involving Model Systems Experiments were conducted to test the influence of triglycerides, total lipids and total phospholipids on the development of rancidity during freezer storage. In addi- tion, the development of WOF was studied using the same experimental treatments. The design of the treatments is shown in Table 1. The levels of total lipids, triglycerides and phospho- lipids added back to the model systems closely corresponded to that removed during the extraction process. Total phos- pholipids were used in the beef and chicken meat model systems at levels of 0.8 and 0.7%, respectively. Total lipids and triglycerides were added to the beef model system at levels of 10.0 and 9.2%, respectively. On the other hand, total lipids and triglycerides from chicken dark meat were added to the model system at concentrations of 5.0 and A.3%, respectively. Total lipids and trigly— cerides were also added to the chicken white meat model system at concentrations of 5.0 and A.3%. Experiment A The primary objectives of Experiment A were to study the influence of triglycerides, total lipids and total phospholipids on the development of rancidity during frozen storage of meat model systems. The experimental model 38 Table 1. Design of Model Meat Experimental Treatments. Meat Code Source Number Composition of Model Meat Systems Beef B1 0.8% beef phospholipids in beef muscle fibers B2 9.2% beef triglycerides in beef muscle fibers B3 10% beef total lipids in beef muscle fibers BA Beef muscle fibers only (control) Dl 0.7% dark meat phospholipids in dark meat muscle fibers *Chicken D2 A.3% dark meat triglycerides in dark dark meat meat muscle fibers D3 5.0% dark meat total lipids in dark meat muscle fibers DA Dark meat muscle fibers only (control) W1 0.7% white meat phospholipids in white meat muscle fibers *Chicken W2 A.3% white meat triglycerides in white white meat meat muscle fibers W3 5.0% white meat total lipids in white meat muscle fibers WA White meat muscle fibers only (Control) a) Muscle fibers without added lipids served as controls. Each experiment was replicated A times. *The same amount of lipids were applied to chicken dark meat and white meat fibers in order to eliminate variations due to lipid levels. 39 systems (Table l) were prepared, stored in polyethylene bags and were held at —18°C for 8 months. Prior to frozen storage, the initial TBA values of the model systems were determined. At the same time, the composition of the fresh lipids in the model systems were also measured. TBA values of the frozen model systems were also determined at 1, A and 8 months of storage. At the end of 8 months storage, fatty acid analysis of the triglycerides and total phospholipids, including that of PC and PE, were carried out. The composition of the phospholipid components was also determined at the end of 8 months storage. Experiment B Experiment B was designed to test the influence of tri- glycerides, phospholipids and total lipids on the develop- ment of WOF in the cooked meat model systems. Thus, the meat model systems, including the control samples (Table l), were packed in 6-l/A inch x 8-1/2 inch retortable pouches (Continental Diversified Industries, Chicago, IL) and heat sealed with a Multi-Vac sealing machine (Busch- W. Germany). The bags containing the treated samples were cooked to an internal temperature of 70°C in boiling water. Following cooking, the bags were opened, and the con- tents thoroughly mixed. The meat was then stored at A°C for A8 hours. TBA values, taste panel scores and lipid (zomposition of the different model systems were then A0 determined. A trained panel of 12 individuals consisting of members of faculty and graduate students in the Depart- ment of Food Science and Human Nutrition served as judges for taste panel and odor evaluations. The scoring system used was as described earlier under methods of analyses. Experiment C The principal objectives of this experiment were to study how length of frozen storage influences the composi- tion and stability of meat lipids as well as its effect on the production of rancidity and WOF. Fresh cuts of beef, chicken dark and white meat were used for this study and were frozen and stored at -18°C for over one year. Basically, the idea was to measure the content of extractible lipids at 0, 8 and 13 months of frozen storage while at the same time determining the levels of malonaldehyde produced by oxidative rancidity in fresh and/or frozen meat as well as in cooked meat. Prior to frozen storage (0 day), randomly selected samples of fresh beef, chicken dark or white meat were analyzed for their initial TBA numbers in the fresh as well as in the cooked state. At the same time, the concentration of total lipids, triglycerides, total phospholipids, including the component phospholipids (LPC, SP, PE and PC), was determined in both the fresh and cooked meat. In addition, the initial fatty Al acid composition of the triglycerides, total phospholipids, PE and PC associated with both fresh and cooked meat were also measured. At 8 and 13 months of frozen storage, randomly selected packages of frozen meat were also removed from the freezer, thawed and evaluated for TBA numbers as well as for extrac- tible lipids and fatty acid composition before and after cooking. This procedure was adopted to effectively relate variations in lipid composition and stability to the de- velopment of rancidity in frozen as well as in cooked meat. Eyperiment D The role of meat pigments, nitrite and non-heme iron on the development of WOF was investigated. The study was divided into two stages. The first stage involved the effects of total meat pigments and nitrite on the develop- ment of WOF, while Stage II was designed to compare the relative contributions of heme and non-heme iron on the development of WOF. In Stage I, lean cuts of fresh beef, chicken dark meat and white meat were used. The meat was cut into pieces, ground first through a 3/8 inch and later through a 3/16 inch plate. Each kind of meat was divided into two groups. Muscle pigments from one group were removed by extraction with deionized water at A°C for 2A hrs with several volumes of water. Then the meat was filtered through cheese cloth A2 until it was virtually devoid of pigments. The unextracted group of samples was used as controls. Four experimental treatments were designed for each type of meat as shown in Table 2. Each experimental treat— ment consisted of 200 g of meat that was mixed with 100 ml of distilled deionized water. In the nitrite treated samples, the level of nitrite was 156 ppm. In Stage II, total pigments were removed from 2.0 kg of fresh beef (LD) as described in Stage I. The extracted meat pigments were concentrated in a Stokes freeze-drier. Then the concentrated extract was divided into lots A and B as shown in Figure 3. The extract from lot A was further subdivided into two equal parts. One part was cooked and the other part used as an uncooked control. Half of both the cooked and uncooked extracts was treated with 2.0% EDTA in order to chelate the non-heme iron. Extract B was treated with 30% H202 to destroy the pigment and release non-heme iron (Wills, 1966; Kwoh, 1970). One part of the H202 treated extract was again chelated with 2% EDTA. The unchelated extracts served as controls. Each extract was added back to the residue and cooked in retortable pouches. The design of the experimental treatments is shown in Table 3. A3 Table 2. Design and Formulation of Treatments. Meat Code Type Number Treatments and Formulations A Beef with pigment, no nitrite Beef B Beef with pigment, plus nitrite C Beef without pigment, no nitrite D Beef without pigment plus nitrite A Dark meat with pigment, no nitrite Chicken B Dark meat with pigment plus nitrite dark meat C Dark meat without pigment, no nitrite D Dark meat without pigment plus nitrite Chicken white A,B,C,E> Same Treatments were used as for dark meat meat Nitrite was added at a level of 156 ppm. 200 g of meat was used for each treatment. Samples were cooked and stored at +A°C for A8 hrs. There were A replicates for each treat— ment. AA 2.0 Kg Ground Beef (through 3/8 and ' 3/16 inch plates) Extracted with deionized H20 I Extract (concentrated by freeze—drying) Treated with 30% H202 _Uncooked Extract Cooked Extract 7 Extract Extract Extract Extract Unchelated Chelated Unchelated Chelated with EDTA with EDTA Extract Extract Unchelat-Chelated ed with EDTA Figure 3. Preparation and design of experiment to compare the effect heme and non—heme-iron on the de- velopment of WOF. A5 Table 3. Experimental Treatments to Compare the Effect of Heme and Non-heme Iron on the Development of WOF. Treatment # Preparation of Experimental Treatments 1 Residue + total meat pigment 2 Residue + total meat pigment (chelated*) 3 Residue + total cooked meat pigment A Residue + total cooked meat pigment (chelated) 5 Residue + H202 treated meat pigment 6 Residue + H2O2 treated meat pigment (chelated) 7 Residue + deionized water ,1— Non-heme iron was chelated using 2% EDTA. Cooking The treated samples were packed in retortable pouches, cooked and stored at A°C for A8 hrs as was described for Experiments B and 0. TBA numbers and/or taste panel evalua- tion (Stage I) of the samples were used to determine the extent of lipid oxidation as influenced by either nitrite, total pigment or non-heme iron. RESULTS AND DISCUSSION Role of Meat Lipids on the Development of Rancidity The role of triglycerides, total lipids and total phospholipids on the development of rancidity during frozen storage of meat was studied by adding them back to lipid- free muscle fibers alone and in various combinations. The experimental design of this study is shown in Table l. The samples were prepared and packed in polyethylene bags and stored at -l8°C for up to 8 months. Prior to storage (0 time), however, the initial TBA values as well as a comprehensive fatty acid analysis of the triglycerides, total phosphilipids and of PC and PE were determined. The samples were tested for development of oxidative rancidity by the TBA test after 1, A and 8 months of frozen storage. TBA numbers and a final comprehensive lipid analysis were carried out at the conclusion of 8 months in frozen storage. Beef Ltpids The TBA values presented in Table A show the effects of beef lipids on development of rancidity during frozen storage. In the control samples (containing no added lipids), the TBA numbers were only 0.A, 0.A2, 0.89 and 1.05 at 0, l, A and 8 months, respectively. The low TBA values indicate that the control samples, which were composed of A6 A7 Table A. The effect of adding beef triglycerides, total phospholipids and total lipids on TBA values of model beef meat systems stored at -18°C. Experimental Treatments Time in Total Frozen Storage Control Triglycerides Phospholipids Total Lipids (Months) (B4) (32) (B1) (B3) 0 0.40 0.38 0.65 0.66 1 0.42 0.82 13.01 20.84 4 0.89 6.33 15.14 21.52 8 1.05 8.31 15.74 20.30 TBA numbers = mg malonaldehyde/kg meat. Bl a 0.8% phospholipids; 82 = 9.2% triglycerides; B3 = 10% total lipids; 34 = muscle fibers only. A8 the extracted meat fibers, did not undergo any appreciable amount of oxidation. Thus, the validity of using the lipid extracted muscle fibers as the basis of the meat model system was verified. In the samples containing added tri- glycerides, the initial TBA value at 0 time was 0.38. It then rose slowly to 0.82 after 1 month in frozen storage but increased rapidly to 6.33 and 8.31 after A months and 8 months in freezer storage, respectively. The TBA numbers of the triglycerides increased most rapidly between 1 and A months of freezer storage and then more slowly between A and 8 months. This indicates a slowing down in the rate of lipid oxidation after a rapid acceleration between 1 and A months storage. The TBA values for the samples containing added phos- pholipids were 0.65, 13.01, 15.1A and 15.7A at 0, l, A and 8 months of freezer storage, respectively. On the other hand, a combination of triglycerides and total phospho- lipids, which represents the total lipids in the original meat sample, gave TBA values of 0.66, 20.8A, 21.52 and 20.38 at 0, l, A and 8 months of freezer storage, respectively. Thus, total phospholipids and total lipids behaved similarly except that the latter gave much higher TBA values, suggest- ing that the triglycerides further accelerated the rate of oxidation. These results would indicate that the phos- pholipids make the greatest contribution to rancidity, but the triglycerides also contribute to the development of A9 rancidity in frozen storage, either alone or in combination with the phospholipids. The increase in TBA numbers during freezer storage (Table A) suggest that unlike the triglycerides, total lipids and total phospholipids do not exhibit a noticeable induction period before lipid oxidation. This is consistent with data reported by El—Gharbawi and Dugan (1965) and Chipault and Hawkins (1971). They observed that oxidation of tissue lipids occurred in two stages. The phospholipids are oxi- dized first and their initial rate of oxidation decreases with time, while after a period of low oxygen absorption, the triglycerides show a rapid rate of oxidation. The trend is shown graphically in Figure A. Analysis of variance indicates that differences between treatments, storage periods and interactions between treat- ments and length of storage were all highly significant (P < 0.001). This suggests that triglycerides, total lipids and total phospholipids are all important in the development of oxidized flavor during freezer storage. However, the results of this study clearly indicate that total phospholipids would contribute more to the development of oxidized flavor during frozen storage than the triglycer- ides. This supports the suggestion of Cadwell gt gt. (1960), Watts (1962), and Greene (1969), who have concluded that oxidative changes during freezer storage are primarily due to autoxidation of the phospholipids. 50 .oomai um omaoum msmpmmm poms moon Hoops mo m03Hm> 42: mammla Looms: .A==U.m a mnoaamu>nolag am.m .. .ooo.o~ 3 1r 0 ... 6851 againoIamoza Lance ax.o ‘ +lllllllllllllr. .boo.o~ moaalh Lance aog .. a éoo.mN [183W OM/UNOWJUBQNHN 881 .: mpswfim 51 The role of beef triglycerides in the development of rancidity may not be important when meat is stored frozen for a short period of time. However, beef triglycerides could play an important role when meat is stored frozen for longer periods. Several researchers (Sulzbacher and Gaddis, 1968; Bratzler gt 1., 1977) have concluded that autoxida- tion of the triglycerides, principally in the adipose tis- sue, is responsible for development of rancidity during frozen storage of meat. Chicken Dark Meat Lipids The TBA values for samples containing chicken dark meat triglycerides, total lipids and total phospholipids are presented in Table 5. The control sample (muscle fibers alone) showed consistently greater TBA values than control beef samples, with values of 1.13, 3.08, 3.23 and 3.A8 at 0, l, A and 8 months of frozen storage, respectively. The higher TBA values for chicken dark meat indicate that the extracted muscle fibers probably contain some unextracted bound lipids. However, the residual lipids in the extracted fibers were not determined. The samples containing triglycerides had TBA values of l.A0, 5.52, 10.75 and 10.36 at 0, l, A and 8 months of frozen storage, respectively. Thus, the added triglycerides substantially increased the TBA values, indicating that 52 Table 5. Effect of adding chicken dark meat tryglycerides, total phos- pholipids and total lipids on TBA values of chicken dark meat model meat systems stored at -18°C. Experimental Treatments Time in Total Frozen Storage Control Triglycerides Phospholipids Total Lipids (Months) (D4) (D2) (D1) (D3) 0 1.13 1.40 1.63 2.05 l 3.08 5.52 11.52 15.05 A 3.23 10.75 12.20 16.86 8 3.48 10.36 13.33 17.33 TBA Numbers - mg malonaldehyde/kg meat. D1 - 0.7% phospholipids; D2 a 4.3% triglycerides; D3 = 5% total lipids; D4 = muscle fibers only. 53 they contributed considerably to lipid oxidation. Chicken dark meat triglycerides did not exhibit the long induction period that was observed for beef triglycerides. This could be related to the higher degree of unsaturation in chicken triglycerides (Katz gt gt., 1966). When total phospholipids were added back to the ex- tracted muscle fibers and frozen, the TBA values were 1.63, 11.52, 12.20 and 13.33 at 0, 1, A and 8 months, respectively. In the samples containing total lipids, on the other hand, TBA numbers were 2.05, 15.05, 16.86 and 17.33 at 0, l, A and 8 months of freezer storage, respectively. Thus, total phospholipids and total lipids from chicken dark meat be- haved in a similar way to those of beef. Thus, like beef the TBA values for chicken dark meat total lipids were higher than for the total phospholipids alone. Further more, analysis of variance for TBA values show that treat- ments, length of frozen storage and the interaction of treat- ment x length of storage were all highly significant (P < 0.001). These results indicate that triglycerides, total lipids and total phospholipids are all important in development of rancidity in chicken dark meat. The results also demonstrated that triglycerides contribute almost as much to development of rancidity as phOSpholipids, although the TBA values for total phospho- lipids were consistently greater than those for triglycerides. 5A . . .oomHI pm omhopm msmumzm poms HmooE poms sumo cmxofino mo mosam> Lonh um.s u. mw iooo N“ ...U chaooozamoza L 0.05). Although the TBA values were slightly higher in the samples containing added phospholipids, the values were all low in comparison to those for beef and chicken dark meat. The rapid increase in TBA numbers observed for beef and 56 Table 6. Effect of adding chicken white meat triglycerides, total phospholipids and total lipids on TBA values of chicken white meat model meat systems stored at -18°C. Experimental Treatments Time in Total Frozen Storage Control Triglycerides Phospholipids Total Lipids (Months) (W4) (W2) (W1) (W3) 0 0.42 0.70 0.78 0.95 1 0.57 0.83 0.94 1.15 4 1.39 1.30 1.98 1.48 8 1.78 1.46 1.95 1.74 W1 2 0.7% total phospholipids; W2 = 4.3% triglycerides; phospholipids; W4 - muscle fibers only. W3 = 5% total 57 .oomai um Uptown msmumzw poms Hmooe poms moans cmxofioo mo mmsfim> aw a canvas Canaan mean on mucosa: HH< oaaaaosnnosa Hope» n ma mocfiaono Hzouumnqmoco u mm mocHEmaocwnum Hzoauwsomosa a mo «casuaooflomha u and oaawaonamoso w\m=posamona wzd .mcoaum:«8pouoo a no some unomohooh spasmoma mm.o“ mm.0n m=.oa sm.on mo.Hn sm.on mm.oh mH.on s=.on mm.on sm.on H=.on o mo.om Hm.m :m.m mm~.H mm.ma mm.: oo.H mm.H mmm.wa wwm.oa om.H mmm.H Hm.on ==.oa mm.ou mm.ou H=.Ha oa.aw mH.ow mm.ow om.on mm.ow =H.Hw m=.o« efl.mm Hw.mfi Ha.a ems.H Hm.Hm m=.ma Hm.m mm.m ssm.mH soo.ma am.m sam.a o my om mm omq me om mm cod no om mm oma Acpcoev owoAOpm poo: moan: coxofico poo: xsmo coxoano «mom couosm no camcoa n.H.mEopm>m poms Hoooe coxowno oco ooon oo Acomauv owoLOpm conooo wcfipso mofiofiflonamoza Hope» ocm Hmzofi>uoCa cw newcono .s manna 60 LYSDPHOSPHRTIOYL CHOLINEILPC) PHOSPHRTIDYL ETHRNOLRHINEIPE) 4.0 10.00 0.00- 3.00 0.00 2.00 fl HG PHOSPHOROUS 0 PHOSPHOLIPID HG PHOSPHOROUS G PHOSPHOLIPIO Wu $2.600 : 4.000 : 0.600 4' 0.000 "‘0 1 2.000 : «foo : 0.600 c 07100 STORRGE TIHEIHONTHS) STORROE TIHE(fl0NTHS) TOTRL PHOSPHOLIPIDSITP) PHOSPHRTIDYL CHOLINEIPC) 40.00 30.00 20.00 1 \i) ”G PHOSPHOROUS G PHOSPHOLIPID HG PHOSPHOROUS G PHOSPHDLIPID 10.000» "‘0 : 27000 : fine ¢ 0.000 , 0.000 0 52.000 ' 4.000 ' 0.000 1 8.000 STORRGE TINEIHONTHS) STURRGE TIHE(HONTHS) Figure 7. Influence of storage at -l8°C on the stability , of phospholipids in meat model systems. ' i Legend: :3 Beef x chicken dark meat A chicken white meat 61 changes in PE appear to be indicative of the extent of lipid oxidation. The greater stability of PE in chicken white meat phospholipids may thus contribute to the low TBA values observed herein. The fatty acid profiles presented later herein verifies this conclusion. Ph0§phatidyl Choline (PC) Highly significant (P < 0.01) decreases (Table 7 and Figure 7) were observed during freezer storage for PC in chicken dark and white meat, but the decrease observed for beef was not significant (P > 0.05). The concentration of PC declined 70.8% in chicken dark meat, 52.6% in chicken white meat and 18.3% in the beef during freezer storage. Corliss and Dugan (1970) and Tsai and Smith (1971) have reported that phosphatidyl choline (PC) does not exert any pro-oxidant effect. However, Acosta gt al. (1966) reported that of the phospholipid fraction, PC was most active in early stages of autoxidation. It is well known (Hornstein gt_al., 1961; Keller and Kinsella, 1973, Body and Shorland, 197M) that PE is much more highly unsaturated than PC, and therefore, would be expected to be more sus- ceptible to autoxidative degradation (Tsai and Smith, 1971). Total Phospholipids (TP) The concentrations of total phospholipid phosphorus at 0 time and at the end of 8 months freezer storage are 62 also presented in Table 7 as well as in Figure 7. Total phospholipids did not change in beef during 8 months frozen storage, but decreased by 56.7 and 39.6% in dark meat and chicken white meat. The apparent stability of PC and total phospholipids observed in beef model meat systems containing added phos- pholipids were not related to the composition of their fatty acid profiles as can be seen later herein. Thus, PC and total phospholipids from beef were more stable during freezer storage than would be predicted from changes in their fatty acid profiles. Changes in Fatty Acid Composition Due to Storage It has been aptly demonstrated by Lea (1953) and Keller and Kinsella (1973) that changes in the fatty acid composi- tion of lipids provide a good indirect measure of the ex- tent of lipid oxidation. Thus, the composition of the fatty acids in triglycerides, total phospholipids and individual phospholipids (PC and PE) were determined prior to frozen storage and after 8 months storage. Beef Triglycerides The composition of fatty acids in beef triglyceride is presented in Table 8. Prior to frozen storage the levels of saturated, mono- di- and poly-unsaturated fatty acids Table 8. Changes in the fatty acid composition of triglycerides during frozen (-18°C) storage of model meat systems (0-8 months).8 Beef Chicken Dark Meat Chicken White Meat Prior to Frozen Prior to Frozen Prior to Frozen Fatty Acid Storage Storage Storage Storage Storage Storage (8 mo.) (8 mo.) (8 mo.) 12:0 --- -—- ..-- ---- ---- ---- 10:0 3.15 2.63 0.50 0.98 0.80 0.82 10:1 ---- ---- ---- ---- ---- ---- 15:0 ---- 0.33 ---- ---- 0.80 ---- 16:0 25.26 20.06 22.06 20.80 25.10 21.914 16:1 3.62 0.30 3.70 6.15 2.77 3.27 16 :2 --- 1 . 68 ---- —--- ---- o. 39 17:0 1.02 0.89 ---- 1.18 ---- 0.05 18:0 22.60 22.08 7.57 8.96 8.30 8.65 18:1 02.19 39.93 39.07 02.91 30.32 39.07 18:2 1.2? 2.63 25.03 10.27 20.93 21.52 18:3I6 --- 0.13 ---- ---- ---- 0.30 18:30:33 0.85 1.30 1.63 0.70 2.90 1.20 20:1 ---- ---- ---- ---- ---- ---- 20:2 ---- —--- ---- ---- ---- 0 . 22 20: 3 an ---- ---- ---- ---- 0 . 17 20:0 ---- ---- ---- ---- ---- 1.96 1 Saturated 52.07 09.99 30.57 35.92 35.08 31.86 S Monoenoic 05.81 00.27 02.77 09.06 37.09 02.30 1 Dienoic 1.27 0.31 25.03 10.27 20.93 21.91 I Polyenoic 0.85 1.03 1.63 0.70 2.90 3.89 ggzziuration 07.93 50.01 69.03 60.07 60.92 68.10 aAs percent of total fatty acids. 60 were 52.07, 05.81, 1.27 and 0.85%, respectively. These values are in good agreement with data presented by Horn— stein et a1. (1967). They reported that beef triglycerides varied from 00.09-60.1% for the saturated fatty acids, 37.53- 50.96% for monounsaturated, 1.10-3.02% for the diunsaturated and 0.10-2.61% for the polyenoic fatty acids. Some changes in the fatty acid profile of the tri- glycerides were observed at the end of 8 months freezer storage. The dienoic acids increased from 1.27 to 0.31% during freezer storage, while the polyenoic acids increased from 0.85 to 1.03%. Consequently, there was a corresponding increase of 0.5% in total unsaturation from the original value at the end of frozen storage. Although there was some increase in unsaturation of the fatty acids during frozen storage of the beef triglycerides, they were relatively stable in comparison to the phospho- lipid fraction (Table 9). It is known that beef tri- glycerides are highly saturated and as such are slow to oxidize (El-Gharbawi, 1965; Chipault and Hawkins, 1971). This probably explains the long induction period observed (Figure 0) in beef samples containing added triglycerides. Chicken Dark Meat and White Meat Triglycerides The initial levels of saturated, mono-, di- and poly- unsaturated fatty acids (Table 8) in chicken dark meat triglycerides were 30.57, 02.77, 25.03 and 1.63%, respectively. 65 The corresponding levels of the same fatty acids in chicken white meat were 35.08, 37.09, 20.93 and 2.90%, respectively. Hence chicken dark meat and white meat triglycerides have a similar composition of fatty acids except for a slightly greater amount of saturated fatty acids in the white meat. Katz gt gt. (1966) reported that the fatty acid composition of the triglycerides in chicken dark meat or white meat varied from 30-32% for saturated, 01-03% for monounsaturated, 25—26% for diunsaturated and 2-3% for the polyunsaturated fatty acids, respectively. Unlike the beef triglycerides, 018:2 and C18z3 fatty acids decreased in chicken dark meat and white meat tri- glycerides during storage. The dienoic acids (principally C18z2) decreased from the original value by 03 and 10% in the dark and white meat triglycerides, respectively. In addition, polyenoic fatty acids decreased from the original value by 56% in dark meat but increased by 30% in the white meat triglycerides during 8 months frozen storage. At the end of storage, total unsaturation had decreased from the original value by 7.72% in the dark meat but increased by nearly 5% in the white meat. Since the unsaturated fatty acids in chicken white meat triglycerides were stable during storage, they probably accounted for the low TBA values (Table 6) obtained for samples containing added white meat triglycerides. The observed decreases in di- and polyenoic fatty acids in 66 chicken dark meat triglycerides, on the other hand, seem to reflect the high TBA values (Table 5) observed in the triglyceride treated samples. Stability of Fatty Acids in Total Phospholipids The fatty acid composition of total phospholipids was measured prior to frozen storage and again at the end of storage. The composition of the fatty acid profiles of beef, chicken dark and white meat samples containing added phospholipids is presented in Table 9. The initial levels of saturated, mono-, di- and poly- enoic fatty acids in beef total phospholipids were 30.98, 37.57, 11.95 and 15.50%, respectively. Hornstein gt gt. (1967) reported the amount of corresponding fatty acids in beef phospholipids to be 28.32-39.59, 23.87-00.32, 11.90— 27.70 and 8.15-20.77%, respectively. Thus, the data pres- ented in Table 9 are in accord with these values. The initial values of the saturated, mono-, di-, and polyenoic fatty acids obtained in chicken dark meat phos- pholipids were 30.27, 21.08, 21.51 and 22.70%, respec- tively. The initial values for the corresponding fatty acids in the chicken white meat samples were 35.67, 27.11, 20.93 and 16.28%, respectively. Thus, the white meat was 5.63% higher in monounsaturates but approximately 6.5% lower in the polyunsaturates than the dark meat. These values generally agree with data reported by Katz gt gt. 67 Table 9. Changes in the fatty acid composition of total phospholipids during frozen storage (-18°C) of model meat systems (0-8 months).8 Beef Chicken Dark Meat Chicken White Meat Prior to Frozen Prior to Frozen Prior to Frozen Fatty Acid Storage Storage Storage Storage Storage Storage (8 mo.) (8 mo.) (8 mo.) 12:0 --- ---- —--- ---- ---- ---- 10:0 1.06 1.03 0.10 1.63 1.50 0.70 10:1 0.52 0.67 -—- 0.05 ---- 0.16 15:0 1.16 2.80 0.37 0.20 1.90 0.20 16:0 18.09 26.15 15.60 20.93 21.90 16.55 16:1 3.61 3.83 1.05 3.66 1.09 2.21 16:2 0.70 1.09 --- 1.22 ---- 0.20 17:0 0.92 3.07 ---- 2.00 --- 0.86 18:0 12.95 10.01 18.16 18.93 10.25 15.29 18:1 33.00 30.05 20.03 35.82 25.62 27.01 18:2 10.52 7.60 21.51 6.92 20.32 21.50 18:3v5 0.37 0.36 0.37 0.37 ~--- 0.20 18:3w3 1.29 1.95 0.01 0.98 0.57 0.70 20:1 --- 0.20 ---- 0.20 ---- ---- 20:2 0.69 --- ---- ---- 0.61 0.30 20:3 2.77 0.60 0.53 0.20 1.20 0.72 20:0 8.51 1.22 17.01 1.97 11.26 9.67 20:5 0.76 0.09 --- ---- 0.03 0.18 22:3 ---- ---- ---- ---- ---- ---- 22:0 0.88 --- 1.23 ---- ---- 1.37 22:5w6 -..- --.. ---- ---- 2.22 0.50 22:5w3 0.92 -- --- ---- 0.60 0.50 22:6 --- --- 2.79 ---- ---- 1-02 5 Saturated 30.98 07.86 30.27 08.17 35.67 33.60 I Monoenoic 37.57 38.79 21.08 00.17 27.11 29.38 I Dienoic 11.95 8.69 21.51 8.10 20.93 22.00 I Polyenoic 15.50 0.66 22.70 3.52 16.28 10.98 ggzziuration 65.02 52.10 65.73 51.83 60.32 66.00 ‘Percent of total fatty acids. 68 (1966). In the present study, the amount of total un— saturation prior to frozen storage was 65.02, 65.73 and 60.32% in beef, chicken dark and white meat, respectively. Thus, there was a close similarity in the fatty acid pro- files for the phospholipids associated with different types of muscle, which is in agreement with reports by Body gt gt. (1966), Ansell and Hawthorne (l960)and Body and Shor- land (1970). Significant losses were observed in the PUFAS assoc- iated with the phospholipids (Table 9). During frozen storage of beef and chicken dark meat samples containing added phospholipids, the Cl8z2 fatty acid decreased by 28.0 and 68.0% from the original value, respectively. On the other hand, it increased by 6.0% in chicken white meat. Arachidonic acid (020:0) decreased from the original value by 86% in beef, 89% in chicken dark meat but by only 10% in white meat. Total PUFAS decreased from the original values by 69.90, 80.52 and 7.98% in beef, chicken dark meat and white meat, respectively. Consequently, the levels of total unsaturation at the end of 8 months freezer storage were 52.10, 51.83 and 66.00% in beef, chicken dark meat and white meat, respectively. Although both beef and chicken dark meat showed a dramatic decline in the amount of PUFAS during 8 months frozen storage, indicating ex- tensive oxidation, chicken white meat showed little change. These results verify the relative stability of white meat 69 during freezer storage and support the observed instability of beef and chicken dark meat samples. The changes observed in the fatty acid profiles of the phospholipids provide good evidence for the involvement of PUFAS in the development of oxidized flavors. In general, significant decreases were observed in the polyenoic acids of the phospholipids in beef and chicken dark meat samples which would reflect their high TBA values (Tables 0 and 5). On the other hand, the low TBA values obtained for chicken white meat samples (Table 6) can be attributed to the great- er stability of their PUFAS. Lea (1953, 1957), Younathan and Watts (1960), Horn- stein gt gt. (1961) and Keller and Kinsella (1973) have concluded that changes in PUFAS of the phospholipids, and especially of arachidonic acid, result in serious flavor problems. Thus, the results of this study provide con- vincing evidence for the involvement of phospholipids in development of oxidized flavor during storage of frozen meat, particularly in beef and chicken dark meat. Changes in the Fatty Acid Profiles of PC and PE In studying the mechanisms of autoxidative degradation of lipids during frozen storage of meat, the composition of the fatty acid profiles in PC and PE associated with total phospholipids was also determined before and follow- ing 8 months of frozen storage. Results are presented 70 in Tables 10 and 11 for PC and PE, respectively. The most remarkable changes in the fatty acids of PC and PE occurred in the polyenes in general, and in the 020:0 and 022:0 fatty acids in particular. The levels of 020:“ in PC and PE for beef declined from 20.30 to 0.20% and from 32.75 to 1.23%, respectively. Thus, a loss of 99 and 96% of C20:0 from the original value occurred during freezer storage for PC and PE, respectively. Sim- ilarly, C20:0 decreased by 96 and 70% in the PC and PE of chicken dark meat. For chicken white meat the levels of 020:0 in PC and PE also declined during frozen storage, the decreases being 76.0 and 50.23% from the original values, respectively. The initial amount of 022:0 had disappeared from both PC and PE by the end of frozen storage in all meat samples. In the beef samples, the levels of PUFAS declined by 59.22 and 60.00% from the original values in PC and PE during freezer storage, respectively. In the chicken dark meat, PUFAS also decreased, with declines of 61.22 and 29.00% in the PC and PE during frozen storage, respectively. Polyenoic acids also declined in the chicken white meat samples during storage with losses of 27.95 and 02.80% from the original values for PC and PE, respectively. The data presented in Tables 10 and 11 clearly indi- cate that polyenoic acids are not stable and undergo auto- oxidative degradation. Lea (1953) reported that unsaturation Table 10. Changes in the fatty acid composition of PC during frozen storage 0-18°C) of model meat systems (0-8 months).a Beef Chicken Dark Meat Chicken White Meat Prior to Frozen Prior to Frozen Prior to Frozen Fatty Acid Storage Storage Storage Storage Storage Storage (8 mo.) (8 mo.) (8 mo.) 12:0 6.05 --- --.. ---- ---- ---- 10:0 5.10 0.36 --— ---- 0.30 ---- 10:1 --- 0.16 --- --- 1.38 ---- 15:0 1.00 1.09 0.93 0.03 1.55 ---- 15:1 ---- 0.52 ---- 1.28 ---- 2.10 16:0 21.97 26.39 19.70 26.00 17.80 18.76 16:1 0.73 1.90 ---- 1.03 --- 1.35 16:2 ---- 0.79 --- 0.51 ---- 0.36 17:0 1.90 1.03 3.35 0.69 1.72 1.05 18:0 10.53 10.06 17.10 18.50 12.07 17.79 18:1 23.73 23.78 15.05 25.70 10.90 18.76 18:2 0.08 7.77 10.78 10.79 10.11 11.50 18:3w6 ---- 0.08 0.02 0.90 0.52 ---- 18:3!3‘ 0.27 1.39 0.35 1.03 0.00 2.00 20:1 -- 0.28 --- —-- --- --- 20:2 0.78 15.03 2.76 3.03 5.52 3.00 20:3 ---- ---- 0.20 ---- ---- 0.80 20:0 20.30 0.20 17.80 0.72 16.66 3.97 20:5 --- 7.13 0.28 8.95 11.89 17.67 22:3 --—- ---- ---- --- ---- ---- 22:0 3.03 -—- 10.78 ---- 5.06 ---- 22:5w6 ---- ---- ---- ---- ---- —--- 22:50:3 ---- 0.00 ---- ---- ---- ---- 22:6 ---— ---- ---- ---- ---- ---- 1 Saturated 06.63 00.13 01.12 05.66 33.52 38.00 1 Monoenoic 20.06 26.36 15.05 28.01 16.32 22.21 S Dienoic 5.26 23.87 13.50 10.73 15.63 10.90 x Polyenoic 23.60 9.60 29.91 11.60 30.53 20.88 Egggiuration 53.36 59.87 58.90 50.30 66.08 61.99 8Percent of total fatty acids; PC - phosphatidyl choline. Table 11. Changes in the fatty acid composition of PE during frozen storage (-18°C) of model meat systems (0-8 months).3 Beef Chicken Dark Meat Chicken White Meat Prior to Frozen Prior to Frozen Prior to Frozen Fatty Acid Storage Storage Storage Storage Storage Storage (8 mo.) (8 mo.) (8 mo.) 12:0 0.08 ---- ..-- ---- ---- ---- 10:0 1.55 1.23 0.25 0.09 1.80 0.30 10:1 --—- 0.30 0.33 0.16 1.56 0.10 15:0 0.33 0.05 1.60 0.65 0.10 0.63 15:1 -- 0.92 ---- 0.72 ---- 0.62 16:0 13.96 21.08 15.73 13.28 13.85 16.60 16:1 1.65 2.01 ---- 0.65 ---- 1.00 16:2 -- 1.68 ---- 1.23 ---- 0.58 17:0 1.09 2.20 2.70 1.08 2.25 2.98 18:0 10.89 17.01 23.23 22.68 13.60 21.36 18:1 19.50 20.80 16.39 22.88 15.92 21.17 18:2 0.80 8.39 11.80 12.82 11.08 10.97 18:3w5 --- --- 0.33 1.15 0.28 0.07 18:3w3 0.37 1.96 0.28 1.32 0.33 0.96 20:1 -- ---- ---- 0.26 ---- 0.58 20:2 1.06 0.92 1.06 3.90 5.97 1.56 20:3 0.37 --- 0.23 0.79 0.28 0.38 20:0 32.75 1.23 18.68 0.83 17.52 8.72 20:5 --- 10.70 ---- 9.73 7.58 3.06 22:3 ---- 0.56 0.35 --- ---- ---- 22:0 6.80 ---- 5.90 0.99 3.79 1.00 22:5w5 ---- ---- ---- ---- ---- 0.36 22:50:3 ---- ---- 0.57 ---- ---- 0.58 22:6 ---- ---- 0.09 0.35 ---- 1.06 1 Saturated 32.70 02.01 03.51 38.18 35.68 01.91 1 Monoenoic 21.15 28.11 16.72 20.67 17.08 27.95 1 Dienoic 5.86 10.99 12.90 17.99 17.05 13.11 S Polyenoic 00.29 10.09 26.87 19.16 29.78 17.03 ggggiurated 67.30 57.59 56.09 61.82 60.31 58.09 8Percent of total fatty acids; PE - phosphatidyl ethanolamine. 73 rapidly disappears at advanced stages of autoxidation. Therefore, the PUFAS appear to be principally responsible for development of rancidity, which is in agreement with earlier reports by Lea (1953, 1957), Hornstein gt gt. (1961) and Keller and Kinsella (1973). Results indicate that PC and PE both play an important role in the develop- ment of rancid flavors in stored meat and meat products. Data presented for TBA values (Tables 0 and 5) and the changes in unsaturation of the PUFAS (Tables 9, 10 and 11) confirm the positive relationship between rancidity and oxidation of the fatty acids. It is well known that poly— enoic acids are extremely reactive and through oxidative degradation give rise to a number of carbonyl compounds, which greatly influence oxidized flavor (Lea, 1953, 1957; Younathan and Watts, 1960). Summary of Results The role of triglycerides, total lipids and total phospholipids on the development of rancidity during frozen storage of meat was studied using lipid-free muscle fibers in combination with added triglycerides, total lipids or total phospholipids, respectively. The experi- mental model systems were held at -18°C for 8 months. Results of this study indicated that added total lipids and total phospholipids greatly increased the TBA values. Oxidation took place in two stages. The total lipids and 70 total phospholipids were the first to oxidize and their rate of oxidation decreased with time. Oxidation of the triglycerides only began after a prolonged induction period. Lipids from chicken white meat were more stable to autoxidative degradation during frozen storage than those from beef or chicken dark meat. It is postulated that the greater amount of oxidation in beef and chicken dark meat may be due to residual meat pigments. Results showed that both triglycerides and phospho- lipids contribute to development of rancidity, but the phospholipids make the greatest contribution. The im- portance of the triglycerides in development of oxidation depends on the degree of unsaturation and the length of frozen storage. The study confirmed the positive rela- tionship between rancidity and oxidation of the PUFAS in both the phospholipids and triglycerides. 75 EXPERIMENT B Role of Triglycerides, Total Ltpids and Total Phos- pholipids on Development of WOF in Cooked Meat Model Systems The experiments to test the effect of triglycerides, total lipids and total phospholipids on development of WOF in cooked meat involved the use of lipid-free muscle fibers. The preparation of the lipid—free muscle fibers was outlined earlier herein, while the treatments are shown in Table l. The prepared samples were cooked in retortable pouches to 70°C internal temperature in boiling water. After storage for 08 hours at 0°C, the samples were tested for TBA values and were also Judged by a trained panel of 12 individuals. Mean TBA values and their corresponding mean sensory scores are presented in Table 12. Results showed that the model systems containing added total lipids gave the highest TBA values, followed by samples containing total phospholipids, triglycerides, and the lipid-free muscle fibers (control), respectively. Although the samples containing total lipids exhibited the highest TBA values, they did not consistently show the lowest flavor ratings. This is consistent with the report by Love and Peerson (1971) suggesting that neutral lipids in association 76 Table 12. TBA numbers and sensory scores for cooked beef chicken dark meat and white meat model systems.1’2 Meat Composition of Mean TBA Mean Sensory Type Treatments Numbers Score B1=0.8% phospholipids 5.76b 2.69a Beef B2=9.2% triglycerides 1.888 3.80b B3=10% total lipids 6.81b 2.603 Bu=control 1.16a 3.75b Chicken Dl=0.70% phospholipids 8.08b 2.97a Dark Meat D2=0.3% triglycerides 6.30a’b 3.65a’b 03=5% total lipids 11.70c 3.25a Du=control 5.78a 3.888"b Chicken wl=o.7% phospholipids 5.03b 2.80a White Meat w2=0.3% triglycerides 2.99a 3.35a W3=5% total lipids 5.53b 3.09a Wu=control 2.18a 0.02b 1There were 0 replicates for each treatment. 2Taste panel score were from 1-5, where l=very pronounced WOF and 5=No WOF. 3All numbers in same column within a meat type bearing the same superscript are not significant at the 5% level. 77 with polar lipids may trap volatile decomposition products of polar lipids and thus reduce their effect on flavor. Analyses of variance for TBA values and sensory scores revealed that the experimental treatments were significantly (P < 0.001) different. Tukey's test for multiple comparisons clearly showed that total lipids and total phospholipids significantly (P < 0.01) increased TBA values while significantly (P < 0.05) reducing flavor scores. Thus, total lipids and total phospholipids are major contributors to development of WOF in cooked meat. Results showed that neither the model system (control) nor the triglycerides alone significantly influenced development of WOF in cooked meat. The results of this study clearly provide evidence for the involvement of total phospholipids in development of WOF in cooked meat. Thus, this study confirms the work of Wilson gt gt. (1976), who first implicated total phospholipids as being responsible for development of WOF in cooked meat. Al- though triglycerides alone did not significantly influence the development of WOF in cooked meat, they did have an additive effect in combination with phospholipids (Table 12). Statistically significant (P < 0.05) correlation coefficients of r = —.57 and r = -.51 were found between TBA values and sensory scores for beef and chicken white 78 meat model systems, respectively. These results confirm the existence of a relationship between WOF and panel scores. They are in agreement with the data presented by Zipser gt gt. (1960), who reported a close relation- ship between TBA values and development of oxidized flavor in cooked meat. In the case of chicken dark meat, however, the cor- relation between TBA values and sensory scores was not statistically significant. The reason for this discrep- ancy may be due to the apparent retention of lipids by the extracted muscle fibers. Chicken dark meat fibers (control) had a higher mean TBA value of 5.78 in compari- son to values of 1.16 and 2.18 for beef and chicken white meat, respectively. The high TBA value for the chicken dark meat control indicates that the extracted muscle fibers probably contain some unextracted bound lipids. Any residual bound lipids may cause a discrepancy in sensory scores for the control samples. There is, however, no evident discrepancy in sensory scores, which suggests the major effect is upon TBA values. However, the relatively high TBA value for the control would decrease the relationship between TBA values and panel scores. 79 Changes in Phospholipid Components Due to Cooking and Storgge at 0°C Quantitative and compositional changes in the com- ponents of the total phospholipids were determined before and after cooking using the phosphorus assay procedure of Rouser gt gt. (1966). Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) combined constitute over 75% of the total phospholipids in meat, in addition to being highly unsaturated (Peng and Dugan, 1965; Body and Shorland, 1970). Hence, the changes in PC and PE will be discussed in detail in order to elucidate the role of phospholipids in development of WOF in cooked meat. The compositional changes in the phospholipid com- ponents as a result of cooking are shown in Table 13. The level of PE relative to total phospholipids prior to cooking was 18.5% for both beef and chicken dark meat but was 23.9% in chicken white meat. These values are in close agreement with data reported by several workers (Lea, 1957; Peng and Dugan, 1965; Lee and Dawson, 1976). The initial proportion of PC relative to total phospho- lipids was 50.7% in beef, 09.1% in chicken dark meat and 56.7% in white meat. These values are in good agree- ment with those presented by Peng and Dugan (1965), David Kova and Khan (1967) and Keller and Kinsella (1973). Results indicated that cooking caused significant (P < 0.01) decreases in total phospholipids. Analysis 80 Table 13. Mean changes in phospholipids due to cooking and storage at 0°C.1’ ’3 Chicken Dark Meat Chicken White Meat Classes Beef Model System Model System Model System of Phospholipids Fresh Cooked Fresh Cooked Fresh Cooked ch4 2.23 0.97 2.55 1.44 1.86a 1.15a 905 3.57a 2.78a 5.81 2.55 7.91 4.00 906 10.57 5.73 15.488 13.48a 18.81 10.86 Others7 3.0 1.16 7.67 10.24 4.58 7.97 Tp8 19.34 10.64 31.51 28.14 33.16 23.98 1Values given are mg phosphorus/g phospholipid. 2There were 0 replicates for each component. 3All numbers in same column within a given meat type bearing the same superscript are not significant at 5% level. 4LPC = Lysophosphatidyl choline. 5PE = phosphatidyl ethanolamine. 6P0 = Phosphatidyl choline. 7Other phospholipids were calculated by difference. 8Total phospholipids. 81 of variance shows highly significant (P < 0.01) inter- actions in the content of total phospholipids between the fresh and cooked state. The significant interactions indicate that PC and PE did not consistently behave in the same manner during cooking. While a significant de- crease in PE was not observed in beef; PC was found to decrease by 05.0% after cooking. Changes in PE and PC in chicken dark meat were opposite to that for beef, i.e., PE significantly decreased while PC was stable during cooking and storage. In chicken white meat, highly sig— nificant (P < 0.01) losses occurred in both PE and PC during cooking and storage. Results showed that total phospholipids from all types of meat significantly decreased during cooking and storage. Phosphatidyl ethanolamine (PE) was less stable to cooking and storage than phosphatidyl choline (PC). This is further verified by the fatty acid composition for PC and PE as will be discussed later herein. Decreases in the component phospholipids may be due to either auto- oxidation, hydrolytic decomposition, lipid browning re- actions or to lipid-protein co-polymerization as outlined by Lea (1957). Products of lipid autoxidative degrada- tion have been associated with the development of off- flavors, principally with WOF (Ruenger gt gt., (1978). 82 Changes in Fatty_Acid Composition as a Result of Cooking Triglycerides The fatty acid profiles of the samples containing added triglycerides were determined prior to cooking and again following cooking and storage. The values are presented in Table 10. For beef samples, the saturated fatty acids decreased from 52.07 to 09.07%, while the monoenes in- creased from 05.81 to 09.29% to give a net increase of 5.02% in total unsaturation. In the chicken samples containing added triglycerides, total unsaturation increased from 69.02 to 71.56% in dark meat, and from 60.92 to 68.99% in the white meat. These results are consistent with reports showing that only minor changes take place in the fatty acid content of the triglycerides as a result of cooking (Chang and Watts, 1952; Giam and Dugan, 1965; Campbell and Turkki,l967). Thus, the stability of the triglycerides is consistent with the low TBA values (Table 12). Total Phogpholipids Compositional changes in the fatty acid profiles in total phospholipids of meat model systems due to cooking and storage are shown in Table 15. In beef the levels of saturated fatty acids were unchanged. The monoenes in- creased slightly, the dienes from 11.95 to 13.12%, while 83 Table 14. Mean fatty acid composition of triglycerides in model meat systems.a Beef Chicken Dark Meat Chicken White Meat Fatty Prior to After Prior to After Prior to After Acid cooking Cooking Cooking Cooking Cooking Cooking 14:0 3.15 1.86 0.54 0.58 0.84 0.63 14:1 ---- 0.80 --- ---— ---— -—-— 15:0 ---— ---- —--- ---- 0.80 ---- 16:0 25.26 32.92 22.46 19.94 25.14 23.08 16:1 3.62 4.20 3.70 4.08 2.77 3.85 17:0 1.02 0.53 --- 0.69 ---- ---- 18:0 22.64 14.16 7.57 7.23 8.30 7.30 18:1 42.19 44.29 39.07 41.27 34.32 39.49 18:2 1.27 1.24 25.03 24.48 24.93 23.94 18:3w6 ---- ---- ---- -——- ---- 1.20 18:ew3 0.85 ~--- 1.63 1.73 2.90 0.51 Z Sat. 52.07 49.47 30.57 28.44 35.08 31.01 Z Mono. 45.81 49.29 42.77 45.35 37.09 43.34 Z Dienoic 1.27 1.24 25.03 24.48 24.93 23.94 Z Polyenoic 0.85 ---- 1.63 1.73 2.90 1.71 Total Unsat. 47.93 50.53 69.43 71.56 64.92 68.99 8Percent of total fatty acids. (1" Table 15. Changes in the fatty acid composition of total phospholipids of meat model systems due to cooking and storage at 0°C.a Beef Chicken Dark Meat Chicken White Meat Prior to After Prior to After Prior to After Fatty Acid Cooking Cooking Cooking Cooking Cooking Cooking 10:0 1.06 0.72 0.10 0.17 1.50 0.87 10:1 0.52 ---- ---- ---- ---- --- 15:0 1.16 1.86 0.37 0.57 1.90 0.69 16:0 18.09 18.73 15.60 16.52 21.90 18.98 16:1 3.61 3.67 1.05 1.26 1.09 2.76 16:2 0.70 0.25 ---- -- --- --- 17:0 0.92 0.80 ---- -- ---- 1.07 18:0 12.95 12.83 18.16 21.03 10.25 10.70 18:1 33.00 30.81 20.03 19.70 25.62 33.87 18:2 10.52 12.66 21.51 19.22 20.32 16.65 18‘3'6 0.37 --- 0.37 0.23 ---- ---- 18:30:3 1.29 0.69 0.01 0.03 0.57 0.85 20:2 0.69 0.21 --- --- 0.61 0.56 20:3 2.77 1.92 0.53 0.52 1.20 0.62 20:0 8.51 9.32 17.01 18.88 11.26 6.90 20:5 0.76 --- ---- --- 0.03 1.62 22:0 0.88 0.02 1.23 0.57 ---- 0.36 22:5w6 ---- --- —--- ---- 2.22 2.15 22:5w3 0.92 1.08 --- ---- 0.60 1.30 22:6 ---- ---- 2.79 0.86 --- ---- 1 Saturated 30.98 30.98 30.27 38.29 35.67 32.35 I Monoenoic 37.57 38.08 21.08 21.00 27.11 36.63 S Dienoic 11.95 13.12 21.51 19.22 20.93 16.65 S Polyenoic 15.50 3.02 22.70 20.83 16.28 10.37 83:280rated 65.02 65.02 65.73 61.05 60.32 67-65 8Percent of total fatty acids. 85 the polyenes decreased from 15.50 to 13.02%; a loss of 13.02% in unsaturation from the original value. This indicates that the polyenes are important in the develop- ment of oxidized flavor. The changes observed in the fatty acid profiles in chicken dark and white meat phospholipids are also shown in Table 15. The level of 018:2 fatty acid decreased in both chicken dark and white meat during cooking. Arachi- domi acid (020:0) increased from 17.01 to 18.88% in chicken dark meat but decreased from 11.26 to 6.90% in white meat. The level of polyenoic acids decreased from 22.70 to 20.83% and from 16.28 to 10.37% in chicken dark meat and white meat, respectively. Although total unsaturation was not drastically affected by cooking and storage, nevertheless, the moderate changes that occurred in both the dienoic and the polyenoic fatty acids may be important in development of WOF. Sev— eral workers (Lea, 1957; Younatham and Watts, 1960; Keller and Kinsella, 1973) have reported that the PUFAS may be involved in the heat induced degradation of lipids which results in off—flavors. Recently, Ruenger gt gt. (1978) have reported that WOF is a result of autoxidation of tissue lipids. Thus, the present study demonstrates that decreases in total phospholipids as well as in PUFAS are related to the development of WOF in cooked meat. 86 Changes in PC and PE in Beef Phogpholipids The fatty acid profiles for PC and PE in beef phos- pholipids are presented in Table 16. Following cooking, total unsaturation in PC increased from 53.36 to 65.00%, mainly at the expense of the saturated fatty acids. Sig- nificant increases were also evident in the dienoic and polyenoic acids following cooking and storage. Specific- ally, C18:2’ 020:0 and 022:0 fatty acids increased from the original value by 106.0, 30.0 and 59.1%, respectively. In PE, however, total unsaturation decreased from 67.30 to 60.67%. Similar to PC, however, the 018:2 and 020:“ fatty acids increased by 103.1 and 10.0% from the original value, respectively. In PE, however, C20:2 and 022:“ decreased by 70.5 and 55.1% from the original value, respectively. These results clearly show that PE is less stable than PC, and therefore, it probably contributes more to development of WOF. Keller and Kinsella (1973) provided data showing that PE was much more highly unsaturated and much less stable than PC. Results of this study clearly revealed that PE is significantly more unsaturated than PC. The levels of PUFAS were 00.29 and 23.60% for PE and PC, respectively. This is in agreement with the data presented by Body and Shorland (1970) showing that the levels of PUFAS were from 17 to 03% and 7-25% in PE and PC, respectively. Table 16. Changes in fatty acid composition of phosphatidyl choline and phos- phatidyl ethanolamine of beef model system.a Phosphatidyl Choline (PC) Phosphatidyl Ethanolamine (PE) Prior to After Prior to After Fatty ACid Cooking Cooking Cooking Cooking 12:0 6.05 0.38 0.08 0.95 10:0 5.10 0.00 1.55 1.00 10:1 ---- ---- ---- 1.08 15:0 1.00 0.32 0.33 2.50 16:0 21.97 10.81 13.96 10.02 16:] 0.73 0.95 1.65 ---- 17:0 1.90 0.76 1.09 2.78 18:0 10.53 17.85 10.89 16.79 18:1 23.73 20.25 19.50 11.56 18:2 0.08 9.23 0.80 9.75 18:3w6 ---- ---- ---- 0.33 18:3W3 0.27 ---- 0.37 0.28 20:1 ---- --—- ---- ---- 20:2 0.78 2.07 1.06 0.27 20:3 ---- 0.38 0.37 0.78 20:0 20.30 27.30 32.75 37.35 20:5 ---- ---- ---- 0.76 22:0 3.03 0.82 6.80 3.05 22:5 ---- --- ---- 0.31 1 Saturated 06.63 30.56 32.70 30.08 S Monoenoic 20.06 21.20 21.15 12.60 1 Dienoic 5.26 9.23 5.86 10.02 I Polyenoic 23.60 35.01 00.29 02.56 Egggéuration 53.36 65.00 67.30 60.67 8Percent of total fatty acids. 88 Changes in PC and PE in Chicken Dark Meat The fatty acid profiles of PC and PE for chicken dark meat phospholipids are presented in Table 17. Total unsaturation in PC increased from 58.90 to 60.99% follow- ing cooking and storage. A slight increase in total unsaturation of PE (1.59%) also occurred. Unlike beef, linoleic acid (C ) decreased in both PC and PE of 18:2 chicken dark meat. Arachidonic acid increased from 17.80 to 21.10% in PC but decreased from 18.68 to 13.95% in PE, representing a loss of 25.32% from the original value. This is in agreement with the work of Keller and Kinsella (1973) who reported a decrease of about 25% in 020:“ fatty acid following cooking of beef. Changgs in PC and PE of Chicken White Meat The fatty acid profiles recorded for PC and PE in chicken white meat are presented in Table 18. In PC, 018:2, 020:0, C20:5 and C22,“ fatty acids decreased by 61.30, 53.60, 27.75 and 23.52% from the original value, respectively. For PE, C18:2 decreased from 11.08 to 8.92%, C20:2 from 5.97 to 1.22% and C20,“ decreased from 17.52 to 3.36% during cooking and storage. The significant loss of C20,“ underlines its vulnerability to cooking, especially in PE. On the other hand, C increased from 7.58 to 9.11%, 20:5 Table 17. Changes in fatty acid composition of phosphatidyl choline and phos- phatidyl ethanolamine of dark meat model system.a Phosphatidyl Choline (PC) Phosphatidyl Ethanolamine (PE) Fatty Acid Prior to After Prior to After Fatty Acid Cooking Cooking Cooking Cooking 10:0 ---- ---- 0.25 0.22 10:1w6 ---- 0.53 0.33 ---- 15:0 0.93 0.92 1.60 1.53 16:0 19.70 15.56 15.73 16.70 16:1w7 —--- ---- ---- ---- 16:2 ---- ---- ---- ---- 17:0 3.35 1.98 2.70 2.70 18:0 17.10 15.75 23.23 20.95 18:1w9 15.05 10.56 16.39 23.00 18:2w6 10.78 8.39 11.80 6.01 18:3w6 0.02 0.53 0.33 0.60 18:3w3 0.35 0.27 0.28 1.70 20:2w6 2.76 3.90 1.06 0.75 20:3w6 0.20 0.20 0.23 0.26 20:0w6 17.80 21.10 18.68 13.95 20:5w3 0.28 2.32 ---- 0.03 22:3w6 ---- ---- 0.35 0.75 22:“"6 10078 13091 509“ Sous 22:5w6 ---- ---- 0.57 0.56 22:6w3 ---- ---- 0.09 ---- 1 Saturated 01.12 30.21 03.51 01.92 1 Monoenoic 15.05 15.09 16.72 23.22 1 Dienoic 13.50 12.33 12.90 7.16 1 Polyenoic 29.90 37.57 26.87 27.70 Total Unsaturation 58.90 60.99 56.09 58.08 aPercent of total fatty acids. Table 18. Changes in fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine of white meat model system.a Phosphatidyl Choline (PC) Phosphatidyl Ethanolamine Prior to After Prior to After Fatty Acid Cooking Cooking Cooking Cooking 10:0 0.30 ---- 1,80 ---- 10:1 1.38 ---- 1.56 ---- 15:0 1.55 0.97 0.10 0.07 16:0 17.80 15.50 13.85 15.02 16:1 ---- 1.72 ---- 3.39 16:2 --- ---- ---— ---- 17:0 1.72 0.07 2.25 3.30 18:0 12.07 9.05 13.60 10.02 18:1 10.90 20.79 15.92 23.13 18:2 10.11 3.91 11.08 8.92 18:3w6 0.52 ~--- 0.28 ---- 18:3w3 0.00 6.00 0.33 6.68 20:2 5.52 9.71 5.97 1.22 20:3 ---- 0.12 0.28 3.36 20:0 16.66 7.73 17.52 3.36 20:5 11.89 8.59 7.58 9.11 22:3 --- ---- ---- ---- 22:0 5.06 3.87 3.79 7.20 22:5w6 ---- ---- ---- 0.35 22:5w3 --- 1.68 ---- ---- 22:6 ---- ---- ---- ---- 1 Sat. 33.52 31.03 35.68 33.25 1 Monoenoic 16.32 22.51 17.08 26.52 1 Dienoic 15.63 13.62 17.05 10.10 % Polyenoic 30.53 32.03 29.78 30.10 Total Unsat. 66.08 68.56 60.31 66.76 8Percent of total fatty acids. 91 while C22z0 increased from 3.79 to 7.20% and 018:3 in- creased from 0.33 to 6.68% during cooking and storage. The Importance of Phospholipids on Develgpment of WOF Holman and Elmer (1907) attributed the tendency of the phospholipids to oxidize to their high content of 020:0, C22:3, C22:0’ 022:5 and C22:6 fatty acids. Thus, the significant changes in some specific PUFAS, which occurred in total phospholipids (Table 15) and in PC and PE (Tables 16, 17 and 18), are of great significance to development of WOF. Greater losses in PUFAS occurred in PE than in PC. Furthermore, PE was found to be less stable to cooking than PC (Table 13). Most of the losses in fatty acids were observed in the 018:2’ C20:0 and 022:“ associated with chicken dark and white meat. Love and Pearson (1971) and Keller and Kinsella (1973) indicated that the loss of C20:0 fatty acids was consistent with its greater propen- sity to undergo autoxidation, especially when associated with PE. Thus, results of this study confirm that PUFAS associated with PE tend to be more labile to heat than those of PC. Hence, PE may be more significant in the development of WOF. Decreases in other PUFAS in addition to arachidonic acid, including that of C fatty acid, 18:2 also appear to accelerate development of WOF. The observation that PUFAS associated with PE are less 92 stable to cooking than those of PC led to another experi- ment. The objective of the second experiment was to study the relative contributions of PC, PE and serum phospho- lipids in development of WOF using beef muscle fibers as the base material. Pure bovine PC, PE and serum phos- pholipids were added back to the model system at a level of 0.0375% either with or without 156 ppm of nitrite. Each of the 8 experimental treatments was prepared using 50 ml deionized water. The experimental design is shown in Table 19. The samples were cooked in retortable pouches to 70°C internal temperature and stored for 08 hrs at 0°C. After storage, TBA analyses as well as taste panel evaluation of the samples were conducted. A panel of 0 trained, high skilled individuals served as the judges. A plot of TBA numbers and sensory scores is shown graphically in Figure 8. A highly significant (P < 0.01) "r" value of -.62 was found between TBA numbers and flavor scores. Analysis of variance for treatment effect upon TBA numbers and sensory scores are presented in Table 20. Differences between experimental treatments were statis- tically significant (P < 0.001) for both TBA numbers and sensory scores. Addition of PE and serum phospholipids significantly (P < 0.01) increased TBA values while de- creasing sensory scores. The addition of PC did not 93 Table 19. The influence of PC, PE, total serum phospho- lipid and nitrite on development of WOF using beef model system.1 Treatment Preparation of Samples 1 Model system only (control) 2 Model system + nitrite 3 Model system + PC Model system + PC + nitrite Model system + PE Model system + PE + nitrite Model system + serum phospholipids OJNGUT Model system + serum phospholipid + nitrite l Phospholipids were added to the model system at a level of 0.0375% and nitrite at 156 ppm. PC = Phosphatidyl choline. PE = Phosphatidyl ethanolamine. Serum phospholipids (lysophosphatidyl choline. 8%; sphingo- myelin, 20%; PC; 53%; phosphatidyl serine, 1%; phosphatidyl inositol, 0%; PE, 6%; phosphatidyl glycerol, 2%; cardio- lipin, 1% and phosphatidic acid, 1%). 90 A = Muscle fibers only B = Muscle fibers only plus nitrite C = PC in muscle fibers D = PC in muscle fibers plus nitrite E = PE in muscle fibers F = PE in muscle fibers plus nitrite G = TP in muscle fibers H = TP in muscle fibers plus nitrite 1.0, _ 5 0.8 b a C "_4 g b O 5 n 2 b a c E” (I (D .1 ll $0.6. b d 0' c .dflh :3 b d oflpc can 5 b d odpcbodn 50.40 b d QOPCbOdn‘ é o P 51.0! oapcbgdn o P b.dn oapcbedn 0.2 . 6, ‘I I’ c h". ‘l " ‘8 ‘I I, t 8". 8' '8 ooP¢b.dn oaPCbedn oo°°P¢b d“ ocflcbsdn A B C D E F G H A B C D E F‘ G H Figure 8. TBA number and sensory scores in cooked beef model meat systems containing purified phos- pholipids and nitrite. ‘ TASTE PANEL SENSORY SCORE 95 Table 20. TBA numbers and sensory scores for cooked meat model systems containing added PC, PE, serum phospholipids or nitrite.1,2 Mean TBA Mean Sensory Experimental Treatment Number Score b + a,b 1. Model system only 0.36:0.07 3.33—0.02 2. Model system + nitrite 0.26:0.06a 0.1110.00b:° 3. Model system + PC 0.30:0.035"b 3.3610515"b 0. Model system + PC + nitrite 0.29:0.07a 0.52:0.27c Model system + PE 0.81:0.13d 2.60:0.58a 6. Model system + PE + b nitrite 0.361007b 3.6110.23 7. Model system + TP 0.6210.l2c 3.29:0.09aab 8. Model system + TP + b b nitrite 0.3010.05a’ 0.00io.03 :0 1 Each treatment was replicated 0 times. 2A significant (P<0.01) "r" value of -.62 was found between TBA numbers and sensory score. Numbers in same column bearing same superscript are not significant at 5% level. 96 significantly influence either TBA numbers or sensory scores. Addition of 156 ppm of nitrite significantly (P < 0.01) reduced TBA numbers and improved WOF scores for all samples, except for the ones with added PC, which were not significantly altered. Sato and Hegarty (1971) showed that nitrite will completely eliminate WOF at 220 ppm and will inhibit development at 50 ppm. Recently, Fooladi (1977) showed that 156 ppm of nitrite effectively prevents WOF in cooked meat and poultry. Zipser gt gt. (1960) proposed that nitrite forms a stable complex with iron porphyrins in heat denatured meat, thereby inhibiting the development of WOF. The inhibition of lipid oxidation in model systems containing only residual pigments suggests that nitrite converted the pigments to catalytically inactive forms and may have also directly reacted with unsaturated fatty acids. Thus, the results are in agreement with the report of Cassens gt gt. (1976) indicating that nitric oxide reacts with unsaturated fatty acids perhaps by chelation. Results of this study have demonstrated that PE is the most important phospholipid component contributing to development of WOF. However, the other phospholipid components also have an additive effect. Thus, results are in excellent agreement with the data presented by Corliss and Dugan (1970) and Tsai and Smith (1971). They reported that PE exerted a much greater pro-oxidant 97 effect than PC. Hence, the present study confirms the involvement of phosphatidyl ethanolamine (PE) in develop- ment of off-flavors, and especially of WOF in cooked meat and meat products. Summary of Results The effects of triglycerides, total lipids and total phospholipids on the development of WOF in cooked meat were studied using lipid-free muscle fibers in combination with added triglycerides, total lipids and total phos— pholipids. In addition, pure samples of bovine PC, PE and serum phospholipids were also added to the model system in a second part of this study. Preparations were heated to 70°C in retortable pouches, stored at 0°C for 08 hrs and evaluated by the TBA test and sensory panel scores. Changes in the fatty acids of the triglycerides, phospholipids, PC and PE were measured using GLC and TLC methods. Results showed that adding total phospholipids to the model system significantly (P < 0.01) increased TBA, while significantly (P < 0.05) decreasing flavor scores. Addition of triglycerides to the model system did not significantly (P > 0.05) influence development of WOF in cooked meat. The triglycerides, however, when combined with phospholipids as total lipids did have a significant 98 influence (P < 0.01) on TBA numbers, while reducing flavor scores. Significant (P < 0.05) correlation coefficients of r = -.57 and r = -.51 were found between TBA values and sensory panel scores for beef and chicken white meat model systems, respectively. Addition of PE and serum phospholipids significantly (P < 0.01) increased TBA values while decreasing sensory panel scores. The use of PC did not significantly in— fluence either TBA values or sensory scores. Addition of 156 ppm of nitrite significantly (P < 0.01) reduced TBA numbers and prevented development of WOF. Cooking caused significant (P < 0.01) decreases in PE of meat phospholipids while PC was rather more stable. In addition, PUFAS in PE were less stable to cooking and storage than those of PC. Thus, total phospholipids are major contributors to development of WOF in cooked meat. The triglycerides enhance WOF development only when combined with phos— pholipids. In addition, PE is the most important com- ponent of total phospholipids contributing to WOF in cooked meat. Changes in the content of PUFAS, especially in those of PE, appear to be directly related to development of WOF. However, nitrite at a level of 156 ppm prevented WOF in cooked meat. 99 EXPERIMENT C Effect of Length of Frozen Storage Time on the Stability of Meat Lipids and on Production of Off-Flavors The principal objectives of this experiment were to study how length of frozen storage of raw meat influences the composition and stability of meat lipids as well as its effect on the production of rancidity in frozen meat and WOF in frozen-cooked meat. Fresh cuts of beef (L-D), chicken dark meat and chicken white meat were frozen and stored at -l8°C for over one year. The levels of lipids were measured at 0, 8 and 13 months during frozen storage. At the same time periods, the levels of malonaldehyde produced during oxida- tion of fresh and/frozen meat as well as in cooked meat held at 0°C or -18°C for 08 hrs were measured. Levels of Extracted Lipids From Muscle Tissues Total lipids were extracted from the meat by the procedure of Folch gt gt. (1957) while the levels of triglycerides and phospholipids were determined using the method of Choudhury gt gt. (1960). 100 Beef Lipids The levels of triglycerides, total lipids and total phospholipids are presented in Table 21. Prior to frozen storage, fresh raw beef contained 13.72% total lipids, which decreased to 9.52 and 9.82% at 8 and 13 months of frozen storage, respectively. The rather high total lipid (13.72%) content of beef may be due to a high level of marbling. Wilson gt gt. (1976) reported a value of 10.79% for total lipids in a highly marbled beef cut. The results also support the report by Pearson gt _t. (1977) that total lipids in beef are highly variable, ranging from lower than 2.0 to over 12.0%. Prior to frozen storage (0 time), the levels of total lipids and triglycerides in the cooked beef held at 0°C and at -18°C were slightly lower than the amount in the fresh uncooked meat. At 8 and 13 months of frozen storage, however, the concentrations of both triglycerides and phospholipids were higher in the cooked than in the fresh meat. Results showed that both total lipids and tri- glycerides significantly (P < 0.01) decreased during frozen storage, regardless of whether it was fresh or cooked. The content of total phospholipids in raw meat were constant during frozen storage, being 0.71, 0.71 and 0.70% at 0, 8 and 13 months, respectively. Results verify the report by Pearson gt gt. (1977) that the phospholipid 101 Table 21. Effect of length of frozen storage, cooking and storage temperature upon beef lipids. Storage Time (Months) 1 Lipids Storage Temp. 5 8 13 Total Lipids Fresh/frozen 13.72i0.06a 9.52:0.60b 9.82:0.93b 4°02 110110.04a 10.01:o.17b 10.45:o.21b’° -18°c3 13.2610.203 10.011022b 10.95:o.42b’c Triglycerides Fresh/frozen Cooked and Held 12.89:0.123 8.70:0.42c 9.09:0.83c +4°c2 113310.05b 8.97:0.11c 9.33:0.04c -18°c3 12.18t0.23b 9.01:0.19c 9.35:0.43‘1’c Phospholipids Fresh/Frozen 0.71:0.03a 0.71:0.09a 0.70:0.13a +4002 0.93:0.02b 0.99:0.02b 0.94:0.01b -18°c3 0.95:0.01b 0.95:0.01b 0.98:0.01b 1Expressed as percentage of total tissue. 2Meat was cooked and held at 4°C for 48 hrs. 3Meat was cooked and held at -18°C for 48 hrs. 4 superscript are not significantly different at P<.05. Values within the same lipid group and in same row bearing the same 102 fraction of tissue is relatively constant although the fat content is highly variable. The results are also in close agreement with the report by Dugan (1971) that the level of phospholipids in meat ranges from 0.5 to 1.0%. Results also showed that cooking significantly (P<0.01) increased the amount of phospholipids in cooked beef. However, the differences in the content of phospholipids for cooked meat held at U°C and -l8°C were not statis- tically significant. Results of this study agree with the data presented by Campbell and Tutkki (1967) and Fooladi (1977), who found that the phospholipid concentra- tion was higher in cooked than in raw meat. A graphic illustration of the variations in beef lipids due to freez- ing and cooking is shown in Figure 9. Chicken Dark Meat Lipids The levels of total lipids, triglycerides and phos- pholipids in chicken dark meat are presented in Table 22 and Figure 10. The amounts of total lipids in the raw meat were 9.12, 6.06 and 7.27% during frozen storage at 0, 8 and 13 months, respectively. When cooked, the levels of total lipids, triglycerides and phospholipids were significantly (P < 0.05) higher than the corresponding amounts in the uncooked meat, regardless of the length of freezer storage. Similar to beef, total lipids and triglycerides significantly (P < 0.01) decreased during PERCENT OF TOTRL TISSUE PERCENT OF TOTRL TISSUE Figure 9 . 1. J (A) Beef TOTRL LIPIDITP) 15.00 5.000% a : 4. t 0 4.000 0.000 12.000 STORRGE TIHEIHONTHS) TRIGLYCERIDEITG) ismo IS- 0 ' 4.000 A 0.000 +123” STURRGE TIHEIHONTHS) PERCENT OF TOTAL TISSUE Legend [3 = fresh/frozen raw meat x = cooked meat held at 4°C cooked meat held at ~18°C TOTRL PHOSPHOLIPIDSIPL) J 0 ' 4.000 0.300 123000 STORROE TIHEIHONTHSI Influence of length of frozen storage and cooking on the levels of beef lipids. 10M Table 22. Effect of length of frozen storage, cooking and storage temperature upon chicken dark meat lipids. Storage Period (Months)4 Lipids1 Storage Temp. 0 8 13 Fresh/frozen 9.12:0.47a 6.06:0.21c 7.27:0.06d Total Lipids +4°c2 10.66:0.20b 7.00:0.19d 8.61:0.0Se -18°c3 10.73.+.o.42b 7.18:0.07d 9.17:0.04f Triglycerides Fresh/frozen 8.20:0.43a 5.15:0.21c 6.42:0.06e +4002 9.53:0.16b 5.89:0.02d 7.55:0.07f ~18°c3 9.64:0.35b 6.03:0.11d 7.99:0.01g Phospholipids Fresh/Frozen 0.82:0.03a 0.83:0.02a 0.85:0.01a +4°C2 0.96:0.01b 1.01:0.13b 0.98:0.00b -18°c3 0.99:0.01b 0.98:0.02b 0.99:0.01b 1Expressed as percentage of total tissue. 2 Meat was cooked and held at 4°C for 48 hrs. 3Meat was cooked and held at -18°C for 48 hrs. 4Values within the same lipid group and in the same row bearing the same superscript are not significantly different at P<.05. 1c: -‘ / Chicken Dark Meat Legend: TOTRL LIPIOITP) g D = fresh/frozen a raw meat I g x = cooked meat 3 held at UOC .— 25 A = cooked meat 5 held at —18°C U U M I.” Q. 5.00 3 3 4 3 * 3 1 0 0.000 0.000 “-000 STORRGE TIHEtHONTHS) TRIGLYCERIDEITG) TOTRL PHOSPHOLIPIDS(PL) 18.00 1.5 U U D D U) (D 9 mac 9 :40 I— .— _J .1 C: C S umo ; a. r— .— IL IL O O 0- 9' I— 2 2 U U U U C K m 7. IAJ a. 0.. 5.0 3 3 3 3 3 3 .8 3 3 ‘ 3 ‘ 3 3 0 6.000 0.000 n.000 0 6.000 0.000 12.000 STORROE TIHEIHONTHS) STORROE TIHEIHONTHS) Figure 10. Influence of length of frozen storage and cooking on the levels of chicken dark meat lipids. 106 frozen storage, while the phospholipids remained relatively constant. The amount of phospholipids in raw chicken dark meat were 0.82, 0.82 and 0.85% at O, 8, and 13 months, respectively. Hence, the levels of phospholipids in the~ raw dark meat were higher than those found in raw beef. Watts (l95u) and Acosta gt gt. (1966) have also reported that the levels of phospholipids are lower in the red meats than in poultry meat. Chicken White Meat Lipids The contents of lipids extracted from chicken white meat are presented in Table 23 and in Figure 11. In the raw meat the amounts of total lipids were 2.58, 1.93 and 1.85% at 0, 8 and 13 months of frozen storage, respectively. The levels of total lipids and triglycerides in raw chicken white meat also decreased during frozen storage as was found in beef and chicken dark meat. Similar to beef and chicken dark meat, the amount of total lipids, triglycerides and phospholipids significantly (P < 0.05) increased during cooking, regardless of the length of freezer storage. The phospholipids in raw chicken white meat amounted to 0.5M, 0.53 and 0.52% at 0, 8 and 13 months of frozen storage, respectively. Phospholipids were higher in both the raw and cooked chicken dark meat (Table 22) than in the white meat (Table 23). This is in agreement with 107 Table 23. Effect of length of frozen storage, cooking and storage temperature upon chicken white meat lipids. Storage Period (Months)4 Lipids1 Storage Temp. 0 8 13 Total Lipids Fresh/frozen 2.58:0.21a 1.93:0.00d 1.8510.09d +4°C2 2.94:0.22b 2.25:0.15e 2.39:0.13e -18°c3 3.24:0.14c 2.31:0.01e 2.30:0.14e Triglycerides Fresh/frozen 2.00:0.14a 1.34:0.01C 1.31:0.06c +4002 2.66:0.25b 1.45:0.13c 1.54:0.06c’d -18°c3 2.48:0.11b 1.56:0.04°’d 1.61:0.09‘“d Phospholipids Fresh/frozen 0.54:0.03a 0.53:0.01a 0.52:0.04a +4°C2 0.64:0.01b 0.67:0.06b 0.62:0.02b -18°c3 0.66:0.0lb 0.67:0.02b 0.60:0.01c 1Expressed as percentage of total tissue. 2 Meat was cooked and held at 4°C for 48 hrs. 3Meat was cooked and held at -18°C for 48 hrs. Values in the same lipid group and in same row and bearing the same superscript are not significantly different at P<.05. Chicken White Meat TOTRL LIPIDtTP) Legend: U 0 a []= iresh/frozen 8 raw meat ... ..J g x = cooked meat 2 held at 4°C 3 ._ A = cooked meat 5 held at -l8°C U K IAJ I. 0 : 4.000 4L 0.000 ; 123000 STORHGE TIHEIHONTHS) TRIGLYCERIDEITG) TOTRL PHOSPHOLIPIDSIPL) 8.000' 1 00 I.“ uJ :3 +> :: U) (D .03 4.0001» 59. .00 u— T— ...l _I (2 Cl: ’5 ’5 .00 h- p- 0. IL. 0 O *_ *_ .70 z z #H“§<:::3 IIJ m U U K a: ILI M L 0. A L A A A A A A A A A A A A V v f ‘7 fl 1 I v W I V Y 1 0 4.000 0.000 12.000 0 4.000 0.000 12.000 STDRROE TIHEIHONTHS) STORRGE TIHEIHONTHS) Figure 11. Influence of length of frozen storage and cooking on the levels of chicken white meat lipids. 109 Peng and Dugan (1965) and Acosta _E._l- (1966), who have reported that chicken dark meat contains higher levels of phospholipids than chicken white meat. Effect of Frozen Storage on the Levels of Extractable Lipids The data presented in Tables 21, 22 and 23 showed that significant (P < 0.01) losses occurred in both total lipids and triglycerides during frozen storage of raw beef, chicken dark meat and white meat. 0n the other hand, the levels of phospholipids in raw meat were not sig- nificantly changed during frozen storage. The results of this study showing decreasing levels of total lipids during frozen storage are in agreement with the work of Zipser and Watts (1962). They reported a 21% decrease in total lipids of oxidizing mullet tissue during five-days of refrigerated storage. They did not, however, report any loss in gross phospholipid content during storage. 0n the contrary, Acosta gt gt. (1966) reported an apparent decrease in the phospholipid content of turkey tissues frozen for 180 days at -25°C. They also reported increased levels of total lipids in all tissues except the liver. Results of this study showed that losses observed in total lipids during frozen storage were principally due to changes in the triglycerides, since the phospholipids 110 were relatively constant. Thus, results of this study are in agreement with the report by Campbell and Turkki (1967), who indicated that neutral lipids, being mostly intercellular, were more rapidly released by the tissue than the intracellular phospholipids. It is not fully understood why total lipids decreased during frozen storage of meat, especially in the raw tissues. It is well known, however, that the phospho- lipids unlike the neutral lipids, are an integral part of the tissue membranes (Kono and Calowick, 1961) and are firmly held in an electrostatic interaction with the pro- teins (Gurr and James, 1971). According to Gurr and James (1971), the neutral lipids are loosely held within the tissues by Van der Waals forces and as such are easily eluted. It will be seen later herein that the variation in the amount of total lipids appear to be positively related to the variation in the levels of malonaldehyde as measured by the TBA test. Composition of Liptds in the Drippings The lipids contained in the drippings of cooked meat were measured immediately after cooking and are presented in Table 24. The total lipids in the drippings were 1.67, 1.37 and 0.05% for beef, chicken dark meat and white meat, respectively. The concentration of 111 Table 2“. Composition of Lipids in Cooked Drippings. Chicken Dark Chicken White Lipids Beef Meat Meat Total Lipids 1.67a 1.37a 0.053a 2.07b 1.77b 0.063b Triglycerides 1.65a 1.36a ..... 2.05b 1.76b _____ Phosphospids 0.00914a 0,0089a _____ 0 012b ----- aExpressed as percentage of fresh tissue. bExpressed as percentage of cooked tissue. 112 phospholipids in the drippings in comparison to the tri- glycerides was extremely low (about 0.01%). Results of this study agree with the report by Camp- bell and Turkki (1967) indicating that the low concentra— tion of phospholipids in the drippings is probably be- cause the phospholipids are firmly held within the tis— sues. They also indicated that the high levels of tri- glycerides in the drippings can be accounted for by the fact they are mainly intercellular, and as such loosely bound by the tissues. Composition and Stability of Phospholipid Components The concentrations of LPC, SP, PE, PC and total phos- pholipids were measured in both the raw and cooked meat during frozen storage, using the phosphorus assay pro- cedure of Rouser gt gt. (1966). The data representing combined mean values from beef, chicken dark meat and white meat are presented in Table 25 and in Figure 12. The combined data were used since the species values were not discordant. Lysophogphatidyl Choline (LPG) The concentrations of LPG in raw meat were 1.25, 1.43 and 1.93 mg phosphorus/g phospholipid at 0, 8 and 13 months of frozen storage, respectively. These values for LPC 113 Table 25. Effect of cooking, storage temperature and length of frozen storage on the stability of the individual phospholipid com- ponents in beef, chicken dark and white meat (combined). Storage Time (Months) Phospholipids2 Storage Temp. 0 8 13 LPC3 Fresh/frozen 1.25:0.69 1.43:0.52 1.93:0.66 +4008 1.54:0.67 1.80:0.39 1.09:0.56 -18°c9 1.91:0.71 1.37:0.85 1.14:0.24 SP4 Fresh/frozen 2.34:0.59 1.88:0.88 1.45:0.60 +4°08 1.69:0.54 1.74:0.57 1.87:0.78 -18°c9 1.95:0.66 1.67:0.54 0.92:0.59 PBS Fresh/frozen 6.6811.08 4.89:0.80 3.77:0.64 +4°08 2.55:1.06 2.64:1.03 2.5810.63 -18°c9 4.09:1.85 3.69:0.99 3.17:0.60 PC° Fresh/frozen 16.70i0.99 14.39i1.94 12.2li1.82 +4°C8 12.89io.73‘ ll.46il.61 10.80:2.09 -18°c9 15.48io.99 11.6012.84 12.23i2.40 Tp7 Fresh/frozen 29.26il.92 24.17i2.82 22.39:1.45 +4°08 24.85io.98 20.52i2.oo 20.62i2.01 -18°c9 26.76:1.84 21.25:3.38 22.28:2.29 1Mean value of 12 determinations; four each from beef, chicken dark and white meat. Values expressed as mg phosphorus/g phospholipid. OQNQUIb 3LPC==lysophosphatidy1 choline. SP = Shingomyelin. PE 8 phosphatidyl ethanolamine. PC - Phosphatidyl choline.a TP - Total phospholipid phosphorus. Meat was cooked and held at 4°C for 48 hrs. Meat was cooked and held at -18°C for 48 hrs. LYSOPHOSPHRTIDYL CHOLINEI LC) Legend: I3= Raw frozen meat x = Cooked meat held at 4°C A = Cooked meat held at —l8°C HG PHOSPHOROUS G PHOSPHOLIPID 0 : 0.0:00 J 0.300 : 1230004 sroanct TIHEIHONTHS) SPHINGOHELINISP) PHOSPHRTIDYL ETHRNOLRHINEIPE) HG PHOSPHOROUS G PHOSPHOLIPID HO PHOSPHOROUS G PHOSPHOLIPIU A A A V 0 ' 4.000 : 0.000 ; 12.3004 0 4.000 : 0.600 f123000 STORRGE TIHEIHONTHS) STORROE TIHEINONTHS) PHOSPHRTIDYL CHOLINEIPC) TOTRL PHOSPHOLIPIDSITP) HG PHOSPHOROUS G PHOSPHOLIPID HG PHOSPHOROUS 0 PHOSPHOLIPID A A 0 : 4.600 0.600 f 123000 : 0 'L 4.000 i 0330 j 123000 ; STORRGE TIHEIHONTHS) STORROE TIHEIflONTHS) L Figure 12. Influence of frozen storage and cooking on the composition and stability of LPC, SP, PE, PC and total phospholipid phosphorus (TP) in meat. 115 comprised 4.27, 4.90 and 6.60% of total phospholipids. The levels of LPG in fresh tissue seem to be quite vari- able. For instance, Davidkova and Khan (1967) reported a value of 1.5% in fresh chicken tissues, while Keller and Kinsella (1973) and Lee and Dawson (1976) reported values of 0.60 and 6.8% for LPC, respectively. The increasing levels of LPG (Table 25) during frozen storage suggest that lipolysis occurred. Several researchers (Awad _t_gt., 1968; Braddock and Dugan, 1972) have reported increasing levels of LPG during frozen storage of meat, which is indicative of lipolytic activity. Phosphatidyl Ethanolamine (PE) Results presented for PE in Table 25 showed that the levels of PE decreased in the raw meat at 8 and 13 months during frozen storage. PE also decreased in the cooked meat held at -18°C for 48 hrs following cooking. The initial concentration of PE in fresh tissue (6.68 mg phosphorus/g phospholid) was 22.83% of the total phos- pholipids. This is in good agreement with values reported by Davidkova and Khan (1967) and Keller and Kinsella (1973). The levels of PE in raw tissues were 4.89 and 3.77 mg phosphorus/g phospholipid at 8 and 13 months of frozen storage, respectively. These values accounted for a loss of 26.80 and 43.56%, respectively, from the initial concentration of PE at 0 time. 116 The levels of PE were significantly (P < 0.05) higher in fresh/raw frozen than in cooked meat, regardless of the length of freezer storage. However, PE was relatively higher in the cooked meat held at —18°C for 48 hrs than in the cooked meat held at 4°C for 48 hrs. Thus, results indicated a higher rate of lipid autoxidation in the cooked meat held at 4°C for 48 hours than either that of the cooked meat held at -l8°C for 48 hours or in the raw frozen meat stored at -18°C from 0 to 13 months. Phosphatidyl Choline (PC) The initial level of PC in fresh tissue at 0 time was 57.07% of the total phospholipids. This value is in good agreement with those reported for PC by Peng and Dugan (1965) and Keller and Kinsella (1973). The concentration of PC in raw meat was found to decline during frozen storage, the levels being 16.70, 14.39 and 12.21 mg phosphorus/g phospholipid at 0, 8 and 13 months, respec- tively. The values at 8 and 13 months amounted to losses of 13.83 and 26.88% of PC from the original value at 0 time, respectively. Levels of PC also consistently declined in the cooked meat held at 4°C for 48 hrs. Although the levels of PC in cooked meat held at -l8°C for 48 hrs declined, the rate of decline was not consistent, the concentrations being 15.48, 11.60 and 12.23 mg phosphorus/g phospholipid at 117 0, 8 and 13 months, respectively. Results showed that like PE, the lowest concentrations of PC were found in the cooked meat held at 4°C for 48 hrs, regardless of the length of freezer storage. Thus, a higher rate of lipid oxidation occurred in the cooked meat held at 4°C for 48 hrs. than for the other two treatments. Total Phospholipids (TP) The concentrations of total phospholipids in the raw meat were 29.26, 24.17 and 22.39 mg phosphorus/g phospho- lipid at 0, 8 and 13 months of frozen storage, respectively. In the cooked meat held at 4°C for 48 hrs, the levels of TP were lower, being 24.85, 20.52 and 20.62 mg phosphorus/g phospholipid at 0, 8 and 13 months, respectively. Sim- ilarly, the levels of TP for the cooked meat held at —18°C for 48 hrs were 26.76, 21.25 and 22.28 mg phosphorus/g phospholipid, respectively, at the same storage periods. Thus, the levels of phospholipids declined consistently during frozen storage of raw meat. This is in agreement with the report of Zipser gt gt. (1962) showing a pro- gressive loss in total phospholipids during refrigerated storage of mullet tissues. Analysis of variance showed that total phospholipids were significantly (P < 0.05) higher in raw than in cooked meat. However, TP tended to be higher in the cooked held at —l8°C for 48 hrs following cooking in comparison 118 to that held at 4°C. This indicates that a higher rate of lipid autoxidation occurred in cooked meat held at 4°C for 48 hrs following cooking. Results of this study (Table 25) have shown that PE, PC and TP were more stable in raw frozen meat than in that cooked and held either at 4°C or at -18°C for 48 hrs following cooking. Results alsoshowed that PE was less stable than PC, especially for the cooked meat held at 4°C for 48 hrs. Thus, results further verify the in- stability of PE, especially in cooked meat. Furthermore, results have demonstrated that phospholipid components were less stable in the cooked meat held at 4°C for 48 hrs following cooking, thus, indicating a higher rate of lipid autoxidation than for the other treatments. The effect of cooking on changes in the amounts of PC and PE is shown in Figure 13. Plate A represents phos- pholipid components from fresh beef tissue while plate B shows the same components following cooking and storage of the tissue. Quantitative differences were evident in three unidentified components (X, Y and Z) and also in PC and PE. This can be seen by comparing plates A and B, which show a reduction in the amount of the unidentified components as well as in PC and PE in the cooked sample. Thus, results of this study further verify the involvement of the phospholipids in development of WOF in cooked meat, since they were shown to be less stable to cooking. 119 Origin Origin Figure 13. Effect of cooking on quanitative changes of PC and PE on thin layer plates. A represents total phospholipids from fresh uncooked beef tissue, while B shows the total phospholipids from cooked beef. Note the changes in size of the spots for PC, PE, and the unidentified spots X, Y and Z. 120 Phospholipid Components in the Drippings Analysis of the composition of the phospholipids recovered from beef drippings is shown in Figure 14. Samples A and C represent drippings from beef, while B is from a standard mixture of known phospholipid com- ponents. Phosphatidyl ethanolamine (PE) was completely absent in the cooked drippings (Figure 14). The absence of PE in the drippings indicates that PE is more tightly bound to the membrane than PC or the phospholipid com- ponents. Thus, the level of PE in the meat would increase during cooking and more PE could be available to react with atmospheric oxygen. The results indicate that PE is more important in development of oxidized flavor than previously believed. Changes in FattygAcid Composition of Lipids During Frozen Storage and Cooking The fatty acid composition of the triglycerides, total lipids, PC and PE were analyzed in fresh and in frozen raw meat as well as in the cooked meat held at 4°C and at -18°C for 48 hrs after cooking. Triglycerides Changes in the fatty acid composition of beef tri- glycerides during frozen storage and cooking is presented Figure 14. 121 O 6 WC £ c Thin layer plate showing the composition of the phospholipid components in the drippings of cooked meat. A and C represent total phospholipids from cooked beef drippings. B shows a standard mixture containing LPC, PC, PE and cholesterol (CHL). X indicates posi- tion of the missing PE in the cooked drippings. 122 in Table 26. The percentages of total unsaturation in raw frozen beef were 54.77, 52.24 and 53.52% at 0, 8 and 13 months, respectively. In the cooked meat held at 4°C, total unsaturation was slightly less, being 53.17, 51.53 and 53.20% at 0, 8 and 13 months, respectively. Similarly, the levels of total unsaturates in the cooked meat held at -18°C for 48 hrs after cooking were 54.89, 52.31 and 51.09% at 0, 8 and 13 months, respectively. Although total unsaturation slightly decreased during frozen stor- age of beef, the difference in unsaturation between the raw frozen and cooked meat does not appear to be sig- nificant. The small changes in total unsaturation re- flected the stability of the saturated, mono- and dienoic fatty acids during frozen storage and cooking. Thus, the data provide further verification Of the stability of fatty acids in the triglycerides in either frozen or cooked meat. The fatty acid composition in chicken dark meat tri- glycerides are presented in Table 27. The percentages of total unsaturation in raw meat gradually increased during frozen storage, the values being 69.47, 71.81 and 73.29% at 0, 8 and 13 months, respectively. In the cooked chicken dark meat held at 4°C, the levels of total un— saturates at 0, 8 and 13 months, were 71.03, 71.52 and 70.25%, respectively. Similarly, the corresponding values for cooked meat held at -18°C for 48 hrs following 1223 .ncaOG beach Have» we uncouon an concasonaoa as.Hm s~.mm ~m.mm Hm.~m mm.am .~.~m mo..m sfl.mm ss..madwumsw Hm.~ mm.~ um.~ mm.~ as.~ can“ sH.s o~.m c°.m ..Muou e~.o. Hm.om um.om ~m.ms NH.ms m..om «p.8m sm.m. mm.am one: u Hm.o. ow.m. o..w. «v.5. s=.ms ms.s. "H.ms mo.e. m~.ms «an n .1..- s~.o ma.o 1.1.. .1..- ”sou so.o ms.o mm.o ---.1 .1--- ---.1 ao.o ca.cc .1... mxmuoa .1..- -...1 .H.o .1--- ----- .1..- ----1 1...- 1.... menses o~.H mm.a mH.H mmha ~H.H wo.a u..~ ...H om.a nuns mo.H. mm.~. mo.as m~.mm mo.mm H..Hs m~.os ms.s. om.o. 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The fatty acid composition of chicken white triglycer- ides are presented in Table 28. While the levels of total unsaturation slightly increased during frozen storage of raw chicken dark meat (Table 27), the reverse was the case during frozen storage of raw chicken white meat (Table 28). The levels of total unsaturation in frozen chicken white meat were slightly less than the values in cooked white meat, although the amount of unsaturates in the latter also slightly declined during frozen storage. Only minor variations were found in saturated, mono-, di-, or poly- enoic fatty acids during frozen storage and cooking. Results showed that only minor changes occurred in the fatty acid profiles of triglycerides during frozen storage and cooking of either beef (Table 26), chicken dark meat (Table 27) or chicken white meat (Table 28). Thus, the results are in agreement with the work of Chang and Watts (1952), Campbell and Turkki (1967) and Igene and Pearson (1978) showing that only slight changes occurred in the fatty acid composition of triglycerides during cooking of meat. 125 .mcuom awash kuou no scooped no coumfisoamom mm.o> mm.o~ mm.m> aa.m> mm.H> Hm.H~ mm.nm mo.a~ ~=.aw poms: Hmuoe mm.~ ww.a Hm.H mm.H sm.H om.H Hw.H NH.H ==.H haom u Hm.=m om.ma ms.mm so.mm m~.mm Hm.mm =~.m~ wm.mH mm.om «a a mm.=a mm.m= mm.>= =m.m: pm.m= 05.»: =m.w= mm.om 03.5: 0:0: u Hm.m~ m>.mm Hp.mm mo.>m m=.mm mH.mm Hm.mm ~m.mm mm.om paw u mm.o m~.o m~.o 11111 snow mm.o Huow No.H mm.H o=.H m=.H om.“ om.H m=.H so.H mH.H :muma mm.o o~.o mm.o mH.o s~.o 11111 mH.o mo.o wm.o wzmuma mm.mm =>.ma m>.mm Nb.mm mm.wm am.~m a~.mH mm.ma mm.om mama mm.mm sa.m= ms.~= Hm.H= om.mm m~.oz a>.o= mm.:a mm.H= Humfl ma.m sm.m pm.m mm.m m~.m ~m.m m~.m hs.= m~.m ouma 11111 1 ma.o m=.o om.o mm.o mm.o 11111 ousa wm.o mH.o 11111 mm.o m=.o 5:.o 11111 Numa HH.= mm.= \so.= Hm.m so.m mo.w ==.m ma.m so.m Huma mH.Hm mm.mm =0.H~ om.Hm mm.mm mm.mm mH.mm mm.mm mm.mm ouma 11111 1 Huma 11111 1 ouma m~.o om.o Hm.o mm.o om.o mm.o Hw.o m~.o mw.o Hana oomH1 003+ use: oomHI oo=+ poo: oomH1 oo=+ use: uoao< nonopm cououm smash zuumm vououm new coxooo cohoum can ooxooo concum can coxooo sauce: ma sauce: m ease: o weansc nooapoozawap» nave sumo «.mconoo can owuaouu couopu coxoano uo :oapfiuanoo pace hunch on» :« someone .NN unnas ..D «1. .ooaoo spade Hmuou go accused on wouoasoamow aa.ms ms.o~ mo.Hh mo.ms m=.=s om.fls mo.ms oH.ms mm.ms some: Hopes mm.m mo.~ oo.m sm.H mH.~ ~H.H wo.H mo.~ om.H sacs a oa.o~ aH.- mo.m~ am.m~ mo.- oo.~m ~m.=~ sa.~m mm.- an a mo.ms as.m= om.~= mm.m= mo.m= m=.~= mm.m= oo.om mm.o= one: a mm.o~ mm.m~ sm.m~ sm.o~ =m.m~ om.m~ sm.=~ om.=~ ~=.o~ uom u mo.H om.o mw.o 11-11 11111 -1111 1111- 11111 -1111 snow -1111 om.o H~.o 11111 11111 11111 mnom -1--- -1--- 1-111 mm.o om.o 111-1 muow -1--- sa.o mo.o 1111- 1---- -111- mH.~ mm.H -1111 Huom om.H ms.a Hp.a mm.H om.~ sm.o mo.H os.H os.H mxmumfi mm.o 11-11 om.o oH.o m~.o m~.o 1-1-1 mm.o o~.o ozmuma mm.m~ mm.H~ mm.s~ mm.mm m~.- ma.~m sm.m~ so.~m mm.- mama mm.mm mfl.mm ~m.~m am.m= sm.== so.H= ma.~s mm.m= sa.mo Huma mm.m mm.w a=.m om.o mm.m ~=.m ~H.~ om.m mm.» ouma mH.o sm.o -.o mm.o mm.o =m.o -11-- 1-111 1111. ouefl -.o m~.o. sm.o Ha.o mm.o ~=.o 1.... 11111 11111 wuwfl mo.= =~.m s~.a H~.= m=.= m~.m H~.= mH.: Hm.= Huoa m~.aa oo.- H~.- ms.mfl m~.ma =o.- mm.sfi o=.mH sm.ma onma mm.o am.o sm.o 1111- ~m.o 11111 m=.o mo.H ~m.o Anna .1111 11111 11111 cum” mm.o sm.o mm.o m=.o =m.o H=.o mm.o wm.o mo.o Huaa 11-1- 11-11 .1111 111-1 .1111 11111 ousfl coma- oo=+ poo: coma- ooz+ ado: coma- ooa+ use: ooao< cououm couchm sauna auuum cohoum can ooxooo vopoum can uoxooo vououm can ooxooo unucoz ma cause: a spec: o a.w:«xooo can omuao»u canopy wcupso nouauoozawapu pave ands: coxoaso «o couuanonsoo owes hogan on» ad nowcazo .om manna 127 Changes in the Fatty Acid Composition of Total Phospho- lipids Beef Phospholipids The fatty acid composition of beef phospholipids is presented in Table 29. The proportions of saturated, mono—, di- and polyenoic fatty acids in the fresh un- cooked beef tissue at 0 time were 32.88, 39.67, 11.95 and 15.50%, respectively. These values are in good agree— ment with those reported by Hornstein gt gt. (1967) and O'Keefe _t gt. (1968). Significant changes occurred in the unsaturated fatty acids in the phospholipids (unlike the beef triglycerides) during frozen storage and cooking. The losses in un- saturation could be largely accounted for by changes in 018:2’ C20:3 and 020:“ fatty acids. In the raw frozen meat, the level of dienoic acid, which was 11.95% at 0 time, decreased to 6.49 and 7.82% at 8 and 13 months of frozen storage, respectively. Similarly, the initial level of 15.50% polyenoic acids in the fresh beef de- clined to 6.80 and 7.39% at 8 and 13 months of frozen storage, respectively. Thus, total unsaturation in the raw tissues declined during frozen storage, the levels being 67.12, 58.41 and 58.40% at 0, 8 and 13 months, respectively. The observed losses in unsaturation would indicate the occurrence of lipid oxidation during frozen Table 29. 128 Changes in the fatty acid composition of beef phospholipids during frozen storage and cooking.a 0 Month 8 Months 13 Months Patty Fresh Cooked and Stored Frozen Cooked and Stored Frozen Cooked and Stored Acids Meat +4°C -18°C Meat +4°C -18°C Meat +4°C -18°C 12 :0 ..... -..- ..... 14:0 ---- ----- -—-- 14:1 1.46 0.72 1.85 2.52 1.27 2.42 1.84 2.03 2.10 15:0 0.52 ---- 0.82 0.50 0.67 0.55 0.53 0.34 0.71 15:1 1.16 1.86 2.06 0.95 0.53 0.62 0.62 0.30 0.98 16:0 18.49 18.73 20.03 27.73 22.65 25.27 26.87 23.93 23.13 16:1 3.61 3.67 .33 5.29 4.00 5.59 3.32 3.38 3.58 16:2 0.74 0.25 .41 0.76 0.60 0.62 0.71 .0.85 0.46 17:0 0.92 0.84 0.62 0.76 0.80 0.91 0.70 0.81 0.75 18:0 12.95 12.83 13.19 12.60 14.51 13.26 13.50 16.56 13.76 18:1 33.44 34.81 35.66 35.92 36.98 38.07 37.41 36.80 35.57 18:2 10 52 12.66 10.10 5.29 7.19 5.39 6.81 6.39 8.95 18:305 0.37 ----- ----- ----- 1.20 0.78 ----- ---- 0.13 18.3..3 1.29 0.68 0.91 0.57 --—-- ---- 0.70 1.35 0.59 20:1 ---------- ---- 0.44 0.67 0.28 ---- 0.20 0.16 20:2 0.69 0.21 0.25 0.44 0.47 0.17 0.30 0.25 0.39 20:3 2.77 1.92 1.73 0.76 1.60 1.14 1.07 1.08 1.46 20:4 8.51 9.32 6.59 3.02 6.33 3.87 4.56 4.83 6.25 20:5 0.76 ----- ----- ----- 0.53 0.18 22 :2 ----- ---- --- 22:3 —--- ----- 0.13 22:4 0.88 0.42 0.63 0.38 0.59 0.24 0.39 22:51.6 .............. 1.57 ---- 0.06 ----- ----- 0.33 22:593 0.92 1.08 0.82 0.88 ----- 0.62 0.47 0.66 ----- 22:6 ----- ---- ----- 1 Sat 32.88 32.40 34.66 41.59 38.63 39.99 41.60 41.64 38.35 s Mono 39.67 41.06 43.90 45.12 43.45 46.98 43.19 42.71 42.39 S Di 11.95 13.12 10.76 6.49 8.26 6.18 7.82 7.49 9.80 z Poly 15.50 13.42 10.68 6.80 9.66 6.85 7.39 8.16 9.46 33:23 67.12 67.60 65.34 58.41 61.37 60.01 58.40 58.36 61.65 aCalculated as percent of total fatty acids. 129 storage. Significant losses occurred in both the dienoic and polyenoic fatty acids of the cooked meat held at either 4°C or at —18°C for 48 hrs at all periods of frozen storage. In the cooked meat held at 4°C, the initial level of total unsaturation was 67.60%, but it declined to 61.37 and 58.36% at 8 and 13 months of frozen storage, respectively. Similarly, the corresponding values for the cooked meat held at —18°C for 48 hrs following cook- ing were 65.34, 60.01 and 61.65% at 0, 8 and 13 months, respectively. Thus, results demonstrated the occurrence of lipid oxidation in the cooked meat throughout storage. Chicken Dark Meat Phospholipids The fatty acid composition for chicken dark meat phospholipids is presented in Table 30. The levels of saturated, mono—, di- and polyenoic fatty acids in the fresh unfrozen tissues were 33.76, 21.99, 21.51 and 22.74%, respectively. Most of the changes in unsaturation during frozen storage and cooking were due to alterations in the levels of 018:1, C18:2’ C20,“ and C22:4 fatty acids. Although the concentration of mono- and dienoic fatty acids gradually increased during storage of the raw frozen tissues, the level of polyenoic fatty acids declined. The initial concentration of PUFAS was 22.74% at 0 time, but it declined to 19.69 and 11.06% at 8 and 13 months, 1.3() Table 30. Changes in the fatty acid composition of chicken dark meat phospholipids during frozen storage and cooking.8 0 Month 8 Months 13 Months Cooked and Stored Cooked and Stored Cooked and Stored Fatty Fresh Frozen Frozen Acids Meat +4°C -18°C Meat +4°C -18°C Meat +4°C -18°C 12:0 ..--- ..-.. _____ 14:0 ---- ---------- 14:1 0.14 0.17 0.16 ----- ----- 0.22 ---------- 0.28 15:0 ----- -—-—- ----- 0.54 0.86 ----- 15:1 0.37 0.57 0.31 ----- ----- 0.88 ---------- 0.23 16:0 15.60 16.52 16.44 12.63 18.44 15.65 17.99 23.27 19.85 16:1 1.05 1.03 0.79 1.61 2.20 2.88 2.57 2.00 2.15 16 2 ----- 0 23 0 17 0 21 0.39 0 19 0 34 0 28 0 23 17 0 --------------- 0.27 0 43 o 51 ---- 0 38 ----- 18 O 18 16 21 03 20 83 18 19 18 44 15 51 12 47 17 27 15 31 18 1 20 43 19 74 19 34 23 94 26 07 28 53 31 75 26 27 29 48 18 2 21 51 19.22 20 11 21 83 20.95 23 26 23 63 18 00 18 82 13 3'6 O 37 0.23 0 35 0 24 0.12 19 0.28 0 13 0 35 18 3W3 0 41 0.43 0.53 0 88 0.45 0 64 1 21 0 88 0 68 20'1 ---------- 0.26 0 13 1.08 ----- 0.19 ----- 0.23 20 2 ----- ---------- 0 96 0.97 1.42 ---- ---- ----- 20:3 0.53 0.52 0.57 0.44 ----- 0.22 0.34 0.75 0.60 20:4 17.41 18.88 15.28 13.86 8.44 8.97 7.90 8.76 10.43 20:5 ----- ---- ----- ----- ---—- ----- 22:3 --- ----- ----- 0.29 ----- ----- 22:4 1.23 0.57 1.84 1.12 0.60 0.42 0.42 0.88 0.74 22:5w6 ---------- ---- 0.21 ---------- 22:5w3 2.79 0.86 0.33 0.24 ----- -“- 22:6 ----- ----- 2.69 2.41 0.56 0.51 0.91 1.13 0.62 1 Sat. 33.76 37.55 37.27 31.63 38.17 31.67 30.46 40.92 35.16 I Mono-21.99 21.51 20.86 25.68 29.35 32.51 34.51 28.27 32.37 5 Di- 21.51 19.45 20.28 23.00 22.31 24.87 23.97 18.28 19.05 I Poly 22.74 21.49 21.59 19.69 10.17 10.95 11.06 12.53 13.42 33:33 66.24 62.45 62.73 68.91 61.83 68.33 69.54 59.08 64.84 aCalculated as percent of total fatty acids. 131 respectively. Results indicate the involvement of the PUFAS in development of rancidity during frozen storage of meat. Nevertheless, total unsaturation in the raw frozen tissues increased gradually from 66.24% at 0 time to 68.91 and 69.54% at 8 and 13 months of frozen storage, respectively. The increasing levels of monounsaturated fatty acids during frozen storage of raw tissues largely accounted for the increased total unsaturation during frozen storage. While the concentration of monoenoic fatty acids rose significantly in the cooked meat, the level of polyenoic fatty acids declined drastically. The dienoic fatty acids were relatively unchanged during cooking. The polyenoic fatty acids in cooked chicken dark meat held at 4°C for 48 hrs amounted to 21.49, 10.17 and 12.53% at O, 8 and 13 months, respectively. Similarly, the corresponding levels in the cooked meat held at -18°C for 48 hrs after cooking were 21.59, 10.95 and 13.42% at the same time intervals, respectively. Results showed that most of the losses in unsaturation occurred in 020:4 in particular, and in the PUFAS in general. Thus, the involvement of the PUFAS in the development of WOF in cooked meat is verified. Chicken White Meat Phospholipids - Most of the changes in the fatty acid profiles of chicken white meat (Table 31) phospholipids occurred in the polyenoic fatty acids. 132 Table 31. Changes in the fatty acid composition of chicken white meat phospholipids during frozen storage and cooking.8 0 Month 8 Months 13 Months Patty Fresh Cooked and Stored Frozen Cooked and Stored Frozen Cooked and Stored Acids Meat +4°c -18°c Meat +4°c -18°c Meat +4°c -18°c 12:0 ---- ---- ..... 14:0 ---- ---------- 14:1 0.65 0.15 ..... 15:0 -------- 0.26 ---- ----- ----- 15:1 2.01 0.57 0.61 0.95 0.67 1.58 ----- ----- ..... 16:0 14.26 18.25 20.45 19.98 20.86 17.85 20.61 22.18 20.13 16:1 0.70 0.84 l 02 1.14 0.94 0.85 0.79 0.80 0.83 16:2 ----- 0.23 ----- 0.19 0.27 0.28 ----- 0.23 0.08 17:0 ----- ----- ----- 0.43 0.36 0.45 ----- 0 l7 ---—— 18:0 14.67 14.78 14.82 13.13 16.36 14.91 17.98 14.84 18.80 18:1 21.44 21.99 21.90 19.26 25.40 22.59 21.17 20.93 19.46 18:2 16.30 17.78 16.44 14.27 15.37 13.56 16.61 18.47 16.70 18:3w6 2.85 0.23 ----- 18:3u3 --- 0.57 0.15 0.14 0.27 9.55 0.28 0.60 0.50 20:1 --- ----- ..... 20:2 0.39 0.34 0.38 4.56 1.35 0.76 ----- 0.23 ----- 20:3 0.79 1.45 1.23 0.48 0.81 0.68 0.51 0.91 0.67 20:4 17.23 15.77 16.34 11.09 13.85 13.44 16.89 15.70 15.47 20:5 ----- 0.44 ---- 0.36 ----- ----- 0.42 ----- 0.37 22:3 1.96 1.59 ..... 22:4 0.52 0.28 1.17 0.52 0.45 0.45 1.61 1.19 1.50 22‘5'6 --.- ---- 1.02 0.79 ----- 0.42 22:5w3 1.46 0.95 0.64 11.65 1.02 ----- 0.31 1.02 0.83 22:6 4.77 3.79 4.59 0.95 2.02 2.03 2.04 2.73 4.24 5 Sat 28.93 33.03 35.53 33-54 37.58 33.21 38.59 37.19 38.93 S Mono 24.80 23.55 23.53 21.35 27.01 25.02 21.96 21.73 20.29 S 01- 16.69 18.35 16.82 19.02 16.99 14.60 16.61 18.93 16.78 3 Poly 29.58 25.07 24.12 26.09 18.42 27.17 22.84 22.15 24.00 832:: 71.07 66.93 64.47 66.46 62.42 66.79 61.41 62.81 61.07 aCalculated as percent of total fatty acids. 133 The level of PUFAS in the fresh tissue was 29.58% at 0 time, 26.09% at 8 months but only 22.84% at 13 months. Lower levels of PUFAS were found in the cooked meat. In the cooked meat held at 4°C for 48 hours the PUFAS com- prised 25.07, l8.42 and 22.15% at 0, 8 and 13 months, respectively. The corresponding levels in the cooked meat held at —18°C for 4- hrs did not change consistently, being 24.12, 27.17 and 24.00% at 0, 8 and 13 months, respectively. Results showed that the PUFAS were less stable in the cooked meat held at 4°C for 48 hours than in either the raw frozen meat or in that cooked and held at —18°C for 48 hours. Results of this study for beef (Table 29), chicken dark meat (Table 30) and for chicken white meat (Table 31) verify the involvement of unsaturated fatty acids, in general, and of PUFAS, in particular, in the development of oxidized flavor in frozen or cooked meat. PUFAS were less stable than either the unsaturated, mono- or dienoic fatty acids, especially in the cooked meat. Thus, the results confirm those obtained in the earlier studies herein (experiments A and B) involving model meat systems, which show that the PUFAS of the phospholipids are major contributors in the development of rancidity during freezer storage or in development of WOF in cooked meat. 134 Fatty Acid Composition of Lipids from Drippings The fatty acid profiles of the triglycerides and phospholipids in the cooked drippings of beef and chicken dark meat are presented in Table 32. The levels of saturated, mono-, di- and polyenoic fatty acids in the triglycerides from beef drippings were 49.85, 47.25, 2.68 and 0.22%, respectively. Similarly, the levels of the same fatty acid in the drippings from beef phospho- lipids were 45.34, 49.66, 3.98 and 1.02%, respectively. The concentrations of saturated fatty acids in the triglycerides and phospholipids of chicken dark meat drippings were 35.97 and 35.27%, respectively. The per- centage of monomeric acids in the chicken dark meat drippings were similar to that found in the beef (Table 32). However, much higher levels of dienoic fatty acids were recovered in the drippings from chicken dark meat than from beef. It is interesting to note that extremely low levels of PUFAS (0.36 and 2.15%) were found in both the triglycerides and phospholipids from chicken dark meat drippings. The low levels of PUFAS in the phospholipids of the drippings suggest that the more highly unsaturated phos- pholipid components are firmly held within the membranes. Results verify the conclusion that phosphatidyl ethanol- amine (PE) is firmly bound to the membranes, and thus is not released during cooking, thereby elevating the levels 135 Table 32. Fatty acid composition or the triglycerides and phospholipids obtained in the cooked drippings from beef and chicken dark meat.8 Beef Drippings Chicken Dark Meat Drippings Fatty Acid Triglycerides Phospholipids Triglycerides Phospholipids 12:0 ---- ---- 1.16 --.. 14:0 --- -- 1.52 0.69 14:1 6.03 2.07 ---- ---- 15:0 1.79 0.95 --- ---- 15:1 0.45 0.30 ---- ---- 16:0 34.21 27.43 26.92 21.93 16:1 5.86 4.96 6.51 6.85 16:2 1.34 0.71 ---- ---- 17:0 0.89 0.59 ---- 1.60 18:0 12.96 16.37 6.37 11.05 18:1 34.91 41.86 40.52 43.40 18:2 1.34 3.27 15.63 10.65 18:3 ---- --- 0.22 0.23 18:3 0.22 0.59 0.14 1.28 20:1 ---- 0.47 1.01 1.71 20:2 ---- --- ---- ---- 20:3 ---- ---- --- 0.64 20:4 ---- 0.43 ---- -—- 1 Saturated 49.85 45.34 35.97 35.27 S Monoenoic 47.25 49.66 48.04 51.96 1 Dienoic 2.68 3.98 15.63 10.62 1 Polyenoic 0.22 1.02 0.36 2.15 Total Unsaturation 50.15 54.66 64.03 64.73 aCalculated as percentage of total fatty acids. 136 of unsaturation in cooked meat. Thus, the development of WOF in cooked meat is enhanced by the high levels of PE retained during cooking. Results of this study (Tables 21, 22 and 23) demon- strated that there were significantly (P < 0.01) higher levels of phospholipids in the cooked meat in comparison to the uncooked meat. Thus, the higher rate of lipid autoxidation in cooked meats is at least in part due to elevated levels of phospholipids. Changes in the Fatty Acid Profiles of PC and PE During Frozen Storage and Cooking of Meat The fatty acid composition of PC and PE in total phospholipids was analyzed in the raw meat prior to freezer storage at 0 time and also following 13 months of frozen storage. At the same time periods, the fatty acid composition of PC and PE was also measured in the cooked meat after being held at 4°C and —18°C for 48 hrs follow- ing cooking. Egg: - The fatty acid profiles of PC of raw and cooked beef are presented in Table 33. The composition of PC in fresh raw beef (at 0 time) comprised of 36.62, 41.63, 10.83 and 10.92% of the saturated mono—, di-, and poly- enoic fatty acids, respectively. These values are in close agreement with the data presented by Keller and Table 33. 1L37 Changes in the fatty acid composition of phosphatidyl choline (PC) in beef phospholipids during frozen storage and cooking.3 0 Months 13 Months Fresh Cooked and Stored Frozen Cooked and Stored Fatty Acids Meat +4°c -18°C Meat +4°c -18°C 12:0 ---- ----- 0.82 --- 0.99 ---- 14:0 0.46 0.65 0.78 ----- ----- 0.29 14:1 0.86 0.65 0.89 ----- 0.58 ..... 15:0 0.51 ----- -—--- 0.69 0.39 0.23 15:1 1.92 1.59 3.48 0.92 1.55 0.75 16:0 20.86 11.67 16.70 21.11 21.86 18.64 16:1 3.04 3.36 3.70 2.20 3.13 1.73 16:2 0.91 0.75 0.43 0.83 .93 .69 17:0 2.13 0.60 0.85 2.06 1.39 0.78 18:0 12.66 19.60 8.88 14.50 13.97 14.39 18:1 34.90 25.66 33.76 28.91 29.60 30.79 18:2 9.92 9.71 9.45 11.56 10.83 8.63 18:3w6 2.63 0.42 0.32 ---------- 0.75 18:37:3 0.53 1.40 1.28 0.55 0.81 0.86 20:1 0.91 --- ---- 0.73 0.70 ----- 20:2 ----- 1.87 1.60 1.10 0.35 1.44 20:3 1.22 1.96 0.96 1.37 0.54 .69 20:4 5.32 14.19 7.04 10.28 6.50 4.03 20:5 1.22 5.92 9.06 2.06 4.33 14.10 22.3 ----- ----- ----- -—-- 0.35 0.36 22:4 ---------- ----- 0.32 0.85 0.22 22:5w6 ---------- ----- 0.46 ---- ----- 22:51:3 ----- ---- ----- 0.35 0.35 0.26 22:6 ---- --------------- ---- 0.37 3 Sat. 36.62 32.52 28.03 38.36 38.60 34.33 1 Monoenoic 41.63 31.26 41.83 32.76 35.56 33.27 I Dienoic 10.83 12.33 11.48 13.49 12.11 10.76 i Polyenoic 10.92 23.89 18.66 15.39 13.73 21.64 Total Unsat. 63.38 67.48 71.97 61.64 61.40 65.67 aCalculated as percent of total fatty acids. 138 Kinsella (1973). The levels of dienoic and polyenoic fatty acids in the PC of raw meat increased during frozen storage, while the monenoic fatty acids in PC decreased by almost 9.0% during frozen storage. At 0 time, the levels of dienoic and polyenoic fatty acids in PC were much higher in cooked meat than in fresh meat. At 13 months of frozen storage, the polyenoic fatty acids of PC in the cooked meat held at 4°C for 48 hrs decreased by 42.53% from the original value at 0 time. On the other hand, the level of polyenoic fatty acids in PC of the raw frozen and in the cooked meat held at -18°C for 48 hrs increased. The variations in the levels of polyenoic fatty acids in PC were principally due to changes in 020:“ and C20:5 fatty acids. The percentage of total unsaturation in PC decreased in both the raw and cooked meat as the length of frozen storage increased. Results showed that the PUFAS of PC were least stable in the cooked meat held at 4°C for 48 hrs, thus indicating much higher rate of autoxidation. The initial levels of saturated, mono-, di- and poly- enoic fatty acids in PE of fresh beef (Table 34) were 35.46, 25.47, 15.66 and 23.42%, respectively. Following cooking (0 time), significant losses occurred in the dienoic and polyenoic fatty acids in PE of the cooked meat. Most of the polyenoic fatty acids were severely affected during cooking, thus greatly decreasing total JJ39 Table 34. Changes in the fatty acid composition of phosphatidyl ethanol- amine (PE) in beef phospholipids during frozen storage and cooking.a 0 Month 13 Months . Fresh Cooked and Stored Frozen Cooked and Stored Fatty Acids Meat +4°C ~18°C Meat +4°C -18°C 12:0 ----- 0.33 0.80 ---- .......... 14:0 ----- 0.72 1.70 ---------- 0.38 14:1 ----- 0.74 1.11 --------------- 15:0 0.70 ---- ---- 0.60 1.35 0.40 15:1 2.55 1.12 ----- 0.35 ----- 0.22 16:0 9.28 31.84 19.15 14.99 17.30 17.59 16:1 1.39 2.65 1.28 1.95 3.05 2.00 16:2 1.74 ----- 0.32 2.40 1.35 1.44 17:0 2.65 0.72 0.64 1.70 2.40 0.72 18:0 22.83 9.51 23.94 20.95 22.26 12.47 18:1 21.53 36.17 29.84 22.38 23.77 28.78 18:2 9.05 8.40 9.31 11.09 8.59 13.91 18:3w5 0.37 ---------- ----- 1.05 0.20 18:3w3 1.14 ---- ----- 0.75 .79 2.20 20:1 ---- ----- ----- ----- ----- ----- 20:2 4.87 0.41 ----- 2.00 0.08 1.80 20:3 1.16 0.43 ---- 2.00 1.50 0.60 20:4 15.08 4.96 11.91 13.99 8.12 4.50 20:5 0.70 2.00 ----- 3.50 7.22 12.23 22:3 1.30 ----- ----- 0.35 0.79 0.16 22:4 0.65 ----- ----- 0.65 0.38 0.08 22:5w6 ----- -------------------- 0.16 22:57:3 1.86 ----- ----- 0.35 ----- 0.16 22:6 1.16 --—-- ---- --------------- 5 Sat. 35.46 43.12 46.23 38.24 43.31 31.56 1 Monoenoic 25.47 40.68 32.23 24.68 26.82 31.00 I Dienoic 15.66 8.81 9.64 15.49 10.02 17.15 1 Polyenoic 23.42 7.39 11.91 21.59 19.85 20.29 Total Unsat. 64.55 56.88 53.78 61.76 56.69 68.44 aCalculated as percent of total fatty acids. 140 unsaturation. Both the dieonic and polyenoic fatty acids in the PE of cooked meat were not as severely altered at 13 months of frozen storage as was the case at 0 time. However, the dienoic and polyenoic fatty acids of PE were found to be least stable in the cooked meat held at 4°C for 48 hrs. This indicated a greater rate of oxida- tion in the cooked meat held at 4°C. Chicken Dark Meat — The fatty acid profiles in PC of raw and cooked chicken dark meat are presented in Table 35. Levels of saturated, mono-, di-, and polyenoic fatty acids in the PC of the raw meat decreased during frozen storage. The PUFAS decreased from the initial level of 18.79% at 0 time to only 8.29% at 13 months. Thus, there was a high rate of PC oxidation in the raw chicken dark meat during frozen storage. The changes in the fatty acids of the PC in the cooked meat were not consistent. However, the dienoic and polyenoic fatty acids were less stable in the cooked chicken dark meat held at 4°C for 48 hrs than in that held at -18°C for 48 hrs following cooking. Results of the changes in the fatty acids of PE for chicken dark meat (Table 36) also clearly showed that the unsaturated fatty acids were less stable in the cooked meat held at 4°C than in the other two treat- ments, which indicate a higher rate of autoxidation in 141. Table 35. Changes in the fatty acid composition of phosphatidyl choline (PC) in chicken dark meat phospholipids during frozen storage and cooking.a 0 Month 13 Months Fresh Cooked and Stored Frozen Cooked and Stored Fatty Acids Meat +4°C -18°C Meat +4°C -18°C 12:0 ---- 0.57 0.26 ----- ----— ----- 14:0 0.41 0.86 0.91 0.34 .......... 14:1 ---- 0.86 0.25 ..... ----- ..... 15:0 0.48 ----- 0.91 0.68 ----- ----- 15:1 0.61 ---—- ---- 1.08 0.92 0.75 16:0 16.42 29.93 12.11 28.47 18.78 18.19 16:1 0.92 0.77 ----- 1.42 1.15 0.93 16:2 0.61 0.29 3.28 1.02 0.69 0.75 17:0 0.54 0.57 ----- .81 0.46 0.93 18:0 20.36 20:19 13.24 25.76 26.87 20.12 18:1 23.62 22.21 11.30 20.44 15.27 12.90 18:2 16.63 18.62 14.63 9.76 12.37 12.34 18:3w5 --------------- 0.34 ----- 0.44 18:3w3 0.61 ----- 10.82 0.95 0.31 0.75 20:1 --------------- 0.51 ----- ----- 20:2 0.61 ----- 11.30 1.42 3.05 13.08 20:3 0.31 ----- ----- --------------- 20:4 10.96 5.13 11.80 3.75 7.33 5.23 20:5 2.88 ----- 0.63 3.25 12.80 12.21 22:3 ---------- 6.81 ---------- 1.38 22:4 1.49 ----- -------------------- 22:51:6 -..-- ------------------------- 22:5w3 0.78 --—-- 1.75 --------------- 22:6 1.76 ----- ----- ----- ---- ----- 1 Sat. 38.21 52.12 27.43 56,06 46.11 39.24 1 Monoenoic 25.15 23.84 11.55 23.45 17.34 14.58 1 Dienoic 17.85 18.91 29.21 12.20 16.11 26.17 1 Polyenoic 18.79 5.13 31.81 8.29 20.44 20.01 Total Unsat. 61.79 47.88 72.57 43.94 53.89 60.76 aCalculated as percent of total fatty acids. 142 Table 36. Changes in the fatty acid composition of phosphatidyl ethanol- amine (PE) in chicken dark meat phospholipids during frozen storage and cooking.a 0 Month 13 Months Fresh Cooked and Stored Frozen Cooked and Stored Fatty Acids Meat +4°C -18°C Meat +4°C -18°C 12:0 ----- 4.49 ----- ----- ----- ..... 14:0 ----- 0.82 0.88 0.41 ----- ----- 14:1 ---------- 0.42 --------------- 15:0 0.99 0.22 ----- --—-— ---—— -..-- 15:1 1.29 ----- 0.55 0.47 ----- ----- 16:0 11.91 21.02 14.52 17.39 13.23 10.83 16:1 0.99 ---- 1.26 1.24 0.59 0.36 16:2 1.32 0.88 0.67 0.44 0.44 0.59 17:0 1.49 ----- 0.84 0.53 0.74 0.59 18:1 15.45 10.32 16.41 14.80 11.03 10.87 18:2 14.56 2.46 9.72 10.07 10.00 9.96 18:3“6 ---------- 0.38 0.27 0.29 0.55 18 : 37:3 ..---- ----- 0.29 1.04 0.69 0.59 20:1 0.74 ----- 0.25 0.21 0.39 ----- 20:2 0.59 2.30 2.78 11.46 4.90 5.93 20:3 ----- ----- 0.55 ----- ----- ----- 20:4 19.56 10.95 12.46 9.24 12.74 15.45 20:5 1.65 ----- 9.09 12.44 11.76 8.86 22:3 0.15 ----- 0.27 0.41 0.66 0.55 22:4 1.32 ----- 1.39 0.30 0.33 0.51 22:5w6 0.17 ----- ----- ---------- ----— 22:5w3 0.33 ----- 0.34 0.18 ----- ----- 22:6 1.22 ----- 2.19 1.07 0.93 2.57 3 Sat. 40.66 73.09 40.97 36.36 45.25 43.21 x Monoenic 18.47 10.32 18.89 16.72 12.01 11.23 S Dienoic 16.47 5.64 13.17 21.97 15.34 16.48 1 Polyenoic 24.40 10.95 26.96 24 95 27.40 29.08 Total Unsat. 59.34 26.91 59.03 63.64 54.75 56.79 aCalculatedas percent of total fatty acids. 143 the former. Chicken White Meat - The fatty acid composition of PC in the raw and cooked chicken white meat phospholipids is presented in Table 37. The level of polyenoic fatty acids as well as of total unsaturation of PC increased during frozen storage of raw chicken white meat. The level of polyenoic fatty acids in PC was lower in the cooked than in the raw meat, irrespective of the length of frozen storage. Results, thus, verify the involve— ment of PUFAS in the development of WOF in cooked meat. The involvement of PUFAS in the development of oxidized flavor is clearly demonstrated in Table 38, which presents the fatty acid profiles of PE for raw and cooked chicken white meat. At each storage interval, the levels of poly- enoic fatty acids of PE in raw chicken white meat were significantly higher than the corresponding levels in the cooked meat. However, the PUFAS were more stable in the PE of the cooked meat held at —18°C for 48 hours after cooking than in that held at 4°C for 48 hrs. Arachidonic acid (020:4) was the fatty acid most severely affected in the cooked meat held at 4°C for 48 hrs. Results of fatty acid analyses for PC and PE clearly demonstrate that the PUFAS were less stable in cooked than in raw meat. However, the PUFAS were less stable in the cooked meat held at 4°C than that held at -18°C Table 37. 1144 Changes in the fatty acid composition of phosphatidyl choline (PC) in chicken white meat phospholipids during frozen storage and cooking.8 0 Month 13 Months Fresh Cooked and Stored Frozen Cooked and Stored Fatty Acids Meat +4°C -18°C Meat +4°C -18°C 12:0 .22 ----- 0.69 ............... 14:0 1.19 0.67 0.80 0.66 0.32 0.71 14:1 0.15 0.49 0.57 --------------- 15:0 ---------- 0.15 0.44 0.57 ----- 15:1 5.03 1.09 0.31 2.05 1.07 1.23 16:0 18.84 26.15 22.90 21.07 21.10 19.72 16:1 0.43 1.28 0.46 2.64 2.14 1.54 16:2 0.44 .18 ----- 0.92 0.70 0.42 17:0 1.31 1.46 0.20 1.03 3.41 0.34 18:0 14.00 8.51 13.55 11.65 19.95 16.61 18:1 18.07 27.00 26.64 24.47 24.78 23.49 18:2 12.20 15.75 20.04 7.88 7.13 9.79 18:3w6 0.33 ---------- .38 ----- 0.22 18:3w3 0.32 ---------- 1.32 1.0 0.42 20:1 0.35 --------------- 0.86 0.34 20:2 2.95 1.82 1.53 0.59 0.86 0.49 20:3 --------------- 0.51 0.14 0.28 20:4 13.68 8.76 9.77 6.15 5.99 13.09 20:5 7.61 .19 2.39 15.92 9.98 6.26 22:3 .06 ---------- 0.37 ----- 1.17 22:4 2.49 0.49 ----- 0.26 ----- 0.28 22:5w6 ------------------------------ 22:5w3 --------------- 1.10 ----- 0.92 22:6 0.66 3.16 ----- 0.59 ----- 2-68 I sat. 35.56 36.79 38.29 34.85 45.35 37.38 S monoeonic 24.03 29.86 27.98 29.16 28.85 26.60 1 dienoic 15.59 17.75 21.57 9.39 8.69 10.70 S polyenoic 24.82 15.60 12.16 26.60 17.11 25.32 Total Unsat. 64.44 63.21 61.71 65.15 54.65 62.62 aCalculated as percent of total fatty acids. 1315 Table 38. Changes in the fatty acid composition of phosphatidyl ethanol- amine (PE) in chicken white meat phospholipids during frozen storage and cooking.3 0 Month 13 Months Fresh Cooked and Stored Frozen Cooked and Stored Fatty Acids Meat +4°C -18°C Meat +4°C -18°C 12:0 -.--- 0.93 -..-- ..... ----- --..- 14:0 ----- 1.16 1.55 0.41 0.53 ----- 14:1 ----- 2.89 0.54 ---------- ----- 15:0 ---- 0.62 0.59 0.22 0.47 ---- 15:1 4.48 ----- 0.67 ----- 1.05 1.09 16:0 11.30 36.08 20.53 15.08 24.73 19.55 16:1 0.37 ----- 1.01 1.22 1.89 1.14 16:2 0.47 ----- 1.13 0.40 0.63 0.33 17:0 2.13 ----- 3.35 0.81 1.42 0.33 18:0 22.40 26.72 28.70 21.53 17.05 22.14 18:1 15.83 10.40 12.57 16.88 16.84 18.25 18:2 10.41 ----- 6.16 11.53 10.70 10.86 18:3w6 0.50 ----- ----- 0.24 ----- 0.33 18:3w3 0.42 1.54 1.26 1.08 0.74 0.57 20:1 0.22 ---- 1.13 ----- ----- ----- 20:2 0.84 18.50 1.26 00.65 1.42 1.09 20:3 0.45 ----- ----- 0.73 ----- 0.68 20:4 22.21 1.16 12.91 12.31 8.84 12.25 20:5 1.57 ---- 3.14 12.99 12.52 8.55 22:3 0.41 ---- ----- 0.43 ----- 0.49 22:4 1.49 ----- 0.89 0.27 ----- 0.27 22:5w6 0.48 -------------------- ----- 22:54:3 0.21 ---------- 1.19 0.37 0.46 22:6 3.81 ----- 2.62 2.03 0.80 1.63 1 Sat. 35.83 65.51 54.72 38.05 44.20 42.02 1 Monoenoic 20.90 13.29 15.92 18.10 19.78 20.48 I Dienoic 11.72 18.50 8.55 12.58 12.75 12.28 S Polyenoic 31.55 2.70 20.82 31.27 23.27 25.23 Total Unsat. 64.17 34.49 45.29 61.95 55.80 57.99 aCalculated as percent of total fatty acids. 146 for 48 hrs following cooking. In addition, the di- and polyenoic fatty acids associated with PC were relatively more stable during freezer storage and cooking than those of PE. The results for beef (Tables 33 and 34), chicken dark meat (Tables 35 and 36) and chicken white meat (Tables 37 and 38) clearly verify the involvement of the unsatu— rated fatty acids, and particularly of the PUFAS autoxida- tion of both PC and PE. Thus, the results confirm the previous studies involving model meat systems (experiments A and B), which clearly demonstrated that PUFAS are in- volved in the development of rancidity in frozen meat and of WOF in cooked meat. There is currently no available data on the effect of freezing and cooking on the fatty acid composition of PC and PE in either chicken dark meat or white meat. In addition, the current information available for beef (Keller and Kinsella, 1973) is not comprehensive. Thus, this study provides a detailed and comprehensive analysis of fatty acid composition of PC and PE in fresh raw/frozen and cooked meat, which is not currently avail- able in the published literature. 147 Effect of Frozen Storage of Meat in the Raw State on its TBA Value Following Cooking and Holding at 4°C or -18°C for 48 Hrs According to deFremery et a1. (1977) extensive con— sumer surveys have indicated that 3/4 of all consumers prefer fresh to frozen meat, yet 2/3 of these consumers froze the meat after purchasing it. The authors cited another study which showed that 63% of consumers froze tflmflx'meat after purchase, even when held for only a few days. This practice is not uncommon in commercial meat processing establishments. The present work rose out of a need to explore the stability of meat during frozen storage, which is of particular relevance in long distance transporting of meat by land or sea. There is currently no information on the effect of frozen storage of meat in the raw state upon development of WOF after cooking. Thus, the main objective of this study was to examine the effect of frozen storage of meat in the raw state on its TBA values following cooking and holding at either 4°C or -18°C for 48 hrs. Lipid antoxidation in raw and cooked meat was measured using the 2-thiobarbituric acid (TBA) test of Tarladgis t l. (1960). The TBA numbers of the fresh raw meat were measured prior to frozen storage at 0 time, and 148 also at 8 and 13 months of frozen storage. At the same time periods, the TBA numbers of the cooked meatheld at either 4°C or -18°C for 48 hrs after cooking were also determined. Beef The TBA numbers for raw frozen and cooked beef are presented in Table 39 and Figure 15. In the raw frozen beef, the TBA values rose slowly from 0.27 at 0 time to 0.31 at 8 months and to 0.41 at 13 months of frozen storage. Thus, raw beef was very stable during frozen storage. The TBA numbers were still well below the threshold levels for rancidity of 1-2 as outlined by Watts (1962). When beef was cooked and held at -18°C for 48 hrs after cooking, the TBA values were 1.63, 2.64 and 0.79 after 0, 8 and 13 months of frozen storage, respectively. The values were significantly (P < 0.01) different from each other. Although the reason for the drop in TBA value at 13 months is unknown, Buttkus (1967) has postu- lated that a reaction between myosin and malonaldehyde may take place during frozen storage and cause a decline in TBA numbers. When beef was cooked and held at 4°C for 48 hrs after cooking, the TBA values were significantly (P < 0.001) higher than those obtained in the other treatments. The 149 Table 39. Effect of length of frozen storage at -18°C and cooking on the level of TBA numbers in beef (LD)1:2:3 Cooked Meat Storage Time Raw Meat Held at -18°C Held at 4°C (Months) Stored at -18°C for 48 hrs for 48 hrs 0 0.27:0.02a 1.631008d 7.26:0.20h 8 03120.03a 2.64:0.21e 6.09:0.16f 13 0.41:0.01b 0.79:0.03C 6.55:0.07g TBA number is expressed as mg malonaldehyde/kg meat. 2Values in the same column or row bearing the same letter are not significantly different at P < .05. 3 Each value represents a mean of 4 replicates. TBA NUMBER (MGMA/KG MEAT) 8.0 7.0 4.0 3.0 2.0 1.0 0.0 F10. 1 l , e -1; FRESH/FROZEN AT ~18°c COOKED, STORED AT 74°C COOKED, STORED AT -18°c 0 8 13 STORAGE TIME (MONTHS) Influence of the Length of Storage Time at —18°C on the TBA Number of Beef Muscle. 151 values were 7.26, 6.09 and 6.55 after 0, 8 and 13 months of frozen storage in the fresh state, respectively. The variation in TBA values is closely related to changes in the amount of total lipids (Table 21) in the cooked meat, i.e., high TBA values and high lipid content were directly related and vice versa. Chicken Dark Meat TBA values for fresh/raw frozen and cooked chicken dark meat are presented in Table 40 and Figure 16. In the raw meat, the TBA numbers rose gradually during frozen storage, the values being 0.36, 1.78 and 2.44 at 0, 8 and 13 months, respectively. Thus, the TBA numbers for raw chicken dark meat would exceed the threshold values for acceptability after 8 months in freezer storage. Results showed that a higher rate of lipid oxidation took place in raw chicken meat (Table 40) than in the raw beef (Table 39). The differences in the rate of oxidation can be largely explained on the basis of a greater amount of lipid unsaturation in chicken dark meat as compared to beef. In cooked chicken dark meat held at -18°C for 48 hrs after cooking, the TBA values were 6.80, 5.42 and 5.73 at 0, 8 and 13 months of frozen storage, respectively. Similarly, the corresponding values in the cooked meat held at 4°C for 48 hrs after cooking were 16.65, 12.22 152 Table 40. Effect of length of frozen storage at ~18°C and cooking on the level of TBA numbers in chicken dark meat. 22: Cooked Meat Storage Time Raw Meat Held at -18°C Held at 4°C (Months) Stored at —18°C for 48 hrs for 48 hrs 0 0.36:0.06a 6.80:0.15f 16.65:0.36k 8 1.78:0.34b 5.42:0.14d 12.221050g 13 2.44:0.51c 5.732010e 13.341055h 1 TBA number is expressed as mg malonaldehyde/kg meat. 2Values in the same column or row, bearing the same letter are not significantly different at P < .05. 3Each value represents a mean of 4 replicates. 153 CHICKEN DARK MEAT 18.0 FRESH/FROZEN AT ~18°c 16.0 COOKED. STORED AT 4°C COOKED, STORED AT -l8°c 14.0 12.0 10.0 8.0 Ah chicken white meat > chicken dark meat. The stability of different types of raw meat during freezer storage was in the following order: Beef > chicken white meat > chicken dark meat. Higher TBA numbers were found in the cooked meat held at 4°C than in the cooked meat held at ~18°C for 48 hrs following cooking, irrespec— tive of the type of meat. Non-consistent but decreasing levels of malonaldehyde were found in the previously frozen raw meat after it was cooked and held for 48 hrs follow— ing cooking. Results showed that changes in total lipids during frozen storage of raw meat were largely due to losses in the triglycerides. The phospholipid content of raw meat was relatively constant irrespective of the time 166 of freezer storage. The concentrations of triglycerides, total lipids and phospholipids were significantly elevated in the cooked meat. PE, PC, total phospholipids and their PUFAS were less stable in the cooked than in the raw frozen meat. Cooked meat held at 4°C for 48 hrs was more susceptible to development of WOF than similar meat held at —18°C for 48 hrs.after cooking. Results suggest that its constituent lipids were not as stable to auto- oxidation. Results verified the involvement of PUFAS in the development of WOF, the stability of different types of meat being in the order of: Beef > chicken white meat > chicken dark meat. 167 Experiment D Influence of Heme Pigments, Non-heme Iron and Nitrite on Development of WOF in Cooked Meat The role of meat pigments, nitrite and non-heme iron on development of WOF in cooked meat was investigated. The study was divided into two stages. The first part was designed to study the effect of meat pigments and nitrite on development of WOF in beef, chicken dark meat and white meat. The second part was designed to compare the relative contributions of heme and non-heme iron on the development of WOF. The first part of this study was conceived to investi- gate the effect of removal of meat pigments and/or the addition of nitrite to control the development of oxidized flavor in cooked meat. The design of the experiment and preparation of samples are presented in Table 2. The proximate composition of lipids and moisture in both the extracted and non-extracted meat samples was determined. This was done to enable a meaningful assess- ment of the effect of pigments on development of WOF. The tissue lipids were left intact while the only variables were removal of the meat pigments and the addition of nitrite. 168 The proximate composition of the meat in terms of total lipids, triglycerides, phospholipids and moisture are presented in Table 43. Total lipids and triglycerides tended to be slightly lower in the pigment extracted meat than in that containing pigment, except for chicken white meat. The amounts of phospholipids were 0.58 and 0.60% in beef with and without pigments, respectively. The corresponding levels of total phospholipids in chicken dark meat were 0.86 and .80%, respectively. The concentra- tion of total phospholipids was considerably lower in chicken white meat than in chicken dark meat, with values of 0.49 and 0.54% in the chicken white meat and with and without pigments, respectively. The concentration of moisture was somewhat higher in all samples after extrac- tion of the pigments. There were only minor differences in the levels of total lipids, triglycerides, phospho- lipids and moisture between samples with and without pig- ments. Thus, the only variable was the presence or ab- sence of the meat pigments. The fatty acid composition of the triglycerides and total phospholipids in meat with and without pigments is presented in Tables 44 and 45. The pattern of fatty acid composition in both the triglycerides and total phospho- lipids is in good agreement with the data presented by Hornstein £3 31. (1961, 1967), Katz gt a1. (1960) and O'Keefe ££.él- (1968). Total unsaturation in the tri- glycerides (Table 44) and phospholipids (Table 45) was 169 .mco«uwcfieaopmp mmefiflasp ucmmmmamm madammm H Hm.om mm.ow mm.w> mH.:> sm.mn Ho.m> oedemaoz am.o ma.o om.o mw.o 00.0 mm.o efiafiaosamona om.H 3H.H em.m om.m Hm.m ms.m mcfismosawase sw.a mm.H se.m om.: mm.m mm.: mpfidaq Hmpoe psmewfim pcmewfim psoewfim pcoewfim pcoewfim newsman pcmgoasoo usospfiz npfiz psonufis npfiz psonpfiz npfiz new: mufinz new: xmmo mwmm H.Am:mmfip nmomm av moHQEmm pmme :fi assumfioe pgm mpHQHH mo mam>mq .m: manme 170 .mefiom shame Hates mo mmmpcmommu m< H Hm.os mm.mm H:.Hs Ho.as, ao.mm mm.am empasspam 7:: Hates mm.am mm.mm me.mm Hm.am mm.m om.m afioemsaoa a oaocmao a mo.ma oo.m: ma.ma os.ma mm.om mm.m: oaosmocoz a mm.mm H:.Hm mm.mm mm.mm mm.m: am.sz empmsssmm a pcmewam ungwfim pcmEmHm pcmewfim unmewfim pacewfim pfio< muumm psonpfiz spas psocpfiz npfiz escapes cuss use: opens 6862 same 686m H.pmmE mpficz 6cm xmmc cmxofico .mmmn CH mopfismomfimfisp map mo COHpHmOQEoo pfiom mpumm mo mhmeesm .z: wanes 171 .mafiom Assam Hmpos mo owmpCmome m< H o>.~m om.mm mm.>m om.>w ow.mm m:.mm cmumASpmm Is: Hapoe ma.ma mm.mH em.mH oo.mH m=.m mo.mfi afiocmsfioa a m:.mm mm.wa Hm.mm mm.wm m=.HH mm.HH oHocwfia m mm.Hm nm.wm we.mm mH.mm mm.mm mm.mm oaocmocoz & om.mm mo.mm :o.mm om.mm om.a= :m.az ompmazumm u pcmeHm newsman pcoewfim pcmEmHm pcoewfim pcmewfim Ufiog mppmm usonpfiz npfi3 psozpfiz npfiz psospfiz npfiz new: mpfinz ummz xmmm H.ummE means one sump smxofino .moop CH mpfiofiaonamona HmpOp map mo coHuHmOQEoo pfiom zppmm mo msmeesm .m: mHnt 172 considerably higher in both chicken dark meat and white meat than in beef. Furthermore, there were no consistent and significant differences in the fatty acid composition of tissues with and without pigments. Thus, any dif- ferences in the rate of lipid oxidation (TBA values) between experimental treatments should be related to the effect of pigments and/or nitrite, which will be discussed later herein. Changes in TBA Numbers and Taste Panel Scores Mean TBA numbers and the corresponding mean taste panel scores for cooked meat are presented in Table 46. Analyses of variance indicated that highly significant (P < 0.001) differences occurred among treatments for both TBA values and taste panel scores. Results showed that the samples without removal of the pigments and without added nitrite (A) had the highest TBA values, and consequently the lowest taste panel ratings. The samples without pigments and without added nitrite (C) had significantly (P < 0.01) lower TBA values than the samples with pigments containing no added nitrite (A). However, taste panel scores were significantly (P < 0.01) higher for the former than in the latter. Thus, meat pigments were clearly the major pro-oxidant in the develop- ment of WOF in cooked meat. .nw.- 7 news mung: cmxonno use Hm.7 7 name game coxonno was.- 4 hmmm "meHm mmhoow Hmflma Ufiw meHmDESC .QmB CmmSme ®5Hw> =9: AHO.OvmV DEMOHMchHmm .m.vm ne nceoHanme no: ene nQHsommedzm eEem mp peBOHHom CESHoo ween CH newness HH< 173 new: annex message news amen ammonno m .mo: 0: m pee mo: peocsogond wcHen H anHz .mIH Eonn mes mmoom Heceq enmeBH o.pmn.: emH.m nom.: ema.n nHm.= n.ema.o mnnsnnc mafia newsman nsonnHz neee oexooo "a 9:6 nH:.: nm:.m nom.: nom.m nHm.o eanan on .nceEwHQ nsoanz nemE Uexooo no one.: enm.H nam.m emm.m nma.= enm.o mnnsnne msHa passena nnHz neeE cexooo Hm eOH.m omm.m emm.H omH.HH em:.m 0mm.H eannH: o: .neeede nnH3 neeE pexooo n< enoom .oz eLOOm .ozz enoom .oz mnceEneenB Hesem chicken white meat > chicken dark meat. Results demonstrated that removal of meat pigments and addition of 156 ppm of nitrite significantly (P < 0.01) inhibited the development of TBA values. Taste panel evaluation confirmed the beneficial effects of removal of the heme pigments and the addition of 156 ppm nitrite to meat for controlling the development of WOF. Thus, results suggested that heme pigments may catalyze lipid oxida- tion. The percentage of bound iron in fresh meat pigment extract was slightly over 90% while the level of free non- heme iron was less than 10%. Cooking, however, released a significant amount of non-heme iron from bound heme pigments, which accelerated lipid oxidation in cooked meat. Thus, the rate of lipid oxidation in cooked meat is due in part to release of non-heme iron during cooking, 186 which then catalyzes lipid oxidation. Although earlier studies have suggested that myoglobin may catalyze lipid oxidation, this study showed that pigments ggt'gg do not greatly accelerate the development of WOF, but serve as a source of non-heme iron in cooked meat. Thus, results showed that non-heme iron was the major pro-oxidant in the development of WOF in cooked meat. Addition of 2.0% EDTA effectively chelated the non-heme iron, and thus, significantly reduced lipid oxidation. REFERENC ES REFERENCES CITED Acosta, S. 0., Marion, W. W., and Forsythe, R. H. 1966. Total lipids and phospholipid in turkey tissue. Poult. Sci. 45:169. Ansell, G. B., and Hawthorne, J. N., eds. 1964. "Phos- pholipids: Chemistry, Metabolism and Function". BBA Libr. Vol. 3, Elsevier, Amsterdam. Arata, A. S., and Chen, T. C. 1976. Quality charac- teristics of convenience chicken products as related to packaging and storage. J. Food Sci. 41:18. Awad, A., Powvie, W. D. and Fennema, O. 1968. Chemi— cal deterioration of frozen bovine muscle at -4°C. 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