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Jantawat has been accepted towards fulfillment of the requirements for PH.D Food Science degree in X g? 9am Major professor Date Lay/m1” WM; 0—7639 macaw K221 ‘AMU’V AUG 73 '97185 37 K2h3 5971mm ’3 5 SE3 5 8 st? 2.997135 as 3&3“- 6121'87fii OVERDUE FINES ARE 25¢ PER DAY _ PER ITEM Return to Book drop to remove this checkout from your record. ‘ W5!%,. 1". .' t'gi', '\.'V / ‘4’ 5-94 FMAA.AA.W 5'0. K308 N3V778FE¢7?; 50 K322 {" b. ' ‘1 ’ i: “if Pi a d mq-..-‘{'bsrscv “fl? I Cd a 3;; "‘ f' “ 1: ~tv r.xv’3 «(3 ‘ 9'". fir,» V.u @413 1* t. I(\ ; r35 3: y. fan LIV" .55.“) Vat“ 5;”. Gib (/ COMPOSITION AND STABILITY OF LIPIDS FROM MECHANICALLY PROCESSED POULTRY MEATS (MPPM) By Pantipar P. Jantawat AN ABSTRACT OF 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 COMPOSITION AND STABILITY OF LIPIDS FROM MECHANICALLY PROCESSED POULTRY MEATS (MPPM) By Pantipar P. Jantawat Lipids from light and dark mechanically processed chicken and turkey meats (MPCM and MPTM), light and dark hand deboned chicken and turkey meat (HDCM and HDTM), their corresponding bone residues and skin tissues were analyzed for total cholesterol con- tents, phospholipid contents (total and fractions) and fatty acid distribution profiles. Small differences were found among fatty acid components of neutral lipids from various tissue samples while phospholipid fatty acid components of MPCM and MPTM resembled more the fatty acids of their corresponding bone or hand deboned meat phospholipids than skin phospholipids. Quantitation and classification of phospholipids in- dicated that the total phospholipid content and quantity of each phos- pholipid class in MPPM were most similar to those found in their bone tissues. Total cholesterol contents revealed that cholesterol content of MPPM lipids more closely resemble cholesterol content of skin lipids or bone lipids than muscle lipids. From quantities of each component found in their composite tissues, models for MPCM and MPTM lipids were set up. Based on fat contributed by each tissue, a value of l:3:6 meat fat: bone fat: skin fat was suggested as a combination for MPCM fat. A model of l:4:5 meat fat: bone fat: skin fat was hypothesized as a MPTM lipid model Pantipar P. Jantawat Storage stability of mechanically processed poultry meats was evaluated in two different studies. The first study involved the effect of inert gases vacuum packed and prefreezing hold time on storage stability of MPPM. MPCM and MPTM were packed along with either N2 or C02 or under vacuum, and frozen at -l8°C either immed- iately after packing or after 72 hrs. holding at 4°C. All treated samples were stored at -l8°C up UJ4 months. Samples were evaluated for oxidative and hydrolytic rancidity by following the decrease in polyunsaturated fatty acid (C l8:3-22:6), the development of TBA reactive substances and changes in total phospholipids. Vacuum and N2 packed samples from every treatment gave signif- icantly lower TBA numbers and higher unsaturation ratios than CO2 packed samples of corresponding treatments. Vacuum packaging was comparable to N2 packaging with respect to the development of TBA reactive substances found in most treatments. Advantages for vac- uum packaging over N2 and C02 packaging were observed in phospholipid losses found in MPTM samples but these advantages were not signifi- cant in MPCM samples. Significantly higher losses of polyunsatur- ated fatty acids, total phospholipids and increases in TBA numbers were found in samples which were held 72 hrs. at 4°C before freezing. The second part of the stability study involved the effect of air at various tension levels on storage stability of MPPM and their lipid extract samples. Air pressures equivalent to 0, 5, l5 and 30 in. of Hg. were assigned to MPCM, MPTM and their lipid extract samples. All treated samples were stored up1x>3 months at -18°C. Changes in phospholipid unsaturation ratios (C l8:3-22:6 / C 16:0), 2-thiobarbituric acid tests and losses in total phospholipids were Pantipar P. Jantawat used to follow lipid oxidation reactions. MPCM samples packed at 5 in. of air were found to be comparable to vacuum packed samples with respect to the development Of TBA reactive substances and loss Of polyunsaturated fatty acids. For MPTM, TBA absorption values of samples packed under vacuum were significantly lower than those found in samples packed at 5, l5, and 30 in. of air after the first month of storage. Significant differences in mean unsaturation ratios were observed between MPCM and MPCM lipid extract samples, but these results were not Observed between MPTM and MPTM lipid extract sam- ples. Significant differences between these two types of stored samples within the same meat species however, were found in their TBA absorption values and total phospholipid contents. To My parents and my husband ii ACKNOWLEDGMENTS My very sincere gratitude is expressed to Dr. L. E. Dawson for his generous and marvelous guidance, advice and encouragement through- out my graduate program. I would also like tO express my appreciation to Drs. L. R. Dugan, Jr., A. M. Pearson, P. Markakis and L. L. Bieber for serving on the doctoral committee. .Special thanks are extended to Dr. C. Cress and R. R. Neitzel for assisting in statistical and computer analyses. Further expressions of gratitude are extended to the Department of Food Science and Human Nutrition for financial assistance throughout the course of this study. Finally, to my husband, Sondate goes an indescribable appreciation for his moral support, understanding and help throughout the years of my graduate studies. iii TABLE OF CONTENTS Page LIST OF TABLES . . .' ....................... LIST OF FIGURES .......................... INTRODUCTION ........................... 1 LITERATURE REVIEW ......................... 4 Composition of Meat Lipids ................. 4 Composition of Poultry Lipids ................ 4 Hand Deboned Poultry Tissues .............. 4 Mechanically Deboned Poultry Meats ........... 7 Cholesterol Content of Poultry Tissues ......... 7 Meat Lipid Oxidation .................... 9 Lipid Autoxidation ................... 9 Oxidation of Tissue Lipids ............... 10 Meat Lipid Prooxidants ................. l0 Stability of Poultry Meats ................. 12 Storage Stability of Ground Poultry Meats ........ l2 Storage Stability of Mechanically Deboned Poultry Meats ......................... 13 Effects of Diets on Poultry Fat Composition and Stability ......................... l7 Effects of Species on Composition and Stability of Poultry Fats ...................... l9 Extraction of Tissue Lipids ................. 2] Separation of Phospholipids from Neutral Lipids and Fractionation of Phospholipids .............. 22 Gas Liquid Chromatography (GLC) ............... 23 Methylation of Fatty Acids ............... 23 Gas Liquid Chromatographic Analysis Of Fatty Acid Methyl Esters .................. 24 Evaluation of Lipid Oxidations ............... 25 Cholesterol Analyses .................... 27 MATERIALS AND METHODS ....................... 29 Materials .......................... 29 Meat Samples ...................... 29 Materials fOr Chromatographic Analyses ......... 30 iv Reference Standards ................... 3l Solvents ........................ 31 Glassware ........................ 32 Preparation of Meat Samples ................. 33 Composition Study .................... 33 Inert Gas Study ..................... 33 Air Tension Study .................... 34 Extraction of Total Lipids ................. 36 Separation of Phospholipids from Neutral Lipids ....... 36 Methylation of Lipids .................... 38 Gas Chromatographic Analyses of Fatty Acid Composition of Polar and Non Polar Lipids ............... 39 Classification of PhOSpholipids ............... 39 Quantitation of Total Phospholipids ............. 40 Phosphorus Determination .................. 40 Phosphorus Standard Curve ................ 4l 2-Thiobarbituric Acid (TBA) Test .............. 4l TBA Absorption Values .................. 42 Cholesterol Analyses .................... 43 Total Cholesterol .................... 43 Extraction of Tissue Lipids ............... 43 Saponification and Extraction of Nonsaponifiable Fraction ....................... 43 Derivatization ..................... 44 Cholesterol Standard Curve ............... 45 Free Cholesterol .................... 45 Gas Liquid Chromatographic Analysis of Cholesterol . . . 46 Statistical Analyses .................... 47 RESULTS AND DISCUSSION ...................... 48 Part 1. Composition Studies ................ 48 Fatty Acids ......................... 48 Fatty Acids from Neutral Lipids ............. 48 Fatty Acids from Phospholipids ............. 52 Classifications and Quantitations Of Phospholipids ..... 57 Cholesterol ......................... 59 Correlations ........................ 62 Part II. Stability of MPPM ................. A. Effects of Inert Gas and Vacuum Packaging on Storage Stability of Mechanically Processed Poultry Meats ...................... 67 Changes in Fatty Acid Composition ............ 67 Z-Thiobarbituric Acid (TBA) Tests ............ 74 SUMMARY AN PROPOSAL F LITERATURE APPENDICES A. on Correlation of Unsaturation Ratios and TBA Page Numbers ........................ 79 Total Phospholipid Phosphorus .............. 8l Effects of Air at Various Levels on Storage Stability of Mechanically Processed Poultry Meats . . . . 88 Changes in Fatty Acid Compositions ........... 88 2-Thiobarbituric Acid (TBA) Tests ............ 94 Total PhOSphOlipid Phosphorus .............. 99 D CONCLUSIONS ...................... l06 0R FUTURE RESEARCH ................... llO CITED ......................... lll ............................ l23 Chromatogram for neutral lipid fatty acids ....... l23 Chromatogram for phospholipid fatty acids ........ 124 Chromatogram for total cholesterol ........... l26 Total Lipid contents of chicken and turkey tissues . l27 Chromatogram for free cholesterol ............ 128 Changes in fatty acid composition of phospholipids from MPCM packed with CO or N2 or under vacuum and stored at -18°C for 4 mofiths .............. l29 Changes in fatty acid composition Of phospholipids from MPTM packed with CD or N2 or under vacuum and stored at -l8°C for 4 mogths .............. l3l Changes in fatty acid composition of phOSphOlipidS from MPCM and their lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at -18oc 000000000000000000 o ..... ‘33 Changes in fatty acid composition of phospholipids from MPTM and their lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at -l8°C ........................ l35 vi Table la. lb. 2a. 2b. 10. ll. 12. LIST OF TABLES Fatty acids of the neutral lipids from chicken tissues . . . Fatty acids of the neutral lipids from turkey tissueS. . . . Fatty acids of the phospholipids from chicken tissues. . . . Fatty acids of the phospholipids from turkey tissues . . . Total phospholipids and phOSphOlipid classes from chicken and turkey tissues ................ Total cholesterol content of chicken and turkey tissues Free cholesterol content Of mechanically processed chicken and turkey meats ................. Comparisons of analyzed values and calculated values of total cholesterol and total phospholipid contents from mechanically processed chicken and turkey meats ..... Mean unsaturation ratios and their Tukey separations for MPCM and MPTM packed under CD2 or N2 or vacuum and stored at -18°C for 4 months .................. Mean TBA numbers and their Tukey separations for MPCM and MPTM packed under CD2 or N2 or vacuum and stored at -l8°C for 4 months ..... . ................. Correlation coefficients between unsaturation ratios and TBA numbers ....................... Mean total phospholipid phosphorus and their Tukey separa- tions for MPCM and MPTM packed under CD2 or N2 or vacuum and stored at 48°C for 4 months ............. Analyses of variance of the unsaturation ratios, TBA numbers and phospholipid phosphorus . .......... Mean unsaturation ratios and their Tukey separations fOr MPCM, MPTM and their lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored at -18°C fbr 3 months . . vii Page 49 50 53 54 58 60 63 89 Table l3. T4. 15. Page Mean TBA absorption values and their Tukey separations for MPCM, MPTM and their lipid extract samples packed at O, 5, l5 and 30 in. of air and stored at -l8 C for 3 Imnms ........................... 95 Mean total phospholipid and their Tukey separations for MPCM, MPTM and their lipid extract samples packed at 0, 5, l5 and 30 in. of air and stored at -l8 C for 3 months ........................... lOO Analyses of variance of unsaturation ratios, TBA absorp- tion values and total phospholipid phosphorus ....... lOl viii Figure 10. LIST OF FIGURES Oxidation of C 18:3 to 22:6 polyunsaturated fatty acids in phospholipids of MPCM packed under N2 or C02 or vacuum and stored at -18°C, either immediately after treatments or after 72 hrs. holding at 4°C ....... Oxidation of C 18:3 to 22:6 polyunsaturated fatty acids in phospholipids of MPTM packed under N2 or C02 or vacuum and stored at -18°C, either immediately after treatments or after 72 hrs. holding at 4°C ....... TBA numbers of MPCM packed under C02 or N2 or vacuum and stored at -18°C, either immediately after treatments or after 72 hrs. holding at 4°C .............. TBA numbers of MPTM packed under C02 or N2 or vacuum and stored at -l8°C, either immediately after treatments or after 72 hrs. holding at 4°C .............. Total phospholipid phosphorus of MPCM packed under C02 or N2 or vacuum and stored at -l8°C, either immediately after treatments or after 72 hrs. holding at 4°C . . . . Total phospholipid phosphorus of MPTM packed under C02 or N2 or vacuum and stored at -18°C, either immediately after treatments or after 72 hrs. holding at 4°C . . . . Unsaturation ratios of MPCM and MPCM lipid extract samples packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at -18°C ................. Unsaturation ratios of MPTM and MPTM lipid extract samples packed at 0, 5, 15 and 30 in. Of air and stored up to 3 months at -l8°C ................. TBA absorption values of MPCM and MPCM lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at -18°C ............. TBA absorption values of MPTM and MPTM lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at -18°C ............. ix Page 70 71 76 77 84 85 90 91 96 97 Figure Page 11. Total phospholipid phosphorus of MPCM and MPCM lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at ~18°C ............ 102 12. Total phospholipid phosphorus of MPTM and MPTM lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored up to 3 months at -18°C ............ 103 INTRODUCTION In recent years, mechanically deboning processes have been used extensively to remove poultry meats from bones. This deboning process has been found to enhance the utilization of poultry meat sources. Chicken necks and backs or frames, turkey racks and spent laying hens which were of low market value, are now being mechanic- ally deboned, and used in emulsified and other processed food pro- ducts. However, it has been found that mechanical deboning alters the lipid and protein composition of the meat. These changes re- sult in flavor instability and formation of some undesirable func- tional characteristics Of the meat. The nature, proportion and unsaturation degree of unsaturated fatty acids present in a lipid system or food will indicate the ap- proximate susceptibility of that product toward oxidative deterior- ation. Generally speaking, the higher the proportion and degree of unsaturation of the fatty acids the more labile the lipid system is to oxidation. The mechanical deboning process was reported to in- corporate heme and lipid components from bone and skin into the re- sulting meat (Froning 1970). Therefore lipids from these sources might also affect flavor quality of the meats and also be respon- sible for stability problems found in subsequent utilization and storage of the meats. Cholesterol,which has been identified as a risk factor in occurrence of premature coronary heart desease has been found to be higher in turkey frankfurters made from mechanically processed meat than those made from the hand processed meat. Thus, the availabil- ity of detailed lipid compositional data for both the meat and its components may help to elucidate the source or sources of this sterol and possibly, the way to minimize it. Many attempts have been made to slow down or prevent oxidative rancidity in mechanically processed meat. In freezing preservation of ground fresh meat, the main consideration in packaging the meat is the exclusion of oxygen. The mechanism of lipid oxidation in fresh meat is complicated due to the presence of heme pigments which are generally accepted as biocatalysts for the meat lipid oxidation. Neil and Hasting (1925) demonstrated rapid conversion of hemoglobin to methemoglobin at intermediate rather than at very high or very low oxygen tension. According to Tarladgis (1961), iron in metmyo- globin or hemoglobin was highly effective in initiating the chain reaction mechanism in the lipid autoxidation process. Storage of meat in an atmosphere containing inert gases has been found to retard bacterial action as well as oxidative changes. Various fresh meats were found to remain palatable during frozen storage much longer when packed in nitrogen or vacuum packed. How- ever, while lowered oxygen tension accelerates oxidation of hemo- proteins to their oxidized forms, lowering of the pH was also found to further accelerate this reaction. Some inert gases such as C02 in the presence of water in the meat tissue, might cause lowering of the meat pH. Thus, the study of behavior of oxygen at various levels as well as the comparison of benefits or disadvantages of some different storage atmospheres might be helpful in improving the meat preservation. Specific Objectives of this study were: 1. To study the composition of mechanically processed poultry meat lipids in comparison with those of the hand deboned meats, skin and their corresponding bone residues. To compare the effect of oxygen at different levels on storage stability of mechanically processed poultry meats. To compare storage stability of mechanically processed meat packaged in the presence of some inert gases with that vacuum packaged. To study the effect of prefreezing hold time on subsequent storage stability of mechanically processed poultry meats. To study and compare the storage stability of meat lipids as they naturally occur, with extracted meat lipids which are freed from other components. LITERATURE REVIEW Composition Of Meat Lipids Meat lipids are classified according to their sites of distri- bution and composition into two major groups. Intermuscular fat is stored as large deposits in adipose tissue or under the skin. This fraction was found to compose mainly Of triglycerides. Another group was found intramuscularly. These muscle tissue lipids are an integral part of such cellular structures as muscle cell wall, mito- chondria and microsomes. They are separated from the depot fat, are highly unsaturated and many of them are combined with protein (Watts, 1961; Love, 1972; Pearson et al., 1977). Terroine (1920) classified structural fats as "element constant" while the depot fats were named "element variable." This classifica- tion was proposed to differentiate the fact that the element varia- ble can either be drawn up to furnish energy for body processes or deposited when there is an ample supply of food and less energy is required. The constant element on the other hand, remains relatively stable in quantity, to preserve the essential structure of the body. Composition of Poultry LLpids Hand Deboned Poultry Tissues Certain fats, such as those of fish, poultry and pork have been reported to be much more easily oxidized than those from other animals such as beef and lamb (watts, 1954). These differences are 5 largely attributed to the total lipid content, phospholipid content and fatty acid compositions of each species. Besides, variations among tissues of the same animal also exist. Nhite chicken meat, the lowest in total lipids, was found to contain almost equal amounts of neutral and phospholipids. Dark meat contained about twice as much Of the total lipids. However, lipid Of dark meat has been found to have only about half as much of the phospholipid content, when com- pared to that found in the white meat. Skin and depot tissue fats were reported to contain low quantitiestyfphospholipids (Katz et al., 1966; Acosta et al., 1966; Lee and Dawson, 1973). Moerck and Ball (1973) reported that chicken bone marrow contained a slightly higher percentage of phospholipids than most other tissues. Although variations were found in phospholipid content of dif- ferent tissues, it was pointed out that the components of phospholi- pids, expressed as a percentage of total phospholipids, are somewhat similar in most animal tissues (Pearson et al., 1977). Phosphatidyl choline and phosphatidyl ethanolamine were found to be the predomin- ant components Of poultry tissue phospholipids. The lesser compon- ents found were sphingomyelin, phosphatidyl serine, phosphatidyl inositol and lysophosphatidyl choline (Peng and Dugan, 1965; Davidkova and Khan, 1967; Nangen et al., 1971 and Lee and Dawson, 1973). Fatty acid composition of neutral and phospholipids from poultry tissues have been studied by various workers (Peng and Dugan, 1965; Machlin et al., 1962; Katz et al., 1966; Marion et al., 1967 and Lee and Dawson, 1973). Predominant fatty acids of the neutral lipids were palmitic acid, stearic acid, oleic acid and linoleic acid. These fatty acids account for approximately 95% Of the total fatty acids of the neutral lipids. Relative quantities of these major fatty acids were shown to be similar among various types of tissue. Predominant fatty acids of phospholipids were palmitic acid, stearic acid, oleic acid, linoleic acid and arachidonic acid. These fatty acids have been found to comprise approximately 75% Of the total fatty acids found in muscle phospholipids. However, the concentra- tion and kind of fatty acids in phospholipids varied among different tissues, and arachidonic acid attributed most to these differences, the quantities of this C 20:4 fatty acid was lower in skin and depot phospholipids than in muscle phospholipids. Difference in fatty acid composition in total lipid of chicken and turkey skins was reported by Miller et al. (1962). Their studies revealed that the largest difference in fatty acid content between turkey and chicken skin was Observed in the eighteen carbon polyun- saturated fatty acids. Turkey skin contained approximately 60% more linoleic and 50% less linolenic acid than did chicken skin. Very few published data are available on the fatty acids of poultry bone marrow. Seigel and Latimer (1971) stated that chicken tibia bone marrow contains high levels of unsaturated fatty acids. Moerck and Ball (1973) found that approximately 91% of fatty acids in triglyceride fraction of chicken bone marrow lipid were comprised of stearic acid, oleic acid, linoleic acid and palmitic acid, while high levels of polyunsaturated, 20 to 24 carbon fatty acids, were found in the phospholipid fraction. Mechanically Deboned Poultry_Meats Compositions of mechanically deboned poultry meat have been subjected to extensive studies by various investigators (Goodwin et al., 1968; Froning, 1970; Satterlee et al., 1971; Dimick et al., 1972; Grunden et al., 1972; Froning and Johnson, 1973 and McMahon and Dawson, 1976). Mechanically deboned poultry meat was found to have higher lipid content than the hand deboned meat. The lipid component from bone marrow and skin was claimed to account for the large in- crease in fat content of mechanically deboned poultry meat. Moerck and Ball (1974) studied lipid oxidation in mechanically deboned chicken meat. They reported that about 1.4% of the total lipid in mechanically deboned chicken meat was phospholipids, while neutral lipids comprised approximately 98.6%. The predominant fatty acids of the triglyceride fraction were palmitic, stearic, oleic and lino- leic acids. The phospholipid fraction contained higher levels of lB-carbon saturated and 20:3 to 22:6 carbon polyunsaturated fatty acids than did the triglyceride fraction. Cholesterol Content of Poultry Tissues A comprehensive review Of available data for cholesterol con- tent Of food, including methods for its determination, was recently prepared by Sweeney and Neihrauch (1976). Mickelberry et a1. (1964) demonstrated that dietary fat in- fluenced moisture, fat, cholesterol content and iodine value of both cooked and uncooked broiler tissue. Further investigation by this group of researchers in 1966 indicated that most of the choles- terol in chicken occurred as free cholesterol and that only a small 8 fraction (2-10%) existed in the form of esters. For total cholesterol content of raw chicken tissues, the fol- lowing values were reported in mg per 100 g of the wet tissue: white meat 57-67; dark meat 82-148; skin 109-472. For cooked tissue, the values were: white meat 82-84; dark meat 91-96 and skin 91 mg per 100 g of wet tissue (Mickelberry et al., 1966; Marion and Noodroof, 1965 and Feeley et al., 1972). Nockles (1973) reported the total cholesterol content of certain tissues of hen in mg per 100 g of dry tissues. He obtained the following values: dry thigh muscle 161; dry liver 1040; and dry adipose tissue, 201. Kritchevsky and Tepper (1961) reported 110 mg per 100 g for total cholesterol con- tent in skinless turkey meat. Neudoerffer and Lea (1968) studied the effects of dietary fat on the amount and proportions of the individual lipids in turkey muscles. They found a total cholesterol content of 84 mg per 100 9 meat and 116 mg per 100 9 meat in breast and dark turkey meat respectively. Hartung et a1. (1973) determined total cholesterol content of cooked commercial broad breasted turkey meat. They obtained 68.3 to 94.2 mg per 100 g for roasted white turkey meat and 73.8 - 130 mg per 100 g for roasted dark turkey meat. Standal et a1. (1970) found a value of 111 mg per 100 9 meat of total cholesterol in smoked turkey. Moerck and Ball (1973), separated chicken bone marrow lipid by using Unisil column chromatography. They reported the free cholesterol content Of femur, tibia and ilium- ischium bone to be 1.4, 1.3 and 1.3% Of total lipid content respec- tively. The values for cholesterol esters for these bones were: 0.3% for femur bone; 0.2% for tibia bone and 0.2% for ilium ischium bone. Only one report was found containing data for cholesterol con- tent in mechanically deboned poultry meat. Values Of 2.5 g choles- terol and 0.5 g cholesterol esters per 100 g Of total lipids were found in mechanically deboned chicken meat by Moerck and Ball (1974). Meat Lipid Oxidation Lipid Autoxidation Mechanism of lipid autoxidation has been reviewed by many authors including: Dugan, 1961; Lundberg, 1962; Shultz et al., 1962; Labuza, 1971; Sherwin, 1972 and Sato and Herring, 1973. The gener- ally accepted autoxidative mechanisms involve a three step series of reactions, as shown in the following scheme (Dugan, 1971): Initiation: RH + initiator + free radical R° Propagation: R° + 02 + ROO° ROO° + RH + ROOH + R° Termination: R° + R° + inert products R° + ROO° + inert products ROO° + ROO° + inert products Initiation step involves the formation of a free radical species. Under sufficient energetic applications, with certain kinds of energy such as light, heat, enzyme, metal and some radiation particles, the labile hydrogen atoms at the double bonds of unsaturated fatty acids can be abstracted from their sites. The propagation step involves the combination of the first Only one report was found containing data for cholesterol con- tent in mechanically deboned poultry meat. Values of 2.5 g choles- terol and 0.5 g cholesterol esters per 100 g Of total lipids were found in mechanically deboned chicken meat by Moerck and Ball (1974). Meat Lipid Oxidation Lipid Autoxidation Mechanism of lipid autoxidation has been reviewed by many authors including: Dugan, 1961; Lundberg, 1962; Shultz et al., 1962; Labuza, 1971; Sherwin, 1972 and Sato and Herring, 1973. The gener- ally accepted autoxidative mechanisms involve a three step series of reactions, as shown in the following scheme (Dugan, 1971): Initiation: RH + initiator + free radical R° Propagation: R° + 02 + R00° ROO° + RH + ROOH + R° Termination: R° + R° + inert products R° + R00° + inert products R00° + ROO° + inert products Initiation step involves the formation of a free radical species. Under sufficient energetic applications, with certain kinds of energy such as light, heat, enzyme, metal and some radiation particles, the labile hydrogen atoms at the double bonds Of unsaturated fatty acids can be abstracted from their sites. The propagation step involves the combination of the first 10 free radical formed with molecular oxygen to form a peroxy radical (R00°). A new free radical is then formed as a result of the reac- tion between the peroxy radical and a nonoxidized unsaturated fatty molecule (RH). Formation of non free radical products was considered to be the termination of the chain reaction. Oxidation of Tissue Lipids Tissue lipid oxidation has been found to contribute markedly to the undesirable flavor changes which occur in stored meat. A sig- nificant quantity of phospholipids have been reported in muscle lipids (Katz et al., 1966; Peng, 1965 and Acosta et al., 1966). Be- cause of their contents of unsaturated fatty acids, these phospho- lipids were found to be very susceptible to oxidative rancidity (Watts, 1954; Younathan 8 Watts, 1959; El-Gharbawi and Dugan, 1965; Love and Pearson, 1971 and Lee and Dawson, 1973). These protein bound lipids have been found to autoxidize much more readily than the free glycer- ide fat. According to Chipault and Hawkins (1971), autoxidation Of meat lipid occurs in two stages. The protein bound lipids oxidize first without an induction period, their initial rate of oxidation then decreases as time progresses. After a period of lower oxygen absorption, the free glyceride fats begin to autoxidize in the auto- catalytic manner characteristic of autoxidation in isolated glycer- ide fat. Meat Lipid Prooxidants Heme pigments have been generally recognized as one of the most important prooxidants for meat lipid oxidation (Watts and Peng, 11 1947; Zipser and Watts, 1961; Love and Pearson, 1971 and Lee et al., 1975). Banks (1944) and Tappel (1953) proposed that preformed lino- leate peroxide was necessary for hematin catalysis. According to Tappel (1962), hematin catalysis involves the formation of lipid- peroxide-hematin compounds, and their subsequent decomposition into free radicals could have a prOpagation effect on the chain reaction step in the autoxidation process mechanism. Tarladgis (1961) hypoth- esized the mechanism of heme catalyzed lipid oxidation. He postula- ted that iron in metmyoglobin was a paramagnetic substance which was in a high spin state. This highly energetic iron could cause the formation of the first free radical of the chain reaction in the autoxidation process. The ratio of hemoprotein to unsaturated fatty acids in muscle tissue has been reported to have a certain influence on the extent of the catalytic activity of the pigments. Kendrick and Watts (1969) studied the acceleration and inhibition Of lipid oxidation by heme compounds. They summarized that, for a maximum catalytic activity, the ratio of quantity of the pigments and unsaturated fatty acid Of the system must exist at an optimum value. According to Hirano and Olcott (1971), the rate of Oxidation of linOleate solutions was catalyzed by low concentrations of heme and heme-proteins and inhi- bited by higher concentrations. Lee et a1. (1975) found that the ratio of relative concentrations Of polyunsaturated fatty acid to hemoprotein in mechanically deboned chicken meat was in the range where heme catalized Oxidation would occur at near the maximum rate. He thus pointed out that this could be one reason why this deboned meat is very susceptible to oxidative rancidity spoilage during 12 . utilization and storage. Non heme iron has also been reported to have an important role in accelerating the oxidation of muscle lipids. Heavy metals (Cu, Fe, Ni, etc.) have been reported to increase the rate of oxidative deterioration in food lipids (Smith and Dunkley, 1962; Ingold, 1962). Wills (1966) pointed out that the peroxidation of fat in tissue mitochondria and microsomal fractions was easily induced by iron. Other investigators also reported similar results (Liu and Watts, 1970; Love, 1972; Uri, 1961 and Heaton and Uri, 1961). Other factors affecting lipid oxidation in meat are light, tem- perature and salts (Watts, 1954 and Dugan, 1961). Sherwin (1968) mentioned enzymes, moisture, heat, light, other oxidized fats and acids as prooxidants of tissue and other lipid oxidative rancidity. Stability of Poultry Meats Storage Stability Of Ground Poultry Meats SatO and Hegarty (1971) stated that any process that causes a disruption in the muscle membrane system could cause exposure of lipid components to oxygen and other prooxidant substances. As a result, an acceleration in the development of lipid oxidation could easily occur. Marion and Forsythe (1964) studied oxidation of lipids in raw ground turkey tissue held at 4°C for 1 week, and reported a rapid increase in TBA numbers in both white and dark meat. Keskinel et a1. (1964) found that a raw ground turkey meat which was held at 5°C upixithree weeks showed a rapid increase in TBA numbers. Dhillon and Maurer (1975b) reported high initial TBA numbers for ground hand deboned turkey meat. Dawson and Schierholz (1976) studied the 13 . develOpment of lipid oxidation in turkey meat products which were either roasted whole or boned,ground and broiled as patties. They found that TBA numbers were highest from ground cooked patties held 7 days at 3°C, followed respectively by ground raw patties held 7 days, freshly roasted meat and freshly ground patties. They then concluded that stability Of turkey meat was influenced by cooking, grinding and storage, and the combination resulted in maximum lipid oxidation. Dawson et a1. (1975) further reported that the high TBA numbers found in turkey patties prepared from ground thigh meat and held at 3°C up to 10 days, could have been effectively controlled by treating with a commercial antioxidant mixture, containing butylated hydroxyanisole, propyl gallate and citric acid. Storage Stability Of MechanicalLy Deboned Poultry Meats Composition and storage stability of mechanically deboned poultry meat have been the subject of numerous studies since 1970. Froning (1976) in his recent review on composition, functional pro- perty and stability of mechanically deboned poultry meat stated that the mechanical deboning process may cause considerable cellular dis- ruption, protein denaturation and increase lipid and heme oxidation in the resulting meat. Besides, oxygen could oftentimes be mixed into the deboned meat during the extrusion process. Various workers have found that the machine deboning process incorporates lipid from skin and bone into the resulting meat. Thus, lower protein and higher fat contents than that found in the hand deboned meat have been observed in various sources of mechanically deboned poultry meat (Goodwin et al., 1968; Froning, 1970; Froning 14 et al., 1971; Satterlee et al., 1971; Dimick et al., 1972; Grunden et al., 1972; Froning and Johnson, 1973 and McMahon and Dawson, 1976). Besides lipids, workers have reported substantial quantities of heme components in mechanically deboned poultry meat. Froning and Johnson (1973) reported a threefold increase in the total heme pig- ments in mechanically deboned fowl meat as compared to those of the hand deboned meat from the same sources. Cunningham and Mugler (1973) and Lee et a1. (1975) reported similar results. Considering all of the mentioned factors, it's Obvious that lipid Oxidation must be considered as one Of the most serious prob- lems involved in storage and utilization of mechanically deboned poultry meat. Maxon and Marion (1970) reported that both lipid oxidation and hydrolytic deterioration occurred in lipids of mechanically deboned turkey meat. They found that besides a linear response of TBA num- ber with storage time, total phospholipids decreased during the 7 days storage at 4°C and in the freezer at -20°C. Schnell et a1. (1971) demonstrated that particle size of mechanically deboned chicken influenced its TBA numbers. According to these workers, smaller par- ticle sizes resulted inlargerVTBA numbers. Dimick et a1. (1972) used carbonyl contents and organoleptic tests to evaluate the qual- ity of mechanically deboned poultry meat. They reported that the quality of mechanically deboned poultry meat could be maintained up to 6 days at 3°C. They also observed that no differences in storage stability were noted where poultry parts were deboned immediately or held 5 days at 3-4°C prior to the deboning. Johnson et a1. (1974), however, noted a significant flavor loss in mechanically deboned 15 _ turkey meat after 12-14 weeks Of frozen storage. Dhillon and Maurer (1975a) studied storage stability of comminuted meat including mechanically deboned chicken meat, centrifuged mechanically deboned chicken meat, hand deboned chicken meat, mechanically deboned turkey meat, centrifuged mechanically deboned turkey meat and ground beef, at ~25°C for 6 months. They observed that highest initial TBA num- bers were found in mechanically deboned chicken meat, mechanically deboned turkey meat and centrifuged mechanically deboned turkey meat. Of all samples studied, they found mechanically deboned chicken meat to be affected the most by frozen storage. Storage stability of products containing mechanically deboned poultry meat was also studied by many researchers. Froning et a1. (1971) incorporated 15% mechanically deboned turkey meat either fresh or after 90 days frozen storage into red meat frankfurters. They reported that the TBA number indicated that frankfurters containing 15% fresh mechanically deboned turkey meat were comparable to all red meat franks in flavor stability. However, after 90 days frozen storage mechanically deboned turkey meat products were significantly inferior. Cunningham and Mugler (1973) reported that the excellent qualities of mechanically deboned chicken meat weiners could be maintained through 6 months storage at -30°C. Dhillon and Maurer (1975a) formulated summer sausages with 50% mechanically deboned chick- en meat and 50% ground beef, 50% mechanically deboned turkey meat and 50% ground beef and 100% ground beef (control). After storage for 6 months at -25°C, quality measurements by TBA numbers and sensory evaluations indicated that there was only a slight decline in quality. Summer sausages containing mechanically deboned chicken 16 _ meat showed the greatest quality loss. However, the products were well accepted and there was no connemt.on any flavor differences. Uebersax et al. (1978a) used various concentrations of mechanically deboned turkey meat in turkey meat loaves. They reported an increase in TBA numbers with increase of mechanically deboned turkey meat sub- stitution for both raw and precooked foil wrapped or vacuum sealed loaves. Mechanism of lipid oxidation and interaction of heme and lipid components in mechanically deboned meat have also been studied. Moerck and Ball (1974) reported that considerable autoxidation de- terioration occurred in the highly unsaturated phospholipid fatty acids of mechanically deboned chicken meat, whereas the triglycer- ide fraction failed to exhibit an apparent oxidation after the meat was stored at 4°C for 15 days. They also found a high correlation between the phospholipid fatty acid oxidation and TBA numbers of the meat. Lee et a1. (1975) destroyed hemoprotiens in a mechanically deboned chicken meat homogenate by treatment with H202. They re- ported that the catalytic function was decreased tO less than 10% Of the original activity. They thus concluded that hemoproteins were the predominant biocatalyst of lipid oxidation in mechanically de- boned chicken meat. Various efforts have been made to alleviate the stability prob- lems found in mechanically deboned poultry meats. Froning and John- son (1973) centrifuged mechanically deboned fowl meat at 20,000 rpm for 15 minutes in a refrigerated Sorval centrifuge (5°C). They re- ported that centrifugation significantly increased the protein con- tent and significantly decreased the fat content of the end products. l7 .Centrifugation in addition, significantly reduced changes in TBA num- bers of mechanically deboned fowl meats. Various additives have been utilized to maintain storage sta- bility of mechanically deboned poultry meat. Butylated hydroxy anisole,commercial mixture of antioxidants containing 20% butylated hydroxyanTSOIe, 6% propyl gallate, and 4% citric acid in prOpylene glycol and food grade phosphates have been found to be effective in maintaining flavor stability of mechanically deboned poultry meat (MacNeil et al., 1973; Moerck and Ball, 1974 and Froning, 1973). MacNeil et a1. (1973) found a rosemary spice extract to be effective in maintaining lower TBA numbers in simulated mechanically deboned poultry meat, after the meat was stored at 3°C for 11 days. Uebersax et al. (1978b) reported that invivo tocopherol supplementa- tion through diet and subcutaneous injections were effective in lower- ing TBA numbers for mechanically deboned turkey meat and mechanically deboned turkey meat substituted loaves. Effects of Diets on Poultry_Fat Composition andetability The lipid contents of the tissue greatly reflect the rearing conditions and diet of the birds. Likewise, the fatty acid content of poultry reflects the fatty acid content Of dietary fat (Marion and Woodroof, 1963; Isaacks et al., 1964; Neudoerffer and Lea, 1968; Edward et al., 1973; and Porter and Britton, 1974). Kummerow et a1. (1948) studied the effect of variations in diet on the characteristics of fat extracted from 18 different groups of immature turkeys. They observed that when compared with control birds, the fat extracted from birds which had been supplemented with 18 . linseed oil was least stable, and those from birds which had been supplemented with choline chloride or ethanolamine hydrochloride was most stable. Klose et a1. (1952) reported that turkeys on low fat diet was roughly estimated to contain 28% total saturated fatty acids and 68% octadecenoic acids. With further studies on quality and stability of turkey as a function of diet, Klose et a1. (1953) postulated that the tendency of fat to deteriorate in frozen storage may be pre- dicted in part from either the induction period or fatty acid compo- sition of the carcass fat. Marion and Woodroof (1963, 1966) studied the effects of diet on fatty acid composition and stability Of chicken broiler carcasses. They reported that the fatty acid composition of breast, thigh and skin responded to the dietary fats by tending to assume the fatty acid composition of the fat in the diet. Feeding of different fats resulted in an increased carcass deposition of the major fatty acids present in each fat. Regarding stability, diets containing coconut Oil or beef tallow produced carcasses with lower TBA numbers than diets containing high protein or menhaden oil. Marion et al . (1967), in a study on the effects of dietary fat and protein on lipid compo- sition and oxidation in chicken meat, Obtained good correlation coef- ficients between TBA numbers and level of each lipid component. Bartov and Bernstein (1977) determined the effect of graded increments of a-tOCOpherOl acetate, in diets containing various fat supplements at different concentrations on stability of abdominal fat and meat of broilers. They Observed that diets containing a- tocopherol acetate significantly improved stability of abdominal fat 19 . and meat in broilers having relatively saturated carcass fat, where- as its beneficial effect was rather limited as the degree of unsat- uration of carcass fat increased. Porter and Britton (1974) observed that feeding chickens with full-fat soybeans resulted in changes of fatty acid composition of their fat. The fat was found to be soft, oily and more susceptible to oxidative rancidity. Effects of Species on Composition and Stability of Poultry Fats No significant variation in major components and fatty acid composition of poultry fat were reported among fat samples from dif- ferent species of poultry (Mecchi et al., 1952; Miller et al., 1962; Marion and Woodroof, 1965; Marion et al., 1970 and Wangen et al., 1971). Pereira (1975), studied fatty acid composition of pro- cessed fat from chicken, turkey and duck, and reported that differ- ences in fatty acid composition due to species difference did exist, but that these differences were expected to be less marked than dif- ferences imparted by dietary fat changes within the same species. Stability variations among species of poultry fat, however, have been reported by many investigators. In the field of poultry products, turkeys are known to be more susceptible to rancidification than chickens, and, due to seasonal production, are generally stored fOr longer time periods (Klose et al., 1952). Nutter et a1. (1943), studied the chemical composition of depot fats in chickens and tur- keys, and reported that turkey fat has a greater tendency toward rancidification than chicken fat. They attributed this phenomenon to the lower content of natural antioxidant in turkey fat. Criddle 20 . and Morgan (1951) reported results in which turkeys on zero and low tocopherol diets were judged rancid after 3 months of frozen storage and those receiving high tocopherol diet did not become rancid until after 9 months storage. Mecchi et a1. (1952) studied the lipid composition of chicken and turkey carcass fat in an attempt to correl- ate composition with rancidity development. They concluded that fatty acid composition differences in chicken and turkey fats were not sufficient to explain the greater tendency of turkey fat toward rancidification. Baker (1958) compared rate of rancidity development in chicken, duck and turkey fats. They reported that both hydrolytic and oxidative rancidity were greater for turkey and duck fats and lowest for chicken fat. Mecchi et a1. (1956 a and b) determined tocopherol content in chicken and turkey fats, and reported about 5 times more tOCOpherol in chicken fat than in turkey fat. They then, postulated that the difference in quantity of this natural anti- oxidant might be responsible for the difference in stability Of turkey and chicken fats. Pickett et al. (1967) demonstrated a re- duction in rancidity development in male turkey carcasses, during 2 months of storage, as a result of an injection of 600 I.U. of tocopherol. Jacobson and Koehler (1970) in studies on rancidity develOp- ment during short time storage of cooked chicken and turkey meat, however, reported only a slight difference in TBA numbers and sen- sory scores between these two types of meat. A somewhat contrary result was found in mechanically deboned poultry meats. Dhillon and Maurer (1975 a and b) studied storage stability of mechanically deboned turkey meat and mechanically de- boned chicken meat, at -25°C for 6 months. Their chicken meat 21 .samples were affected more than meat from turkey, during frozen storage, as indicated by TBA increase and taste panel results. When these 2 types of meat were formulated separately at 50% level with ground beef in sunmer sausages, those sausages containing chicken meat showed greater quality loss. They speculated that this differ- ence may have been particularly due to higher fat-water combination in mechanically deboned chicken meat which was composed Of just backs and necks. Extraction Of Tissue Lipids For quantitative extraction of tissue lipids, a polar solvent, usually in combination with a non polar solvent is necessary. Nelson (1975) stated that covalently bound lipids must be subjected to a hy- drolysis procedure before they can be extracted with organic solvents of any polarity. The polar solvent is able to disrupt the hydrogen bonding of protein bound lipids, thus allowing free access of non polar solvent to the lipids. Folch et a1. (1957) used 2:1 chloroform-methanol mixture to extract tissue lipids and aqueous solutions for their purifi- cations. Ostrander and Dugan (1961), evaluated 4 lipid extraction methods and proposed a modification of the method by Bligh and Dyer (1959), using methanol -chloroform-water as the extracting sol vent, to be most satisfactory in extraction ofmeat lipids. Radin (1969) also reported that chloroform-methanol appeared to be a good solvent combination for extracting wet tissue. Sheppard et a1. (1974) compared 8 lipid extraction methods by analyzing 8 different food samples. Based on the recovery of total lipid, fatty acid distribution and triglyceride recovery, they found 2 methods to be satisfactory: a 4N HCl digestion followed by ethyl 22 . ether extraction and another one, a 2:1 chloroform: methanol extrac- tion. In their recent study, Hubbard et a1. (1977) used 7 extraction methods to compare the efficiency of total lipid extraction from samples of 8 different food products. They reported that, on the basis of total lipid recovered and amounts of fatty acids and sterols present, the chloroform methanol method described by Folch et a1. (1957) was selected as the most effective method. Separation of Phospholipids from Neutral Lipids and Fractionation Of Phospholipids Silicic acid column chromatography has been used by many workers to separate polar lipids present in animal tissues (Hanahan et al., 1957; O'Brien and Benson, 1964; Katz et al., 1966; Peng and Dugan, 1965 and Nelson, 1975). This separation method was found to be use- ful in separation of a sample into major lipid classes such as neu- tral lipids, glycosphingolipids and phospholipids. Rapid growth of thin layer chomatography (TLC) as a tool for lipid separation has occurred in the past decade. It has advantages over the silicic acid column separation method with respect to res- olution, Speed of separation, and generally simpler procedures (Nel- son, 1975). Silicic acid column chromotagraphy, however, was found to be useful as an adjunct to TLC. It was reported to be a superior technique in preparing lipid samples for further analysis by TLC (Nelson, 1975). Two dimentional TLC on silica gel plate was found to be the most convenient and reliable method, currently available for separ- ation of a complex mixture of phospholipids. Skipski et a1. (1962) separated phospholipids and cerebrosides on silica gel G plate using 23 _a mixture of chloroform, methanol, acetic acid and water as develop- ment solvents. Parker and Peterson (1965) found a special washed silica gel-H plate and solvent mixture containing chloroform, methanol, acetic acid and water to be a rapid and accurate micro method for fractionation of phospholipids prior to their quantitative determin- ations. Rouser et a1. (1966) used a two dimensional TLC with solvent pairs: chlorofOrm-methanol-water and n-butanol-acetic acid-water; andcflfloroform-methanOl—28%aqueous ammonia followed by chloroform- acetone-methanol-acetic acid-water to improve separation of phospho- lipids prior tO analysis of their phosphorus contents. Nelson (1975) concluded that TLC is a precise, accurate and reproducible method for fractionation of complex phospholipids. Many methods including gravimetric, colorimetric and titrimetric have been used in phosphorus analyses. Among these, colorimetric methods are usually preferred for their adaptability to the micro- method and for their simplicity. Colorimetric methods described by Morrison (1964), Parker and Peterson (1965) and Rouser et a1. (1966) are among those that are generally accepted and used currently. Gas Liquid Chromatography (GLC) Methylation of Fatty Acids Purposes of fatty acid esterification prior to GLC analysis are to prepare volatile esters from relatively non volatile compounds and to reduce the polarity of the compounds. Two techniques are commonly used in fatty acid derivatization: direct interesterifica— tion reaction and liberation and isolation of fatty acids from lipids by saponification, acid hydrolysis or'enzymatic hydrolysis and 24 subsequent esterification of the liberated fatty acids Choices of reagents and methods should depend on the type of compounds one wishes to analyze (Anon, undated). For fatty acids containing eight or more carbons the BF3 - MeOH or BCl3 - MeOH re- agents will give good results. For low molecular weight fatty acids BF3 - BuOH reagent is preferable. For fatty acids. triglycerides phospholipids etc., which are difficult to esterify, the saponifica- tion-esterification procedure will do the job. Commonly used fatty acid esterification methods at present, include those described by Morrison and Smith, 1964; McGinnis and Dugan, 1965 and Metcalfe et al., 1966. Gas Liquid Chromatographic Analysis of Fatty Acid Methyl Esters A gas liquid chromatographic technique was introduced in 1952 by James and Martin, as a method for carboxylic acid separation. Since then, it has been developed and used widely as a reliable tool in many analytical fields, including lipids. Choice of liquid phase in GLC analyses of lipids depends on composition of sample. For an efficient, normal separation, liquid phase should be similar in chemical structure to component of mixture. Orr and Callen (1958) recommended polyester type liquid phase to shorten analysis time and to provide a good resolution in separation of esters of common and polyunsaturated fatty acids. Of these types, adipate and succinate polyesters of diethylene glycol are most widely used. Kuksis (1965) recommended various silicone polymers, of which the most popular has been SE-30, for a high temperature separation of natural triglycerides. 25 Mehlenbacker (1960) pointed out that elution characteristics of individual compounds in GLC analysis depends on type and amount of liquid phase, temperature, gas flow rate, and type of compound analyzed, while resolution of chromatographic peaks was related to column efficiency and solvent efficiency. Seino et a1. (1973) studied influence of Operating conditions on determination of fatty acid methyl esters. They found that sample size and flow rate of carrier gas had significant influences on the analytical values. Identification of various peaks on fatty acid GLC chromatogram can be done in many ways. Retention volume or time which is charac- teristic of the samples and liquid phase can be used to identify samples. James (1960) introduced an identification method in which retention times of standard fatty acids were plotted versus their chain length on semilogarithmic graph paper. Since a plot of the log of the retention times is proportional to some increasing property of the homologous series, identification of members of each homolo- gous series can thus be obtained. This method is advantageous in that only 2 or 3 compounds are needed to establish the slope of the line and thus can be used to identify other members of the same series. Evaluation of Lipid Oxidations Numerous methods are available for evaluation of lipid oxida- tion. however, none of them has been found to be ideal. The choice of method used, thus, will depend normally, on factors such as: types of products to be analyzed, nature of problems and equipment availability. According to Erickson and Bowers (1974), objective methods 25 Mehlenbacker (1960) pointed out that elution characteristics of individual compounds in GLC analysis depends on type and amount of liquid phase, temperature, gas flow rate, and type of compound analyzed, while resolution of chromatographic peaks was related to column efficiency and solvent efficiency. Seino et a1. (1973) studied influence of operating conditions on determination of fatty acid methyl esters. They found that sample size and flow rate of carrier gas had significant influences on the analytical values. Identification of various peaks on fatty acid GLC chromatogram can be done in many ways. Retention volume or time which is charac- teristic of the samples and liquid phase can be used to identify samples. James (1960) introduced an identification method in which retention times of standard fatty acids were plotted versus their chain length on semilogarithmic graph paper. Since a plot of the log of the retention times is proportional to some increasing property of the homologous series, identification of members of each homolo- gous series can thus be obtained. This method is advantageous in that only 2 or 3 compounds are needed to establish the slope of the line and thus can be used to identify other members of the same series. Evaluation of Lipid Oxidations Numerous methods are available for evaluation of lipid oxida- tion. however, none of them has been found to be ideal. The choice of method used, thus, will depend normally, on factors such as: types of products to be analyzed, nature of problems and equipment availability. According to Erickson and Bowers (1974), objective methods 26 available for determination of lipid oxidation can be classified into 4 groups: 1) methods based on lipid compositions; 2) methods based on absorption of Oxygen; 3) methods based on the intermediate forma- tion of lipid peroxides and 4) methods based on the measurement Of one or more Of the final reaction products or classes of products resulting from peroxide decompositions. In most lipid composition studies, fatty acid analyses will be done. Quantity of unsaturated fatty acids as well as their de- grees of unsaturation in a lipid system, in general, will indicate the approximate susceptibility of that system toward oxidative de- teriorations. Decreases in degree of fatty acid unsaturation as storage time of fat increases have been observed. And, in some cases this reduction can be positively correlated with other methods used to measure the progress of lipid oxidation (Lee and Dawson, 1973 and Moerck and Ball, 1974). Measurement of products formed subsequent to decomposition of peroxides are also used to measure oxidative rancidity in animal tissues. Sinhuber et a1. (1958) reported that condensation products of one molecule of malonaldehyde with 2 molecules of 2-thiobarbituric acid (TBA) reagent was responsible for the red color developed in rancid salmon oil. Tarladgis et a1. (1960, 1964) re- ported that free malonaldehyde was produced during the oxidative breakdown Of unsaturated fatty acids from food products. According to these researchers, malonaldehyde itself did not contribute to the rancid Odor in food. However, good correlations between TBA test and rancid flavor development have been reported by many workers (Younathan and Watts, 1959; Jacobson and Koehler, 1970; Webb et al., 27 1972 and Johnson et al., 1974). Another method which is generally accepted, in this group is carbonyl-test (Henicketal., 1954; Dugan, 1955 and Lea and Swoboda, 1958). An advantage Of using TBA test to determine rancidity development in animal tissues is that the test can be applied directly to the products without prior extraction Of lipid. Cholesterol Analyses Steps necessary to determine the cholesterol content of pro- ducts are extraction of lipids, Separation of cholesterol from lipids and interfering materials and detection and measurement Of isolated cholesterol (Sweeney and Weihrauch, 1976). In general, any lipid extraction method that will satisfactorily extract lipids from muscle tissue should be suitable for extraction of tissue cholesterol. Sweeny and Weihrauch (1976) stated that a mixture of polar and non polar solvents gave better cholesterol ex— traction from dairy products or from any other food products in which part of the cholesterol may be bound to a lipoprotein or some other substance in food. Hubbard et a1. (1977) reported that the method described by Folch et a1. (1957) was the most effective method among 7 other methods they evaluated, in extraction of cholesterol from some food products. Separation of cholesterol from lipid and interferring materials can be done in various ways. Digitonin was used by many workers to precipitate free cholesterol from lipid extracts (Sperry and Brand, 1943; Hunter et al., 1945; Sperry, 1963 and Tu et al., 1967). Kabara and McLaughlin (1961) and Edwards et a1. (1964) reported that 28 tomatine was more specific than digitonin for cholesterol precipi- tation. Various chromatographic methods have been used to separate cholesterol. Moerck and Ball (1973, 1974) used a mixture of differ- ent polar solvents to elute cholesterol and cholesterol esters from Unisil columns. Thin layer chromatography (TLC) has been used by many investigators to separate cholesterol and cholesterol esters (Skipski et al., 1968; Thorpe et al., 1969; Tattrie, 1972 and Teich- man et al., 1974). The most commonly used adsorbent in TLC isolation of cholesterol is silica gel G. Comnon solvents used for developing the chromatograms include: ethyl ether -petroleum ether, hexane- ethyl ether-acetic acid, chloroform-methanol and chloroform-methanol- water. Gasliquid chromatography has been recently used widely for both sterol separation and determination. According to Sweeney and Weihrauch (1976), it has greater specificity and more accuracy for cholesterol analysis than colorimetric methods, especially when the sample being assayed contains sterols and other interfering materials. MATERIALS AND METHODS Materials Meat Samples All meat samples were Obtained from a commercial poultry proc- essing plant in Michigan. Mechanically processed meats were processed through a Behive mechanical deboning machine (Model AU968MF, Behive Machinery Co., Sandy, Utah). Composition Study - Light hand deboned poultry meat (HDPM) was processed from breast meat while dark hand deboned meat was Obtained from thigh and drumstick meats. Light and dark skins were Obtained from their corresponding light and dark meat pieces. Light mechan- ically processed poultry meat (MPPM) was processed from hand boned breast racks whereas the dark MPPM was from back racks and portion of necks. Bone samples, both light and dark, were separated by the deboning machine from either white or dark machine processed meats. Chicken meat and products were obtained from freshly dressed (not frozen) adult hens (fowl), and further processing was accomplished as soon as major muscles were removed from carcasses for use in other products. Turkey meat and products were obtained from previously processed and frozen birds. The original source and length of stor- age of these turkeys is unknown. Turkeys were thawed in cold water prior to hand boning major muscle and machine deboning of remaining bony portions. All samples were packed in cryovac bags. The closed 29 30 bags were then packed along with dry ice and ice in an insulated box, and transferred to the Food Science Laboratory. Stability Study - Both mechanically processed chicken and tur- key meats were processed from whole carcasses. Meat samples were packaged in 18 kg corrugated board boxes, lined with plastic sheets. Temperature Of meat at time of receiving at laboratory ranged from 4-7.5°C. Materials for Chromatographic Analyses Stainless steel columns (0.32 cm x 1583 m) packed with 10% Diethylene glycol succinate-phosphoric acid (DECS-PS), on 80-100 mesh supelcoport, were Obtained from Supelco Inc. The columns were conditioned by temperature programming from 50°C to 190°C at 2°C/min., for 48 hrs. The column flow rate during the conditioning period was 20 ml/min. For sterol analysis, a silane treated glass column (0.635 cm x 1.83 m) packed with 3% 0V-17 on 100-200 mesh gas-chrome Q, was ob- tained from Applied Science Laboratories Inc. The column was condi- tioned prior to use by programming from 50°C to 300°C, at 2°C/min. for 48 hrs. Column flow rate during conditioning was 80 m1/min. Silica gel G and H plates were purchased from Applied Science Laboratories Inc. or from Supelco Inc. All plates were activated at 105°C for 1 hour before use. Silicic acid (100 mesh) was Obtained from Mallinkrodt CO. Inc. Preparation of the column was as follows: Silicic acid was washed several times with deionized distilled water to remove the fines. Water was removed fronl washed acid 31 by passing the slurry through BUchner funnel fitted with # 1 filter paper. The precipitate was then rinsed on the filter, twice, with anhydrous methanol. Washed silicic acid was activated at 105°C for 24 hours. A 25 9 sample was then dispersed in chloroform and poured into a 1.5 x 30 cm glass column fitted with a sintered glass disc and Teflon stopper at its lOwer end. Silicic acid was allowed to settle and chloroform was drained from the column by applying a slight suction. A 1 cm layer of granular anhydrous sodium sulfate was then placed on top of the acid column. The column was rinsed several times with chloroform prior to use. Reference Standards Standard cholesterol (99 + % pure) and 5a-Cholestane (99 + % pure) was obtained from Applied Science Laboratories, Inc. Mixture of fatty acid methyl esters were obtained from Supelco Inc. and Applied Science Laboratories Inc. Standard phosphorus solutions were made from dried potassium dihydrogen phosphate primary standard (Fisher Scientific Company). Phospholipid standard inixtures were Obtained from Supelco Inc. Solvents Analytical reagent grade chloroform and methanol were freshly redistilled prior to each use to remove any contaminating peroxides. When the redistilled chloroform required storage for more than 1 day, a 0.25% methanol by volume was added to prevent its decomposi- tion. All other solvents, unless otherwise specified, were a 32 'Distilled in Glass' high purity solvent grade Obtained from Burdick and Jackson Laboratories. All solvents were stored at 3°C. Glassware All glassware which might come in contact with sterol and fatty acid methyl esters was subjected to a special treatment. First the glassware was washed through a normal laboratory washing proced- ure. After drying, they were soaked in concentrated sulfuric acid overnight. After soaking, they were rinsed copiously with water and distilled water. Before use, all surfaces which would come in con- tact with the sample were rinsed twice with either chloroform or a 2:1 chloroform methanol solution. All screw caps used in sterol and fatty acid analyses were cleaned by using a sonic type cleaner. After cleaning, they were rinsed with distilled water and a 2:1 chloroform methanol solution. Teflon pads which were soaked and rinsed well with chloroform were used as liners for all screw caps. Silane treated glass centrifuge tubes and their stoppers used to derivatize cholesterol were prepared and cleaned according to AOAC (1976) method. Glassware used for phosphorus determinations was cleaned by rinsing with water. After each use, they were soaked with conc. sulfuric acid overnight. After soaking, they were rinsed well with distilled water, followed with deionized distilled water and were dried prior to use. 33 Preparation of Meat Samples Composition Study Hand deboned meats, skins and bone residues were ground through a Hobart meat grinder, fitted with a 3 mm hole plate. A11 ground tissues including mechanically processed meats were hand mixed, to assure their homogeneity, then vacuum packed at about 150 9 level, in # 13 IKD plastic bags and stored at -18°C. Inert Gas Study Mechanically processed chicken and turkey meats were individ- ually mixed at medium speed in a Hobart mixer (Model K-5A), under N2, for 2 minutes. Mixing was performed to Obtain homogeneity within each batch of meat. After mixing, the meat was packed in 100 g por- tions in 170 x 100 mm2 # l3-IKD plastic bags. One third of the total packageswere then vacuum sealed by using a Kenfield vacuum packaging sealer. The rest of the packages were then flushed with either N2 or C02 gas and sealed so that a constant and appropriate volume of either of the gases was confined within each bag. After sealing, half of the packages from each treatment were randomly selected and stored in a walk in type freezer at -18°C. The remaining packages were transferred to a home Style refrigerator and held there for 72 hours. Temperature variations during this holding period ranged from 2.5-5°C. At the end of the holding period, all packages were transferred to the freezer and stored at -18°C, for 4 months. Treatment diagram is illustrated as follows: 34 MPPM l n MPCM MPTM packing packing 111- I‘ll vacuum N2 CO2 vacuum N2 CO2 freezing Hold 4°C, 72 hrs. prior freezing \¢/ stored -18°C,4 months At the end Of each specified interval, samples were randomly selected for analyses. All samples were partially thawed at room temperature for 1 hour, prior to analyses. Air Tension Study All meat samples were mixed at a medium speed, on a Hobart mixer (Model K-SA), under N2 for 2 minutes. After mixing, each meat item was vacuum packed in a # 13 IKD plastic bag in such a manner that they were uniformly spread in a 2.54 cm thickness layer within each bag. After sealing, they were held in the freezer at -l8°C overnight. Frozen meats were then cut into 5.08 x 5.08 x 2.54 cm3 by using a Ho- bart meat saw (Model 5212). Each meat block was wrapped with a piece Of low density polyethylene sheet and arranged into a prepared storage chamber. Lipid extracts from 100 g Of meat, stored in a 30 x 35 35 mm wide mouth bottle type glass vials, were arranged within the same storage chamber. A low density polyethylene wrap was selected to help minimize moisture loss from the meat surface and to minimize the interfering effect of the transportation of microclimatic 02 into the meat block during storage. After the chamber was closed, an assigned quantity of air pressure was created within each chamber. The chamber was first evacuated by using a Duo-Seal vacuum pump (model 1400). After complete evacuation, a known quantity of air was injected back into the chamber, and the chamber was sealed. A U-type vacuum guage was used to assure the appropriate quantity Of air within each chamber. The closed chamber was then placed in a cryovac bag and the bag was sealed with a Tipper Clipper bag sealer (model 72105). Storage chambers were stored in the freezer at -18°C for 3 months. At one month intervals, the products in one chamber from each treatment were randomly selected for chemical analyses. Treatment diagram for this study is as follows: MPPM r l I MPCM MPTM I 4 l I l . 1‘1 Meat Lipid Extract Meat Llpld Extract M Packing J 1 Air Pressure 1 0H 5" 15H 30" Storage -18°C, 3 months 36 Extraction of Total Lipids The method described by Folch et a1. (1957) was used to extract total lipids from all samples. A weighed quantity of ground tissue containing at least 0.5 g of fat was homogenized at high speed in a Vortex homogenizer with a volume of 2:1 chloroformzmethanol (about 20 times the sample weight) for 2 minutes. The extracting solvent and tissue residue were then transferred to BUchner funnel fitted with # 1 Whatman filter paper and the filtrate transferred to a 500 ml separatory funnel. The residual cake and filter paper were re-ex- tracted for 1 minute with an additional solvent system of about 5 times the sample weight. The extracting solvent was filtered and collected in the separatory funnel. The crude extract was washed with 0.2 its volume with a 0.74% potassium chloride solution and allowed to stand at ~18°C overnight to facilitate its separation. The chloroform layer was collected by passing the solution through a glass funnel containing about 20 g of anhydrous sodium sulfate into a glass stoppered round bottom flask. The water layer was washed 2 more times with 20 m1 portions of chloroform, and the solvent layers were combined. Solvent was evaporated at 20°C by using a rotary vacuum evaporator (Rinco Instrument Co.). After drying, traces of chloroform were further evaporated under a N2 stream. The total lipid was stored in a vacuum desicator at -18°C when not used immediately. Separation of Phospholipids from Neutral Lipids Composition Study - Column chromatography was used to separate phospholipids from neutral lipids in this study. A 0.25 9 sample of lipid in chloroform, was added to the column. 37 Elution of each fraction was performed under a gentle pressure. A stream of nitrogen was used to flush the sample from the top of the column throughout the elution period. The sample was eluted from the column at a rate of 2 drops per second. A 15 ml portion of sol- vent was used for each elution until the total volume of solvent was 20 times the lipid sample weight. Neutral lipids were eluted with chloroform while methanol was used as eluant for phospholipids. Purity of each separating fraction was checked by thin layer chroma- tography. A 0.25 mm thickness silica gel G plate was used. A sol- vent system consisting of petroleum ether, ethyl ether and acetic acid (90:10:l by volume) was used to check purity of phospholipids. Purity of neutral lipids was confirmed by using a mixture of chloroform methanol-water (65:25z4 by volume) as the developing solvent. The solvent was evaporated from each lipid fraction on a Rinco rotary vacuum evaporator. Last traces of solvent were removed under a N2 stream. Phospholipids from each separation were quantitated by adding accurately 5 m1 of 2:1 chloroform methanol into the concentrated residue. After thoroughly shaking, a 1 ml aliquot was taken and dried in a forced air oven at 105°C to a constant weight. The remaining solution was stored under N2 in a 4 m1 Teflon lined screw cap vial at -18°C until use. Stability study - Thin layer chromatography was used to separ- ate phospholipids from neutral lipids for both stability studies. A 0.50 mm thick layer silica gel G plate was used for this separation. Fat samples dissolved in chloroform were applied under N2 along the bottom of the plate in several spots, 0.5 cm apart. A 10 ul 38 microsyringe was used for application of the sample. The plate was developed in a saturated chamber, by using chloroform as a developing solvent. After each development, the lipid bands were visualized by spraying with 0.5% iodine in methanol solution. Iodine was allowed to sublimate from the plate under a N2 stream, prior to the removal Of lipids from the plate. Phospholipids, were then scraped from the bottom of the plate and transferred into a 20 m1 screw cap test tube with the aid of several 2 ml portions of 2:1 chloroform methanol sol- vent. The remaining neutral lipids were then scraped into another tube. Diethyl ether was used as an eluting solvent for the neutral lipid. After elutions, solvents were evaporated from the sample under N2 stream, and the samples were subjected to methylation for GLC an- alysis. Methylation Of Lipids Methylation of lipids was performed according to the method described by Morrison and Smith (1964). Solvent was evaporated from lipid sample under N2. One ml of 14% boron trifluoride in methanol was added under NZ to the polar lipid residue in a 10 x 125 mm test tube. The tube was sealed with Teflon-lined screw caps and heated for 10 minutes in a boiling water bath. The non polar lipid residue was treated in the same manner except that 0.2 ml of benzene was added to the sample prior to heat- ing and the heating time was 30 minutes. After heating, samples in test tubes were cooled to room temperature and the methyl esters were extracted. A 3.0 m1 portion of petroleum ether and 1.5 ml of dis- tilled water were added to the sample in the tube and the tubes were 39 shaken briefly on a test tube shaker and let stand for 2 minutes. The upper petroleum ether layer was removed. Re-extraction was per- formed with an additional 1.5 ml of petroleum ether. The methyl es- ter solution was stored under N2 at -18°C in a 4 ml Teflon lined screw cap. Fatty acid methyl ester solutions were concentrated under N2 prior to injection into the GLC. Gas Chromatographic Analyses of Fatty Acid Composition of Polar and Non Polar Lipids Gas chromatographic analyses were performed by using an F&M model 810 dual column gas chromatograph, equipped with a flame ioniza- tion detector. Helium was used as the carrier gas ata flow rate of 30 ml per minute. The hydrogen flame was fed with 35 ml per minute hydrogen and 350 ml per minute compressed air. Temperature was programmed from 150 to 190°C at a rate of 4°C per minute. Tempera- tures of detectors and injection ports were maintained at 255°C and 250°C respectively. A sample of 2.0 p1 was injected for each anal- ysis. . Fatty acid methyl esters were identified by comparing their relative retention times (relative to methyl palmitate), using a plot Of logarithm of the relative retention time versus the number of carbon atoms. After peak identification, the percentage of each fatty acid ester was calculated by dividing the area of each indi- vidual peak by the total area of all peaks. Classification of Phospholipids Phospholipid solutions obtained from silicic acid column sep- aration were applied on 0.5 mm, thick layer silica gel G plates, 40 along with standard materials. The plates were developed in a chamber saturated with a mixture of solvents containing 65:25:8:4 chloroform: methanol:acetic acid: water (by volume). After each development, spots were visualized with iodine vapor and located. Identifications were made by comparing the Rf values of the unknown spots with those of standards. Various quantities of spots were then scraped directly into 30 m1 Kjeldahl flasks. Adjacent areas of blank silica gel corresponding in size to the areas containing phospholipids were also scraped into digestion flasks to serve as blank determinations for the analysis (Skipski et al., 1962). Quantitation of Total Phospholipids Separation of phospholipid from neutral lipid was done by the thin layer chromatographic method previously described under the title “Separation of Phospholipids from Neutral Lipids." After all phospholipid spots were visualized and located, they were scraped into a 30 m1 Kjeldahl digestion flask. Blank samples were obtained in the same manner as those described for phospholipid fractionation. Phosphorus Determination The total phospholipids and their fractions were quantitated by analyzing the phospholipid phosphorus using the method described by Rouser et a1. (1966). Samples in Kjeldahl flasks were digested with 0.9 m1 of 72% perchloric acid, using a low heat setting on an electrically heated digestion rack (Laboratory Construction Co., Model A). The flasks were shaken occasionally during the digesting period. After 30 minutes, each sample was allowed to cool and the flask side was 41 rinsed with 5 ml of deionized distilled water. One ml of 2.5% ammonium molybdate was added to the sample. After swirling, 1 m1 of freshly prepared 10% ascorbic acid was added and the content of the flask was mixed and transferred to a 10 ml centrifuge tube. The flask was rinsed with 2 m1 deionized distilled water and the rins- ings were combined. The tubes were then heated in a boiling water bath for 5 minutes. After cooling, the absorbance of the superna- tant solution was read at 820 nm using Bausch & Lomb Spectronic-ZO. Water was used to adjust the zero reading and a mean value from sev- eral blank determinations was subtracted from the sample reading. Phosphorus content of each sample was determined directly from a standard curve. Phosphorus Standard Curve A solution containing 0.1 mg of phosphorus per ml, was made by diluting 0.4393 g of dried potassium dihydrogen phosphate primary standard to one liter with deionized distilled water. Various amounts Of standard solution to contain 2-10 pg of phosphorus were pipetted into Kjeldahl flasks, and further treated in the same manner as those for each food sample. Concentrations of phosphorus in micrograms were plotted against their absorbances. 2-Thiobarbituric Acid (TBA) Test A 10 9 portion of mechanically processed poultry meat was hom- ogenized with 50 ml of distilled water, at medium speed for 2 min- utes, in Virtis homogenizer. The resulting mixture was then quanti- tatively transferred, with the aid of 47.5 ml distilled water, into a 500 m1 distilling flask. Two and one-half m1 of 4N HCl were added 42 to lower the pH to about 1.5. A few glass beads were added and the mixture sprayed with Dow Corning antifoam. After thorough mixing, the flask was connected to the distilling apparatus. The distilling unit was composed of a 30.5 cm long distilling column connected to the condensor with a bending shoulder, and a 50 m1 graduated cylinder served as a receiver. Distillation was continued at a rate in which 50 ml of the distillate was collected within 10-15 min., subsequent to boiling. Two aliquot portions of the distillate were pipetted and trans- ferred to the reacting tubes. Accurately, 5 m1 of 0.02 M Z-Thio- barbituric acid in 95% redistilled glacial acetic acid were added and the tubes were capped. After thoroughly mixing and heating in a boiling water bath for 35 min., they were cooled in cold water for 10 min. The absorbance was determined at 532 nm against a reagent blank in which 5 ml of distilled water was used in place of the dis- tillate. The TBA number was calculated by multiplying the mean absorb- ance by 7.8, distillation constant, (Tarladgis et al., 1960) and reported as mg TBA reactive substance per 1000 g of meat. TBA Absorption Values A quantity of lipid extract equivalent to 10 g of mechanically processed poultry meat (2.0 g for turkey lipid extract and 2.3 g for chicken lipid extract) was weighed into 500 m1 distilling flask. Fifty ml Of distilled waterwere added and the flask was stoppered and shaken for 2 min. on a test tube shaker. An additional 47.5 ml distilled water were used to rinse the flask stopper and then combined 43 with the contents of the flask. Further treatments were done in the same manner as those for the tissue determination. TBA numbers of lipid extract were reported as TBA absorption values. Cholesterol Analyses Total Cholesterol Cholesterol analysis was done according to the method described in AOAC (1976). Extraction of Tissue Lipids Tissue lipids were extracted and purified by using the method described by Folch et a1. (1957). Saponification and Extraction of Nonsaponifiable Fraction An accurate volume Of chloroform-lipid extract to contain 250- 500 mg of fat was filtered through a glass funnel containing a pledget of glass wool and about 25 g anhydrous sodium sulfate. After several rinsings with chloroform, the solvent was evaporated to dry- ness on a Rinco rotary vacuum evaporator at 20°C. The residue was dissolved in 70 m1 of petroleum ether and filtered through Whatman no. 1 filter paper containing about 20 g anhydrous sodium sulfate into a 300 ml glass stoppered Erlenmeyer flask. The round bottom flask was rinsed with several 10 ml portions of petroleum ether and the rinsings combined. Petroleum ether was then evaporated to dry- ness on a vacuum rotary evaporator at 25°C. The fat residue was saponified with 8 m1 conc. KOH solution (60 g KOH in 40 ml water), and 40 m1 of reagent alcohol (a mixture of ethyl alcohol: methyl alcohol: isopropyl alcohol, 90:5:5) on a 44 negnetic stirrer hot plate. The sample was gently stirred by means of a magnetic stirrer bar throughout the saponification process. A 1 meter long glass column was attached to the reacting flask during saponification to prevent an excessive loss of the solvent. At the end of the digestion, 60 ml of reagent alcohol were introduced into the flask through the column. After cooling, 100 ml of benzene (accur- ately measured) were added to the sample and the flask was shaken vigorously for 30 sec. The flask contents were then transferred into a 500 m1 separatory funnel and the benzene layer was separated by shaking with 200 m1 of 1 N KOH solution. After the aqueous layer was discarded, the benzene layer was washed with 40 ml of 0.5 N KOH solution, followed by 4 x 40 m1 portions of distilled water. The washed benzene solution was then filtered through Whatman # 4 filter paper containing about 15 g of anhydrous sodium Sulfate into a glass stoppered flask. An additional 20 g of anhydrous sodium sulfate were added into the flask. After vigorously shaking, the flask was al- lowed to stand for 15 minutes. A 50 m1 aliquot portion of benzene was then pipetted into a 100 ml round bottom ground-glass-jointed- flask and evaporated to dryness at 30°C on a rotary vacuum evaporator. After drying, 3 m1 of acetone were added and the mixture was re- evaporated to dryness. The residue was then dissolved in 3 m1 dimethylformamide. Derivatization An accurate 1.0 m1 portion of the extracted nonsaponifiable solution in dimethylformamide solution was derivatized in a silane treated 15 ml centrifuge tube. Each sample solution was shaken vigorously on 45 a test tube mixer with 0.2 m1 hexamethyldisilazane and 0.1 ml tri- methylchlorosilane. After standing 15 min., 1 ml of 5 a-cholestane internal standard solution (0.2 mg per ml 5 a-cholestane in n-heptane) and 10 m1 of distilled water were added into the tube, and the tube was shaken for 1 min. Duplicate 2 pl heptane layers were injected into the GLC equipment. Cholesterol Standard Curve A cholesterol standard solution in dimethylformamide was made to contain 2.0 mg of cholesterol per one ml of the solution. Further dilution provided a concentration range from 0.05 to 1.25 mg/ml. A 0.2 mg per 1 ml solution of 5 a-cholestane in n-heptane was also pre- pared. Derivatization Of cholesterol standard solutions was then per- formed on a 1 ml aliquot of each standard dilution, in the same manner as that described for sample derivatization. Duplicate 2 pl of heptane layer of each dilution was injected into the GLC equipment. Peak area ratio at each concentration, calculated by dividing choles- terol peak area by 5 a-cholestane peak area, was then plotted against standard cholesterol concentrations. Free Cholesterol TLC separation of total lipids was performed on a 0.5 mm thick layer silica gel H plate. A solvent system containing 90:10:l hexane- ethyl ether-acetic acid was used. The plate was developed in a cham- ber saturated with solvent. A 0.5% iodine in anhydrous methanol was used as visualizing agent. Separated free cholesterol was identified by comparing its Rf value with that of pure cholesterol standard. A row of cholesterol spots was then scraped into a test tube with the 46 aid of 5 ml chloroform. Additional 3 x 5 m1 portions of chloro- form were used tO elute the samples out of the silica gel powder. The filtered solvent was then evaporated under N2 stream at 45°C. The dried residue were dissolved in 1 ml of dimethylformamide and sub- jected to derivatization. Standard curves for free cholesterol were prepared by spotting known quantities of cholesterol standard solutions on silica gel H plates. The plates were then developed with the same kind of solvent and procedure as those described for the samples. Further treatments followed the same manner as those for the standard until completion of derivatization. Duplicate 2 pl of standard materials and sample were injected in to the GLC equipment. Gas Liquid Chromatographic Analysis of Cholesterol Analysis of cholesterol was accomplished by using an F & M model 810 dual column gas chromatograph, equipped with a flame ioniza- tion detector. Nitrogen was used as the carrier gas at a flow rate of 35 ml per min. A 35 ml per min. hydrogen and 350 ml per min. of compressed air were used to feed the hydrogen flame. A temperature programming Operation was performed from 240 to 275°C at a rate of 6°C per min. The detector and injection port temperatures were kept constant at 300 and 275°C respectively. The emerging peaks were ident- ified by comparing their retention times with those of standards. Peak areas were determined for both sample and internal standard. The relative area ratio of sample to internal standard was then used to determine cholesterol concentration directly from a standard CUTVE. 47 Statistical Analyses Statistical analyses were performed by using a Michigan State University computer program identified as Analysis of Variance Pro- gram on MSU STAT System Package and run on a Control Data Corporation (CDC) 6500 computer at MSU computer laboratory. RESULTS AND DISCUSSION Part I. Composition Studies Lipids from light and dark mechanically processed poultry meats (MPPM), their corresponding hand deboned meats (HDPM), bone residues and skin tissues were analyzed for total cholesterol contents, phos- pholipid distributions and fatty acid compositions. Attempts were made to correlate components of MPPM lipids with those found in HDPM, bone residues and/or skin tissue lipids. The purpose of this study was to determine the source or sources of fats which entered into MPPM during the machine deboning processes. Fatty Acids Fatty acid distribution profiles for composite tissue samples including neutral and phospholipids from light and dark chicken and turkey tissues are shown in Table l and 2 respectively. Examples of chromatograms for neutral and phospholipid fatty acids from gas liquid chromatographic analyses are shown in appendices A and B. Fatty Acids from Neutral Lipids Ten different fatty acids were identified and quantified from neutral lipids of dark and light chicken and turkey tissues. The most prevalent fatty acids found in this fraction for all types of tissues examined were the 16-carbon atom fatty acids (palmitic and palmitoleic acids) and 18 carbon atom fatty acids (stearic acid, 48 49 .0005 vwconmu 0:010 .0005 ummmmuoga »_qu_cmcumzv .mcowuchELmumn v Eocm mco0000>ou ugmucoum 0:0 c0m2u .mccon mpnzou we Logan: “mcongmu mo consaz a .00000_ _0L0=mc :0 00000 a00mm _00o0 yo mmwucmugoa ma um00~aupmuo 00.0 00.0 00.0 00.0 00.0 00.0 00._ 00.0 000000 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 0:000 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 0:00:02 00.0 00.0 00._ 00.. 00.0 00.0 00.0 00.. 00000 000000000000 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 000000000000 00000 00.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 0000000000 _0000 - - 0 0 0 0 0 0 0000 - - - - - - - - mn0m 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 mm._ _N.0 0 00.0 00.0 0 00._ 00.0 0 00.. 00.0 0 00.0 0”0_ - - - - - - - - 0H0N 00.0 0 00.00 00.0 0 00.00 00.0 0 00.00 00.0 0 00.00 00.0 . _m.00 00.0 0 00.00 00.0 0 00.00 00.0 0 00.00 Nu00 00.0 0 00.00 00.0 0 00.00 00.0 0 00.00 0_.0 0 00._m 00.0 . 00.00 00.0 0 00.00 00.0 0 00.00 00.0 0 00.00 .000 .0.0 0 00.0 00.0 0 _N.0 00.0 a _0.0 00.0 N 00.0 00.0 . 00.00 00.0 a 00.00 00.0 n 00.0 00.0 0 00.0 0H0_ 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 0_.0 0 00.0 00.0 0 0_.0_ 00.0 . 00.0 00.0 a 00.0 00.0 0 00.0 _u0_ 00.0 . 00.00 00.0 0 _0.0N 00.0 0 00.00 00.0 . 00._N 0_.0 0 00.00 00.0 H mm._m 00.0 0 00.00 00.0 0 .0.00 0000 0_.0 0 00.0 00.0 m 00.0 0_.0 . 00.0 0_.0 n 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 00.0 0 00.0 0”0_ 00.0 . 00.0 0_.0 . 00.. 00.0 . 00.. 0_.0 . 00.0 00.0 0 00.0 00.0 a 00.. 00.0 0 00.0 00.0 0 _0.0 0mm, 000000 0000002 00 0000< 00000 0000000 0000 0:000 xc00 0:000 0c00 0:000 0000 00000 0000 0000000 0:00 020: 020: 0.0 000< x0000 mn_u< 0000; we mocsom 0.mmamm_0 cwxuwgu soc» mu_q_0 .0L0sw: 0:» mo 00000 x0000 .00 m_omp Fatty acids of the neutral lipids from turkey tissues.a Table lb. Source of Fatty Acids Dark C op x W H a: 05 up _I .8 L 0U D Q! 3 '0 up m U a: 0 3% Light 110Me Dark Light MPMd Dark Light Fatty Adid b’c ipids Turkey Fatty Acids of Neutral L N N C +1 1.53 z 0.35 3.06 1.46 t 0.21 3.10 t 0.09 0.02 fl 2.57 1 0.19 0.56 +1 #1 +l +1 OCO 00 +1 +1 NLD C F- 0') +1 +1 50 tooxomcn “NMMN 00000 +1 +1 #1 +1 +1 N¢$O¢5 H ‘J 1‘6 u. mMONm CONDO 00000 +1 +1 +1 +1 +1 Qv—QNU) DOV—CO N t 0.06 0.40 t 0.08 1.83 0.71 1.24 t 0.12 0.61 0.31 0.30 z 0.05 2.23 0.60 t 0.04 2.91 16.60 0.76 t 0.13 0.57 1 0.07 0 14: Unknown 1 1x 0 +1 0.19 +1 0.13 +1 0.14 +1 2 0.09 meta MON COO +1 +1 +1 @010 Pro COO +1+1+1 oooao WON QNO ,— ano NO!— 000 +1 +1 +1 Pwe MEN 0 l O r~r-c: ,_ O‘MN Or—O cacic: +1 +1 +1 .—10 $010 mr-O ,— 0Q!— vr-O COO +1 +1 +1 10m wN -—°o POM Nr-O ODD +1 +1 +1 NON OLDN ‘Dr—O .— meow 1"’700 COO +1 +1 +1 Q¢c>c>c>c>c>c>c>c>c>c>c>c> c>c>ES <3 +1+1+1+1+1+1+1+1+1+1+1+1+1u+1+1+1H+1 NmQGNF-r-LDONNQLO mmN 1", memmcmeMNON mow r- r-NOOI—Q‘OQOOOF-Ln vo—M '— '— r—r—r— F'- r— N EOPogo—NMON VmQWNOON DOWN owmwwOOOOONNNVQ EPr-F-‘SP-r—r—F-NNNNNNNNNN C C D D 45.99 52.48 31.31 64.91 34.05 62.69 32.49 62.72 31.53 64.49 32.90 63.64 34.60 61.96 Total Saturation Total Unsaturation Unsaturation Ratio 1.14 2.07 1.84 1.93 2.05 1.79 v—OQNMI‘N MQNNNN OWNMOI— r—r— '— mmOQV’F— OmNNov— N'— '- LDO‘NC) 1~D mv—r—m to Nmr-Qr—r— v—t— N IDMKOF-Lno‘ mQQNww mmr—Or—r— I—r— N Newt—RN tomcnmmm \OC‘OOI—m F'— N $1.150“)qu O‘NKDN mmF-ONG '_"_' N MONQQU‘) meMCQ [\Nr—Nr—N F'P— N LDLDCDQQN \ONNLDO‘N NNr—O‘F-M r—o— '— Q10 0) CCQJ C 0.10011: Q10) C 10160 O C Q) LHIU C Gav-H C x O'F ‘- CJQJ Q1 {OPP—Q: ipids. aCalculated as percentage of total fatty acids in phosphol b Number of double bonds. Number of carbons CMeans and standard deviations from 4 determinations. 55 by various workers (Marion and Woodroof, 1966; Katz et al., 1966; Lee and Dawson, 1973 and Moerck and Ball, 1974). There were no apparent variations in the fatty acid compositions of light and dark tissue phospholipids. Slightly higher quantities of arachidonic acids and slightly lower quantities of palmitic acids were found in almost all the phospholipids from dark tissues when com- pared to those from the light ones. Most of the dark tissue phospho- lipids also had slightly more unsaturated molecules than their cor- responding phospholipids from light tissues. The only exceptions are phospholipids of hand deboned meat and of turkey skin, where these are nearly equal. In general, the relative distribution of fatty acids in phospholipids of these two types of tissue are almost ident- ical. These results are true fbr all types of tissue items examined. The greatest difference between phospholipid and neutral lipid fractions were the high levels of C20 to 24 polyunsaturated fatty acids and low levels of oleic acid and linoleic acids in phospholipids. These results corroborate previous findings by Marion and Woodroof (1966), Katz et al. (l966), and Lee and Dawson (1973). Moerck and Ball (1974) explained that, since arachidonic acids found in poultry meat were synthesized from linoleic acids, the phospholipid fractions which were higher in arachidonic acids thus, were lower in linoleic acid than the triglyceride fractions. The most obvious difference among phospholipids from different types of tissue are the quantities of arachidonic acids and the levels of the total polyunsaturated 20 to 24 carbon fatty acids. The percentage of arachidonic acid is lowest in phospholipid of skin tissues. This result is true for both chicken and turkey. Unusually 56 high levels of arachidonic acid were found in phospholipids from bone residues of dark meat of both chicken and turkey. Dietary fat might be responsible fOr this result. As mentioned in the literature review section, many workers have found that dietary fats affected composi- tion of poultry body fats. Thus, a high percentage of unsaturated molecules might be expected in fat from birds fed with diets contain- ing high levels of unsaturated fats. The C20 to 24 polyunsaturated fatty acids werelowest in skin tissues, highest in hand deboned tissues and comparable between machine processed meats and bone tissues. The low percentages of polyunsaturated C20 to 24 fatty acid in skin tissues accounted for the high levels of saturated fatty acids fbund in this tissue. As is evident from the table, highest levels of palmitic and stearic acids were found in skin phospholipids of both chicken and turkey. Unlike tetraenes, the quantities of pentaenes and hexaenes were highest in hand deboned tissue phospholipids, instead of bone or machine processed tissues. These results, although not expected, can be reasonably explained. Both machine processed meats and bone samples were ground and subjected to high tension treatments'during the deboning process. According to Holman (1954), the increase in each double bond in a polyunsaturated fatty acid could increase the rate of autoxidation by a factor of 2. Thus, during the processing period, followed by subsequent handling and storing, pentaenes and hexaenes which are most vulnerable to oxidative attack might undergo oxida- tion. As a result, lower levels of these acids were then quantitated at time of analyses. When comparing the unsaturation ratios of all types of tissues, 57 it is obvious that, except for skin tissue, there are small differences among phospholipid fatty acids of MPPM, HDPM and bone tissues. These results, as well as relative quantity of individual major fatty acids, found in each tissue, suggest that fatty acid distributions in phos- pholipid fractions of MPPM resemble more those of the hand deboned meats or bone tissues than skins. Classifications and Quantitations of Phospholipids Total phospholipid phosphorus compounds including their classi- fications and quantities are shown in Table 3. Light hand deboned chicken and turkey lipids contained the highest amount of total phospholipids, and the smallest values were found in skins from dark meat from both Species. For all tissues investigated, lipids from light tissues contained a higher proportion of phospholipids than those of the dark ones. This result corrobo- rates those findings by Katz et al. (1966). Five classes of phospholipids were identified and quantified from chicken and turkey tissues. Phosphatidylcholine, phosphatidyle- thanolamine, sphingolipid and phosphatidylserine are predominant com- ponents of muscle and bone phOSpholipids and comprise approximately 90% of total phospholipids. Traces of lySOphosphatidylcholine were also fOund in these tissue phospholipids. Skin phospholipids, on the other hand, was fOund to be richer in lysophosphatidylcholine and only traces of phosphatidylserine were detected in skin phospholipids of both chicken and turkey. Davidkova and Khan (1967) and Lee and Dawson (l973) re— ported similar findings for chicken muscle and skin phospholipids. The most prevalent of the phospholipids fbund in all types of tissue was phosphatidylcholine. This component accounted for .mumwe ummmmuoca xp_muwcmzumz .muows coconut ucoxu n .mcowuecwELmuou c scam mco_um_>mu ucmucmum vac mccmzo mo.o ~_.o mm.o mm.o _o._ mw._ om.o ev.o ocwEopocozum_xu_uocqmocm u a mo.o mo.o mF.o m_.o mo.o __.o mewgmmpxu_uwcqmoga up.o om.o mm.o om.o mm.~ -.m _m.o wa.o cw_ozu_xc_uogamoca mo.o mo.o op.o m_.o mm.o oo.o N_.o om.o cw_mxsomcwnam mo.o oo.o u u u a u u mcw_ocu_»nwumzamogaomx4 moo.o H om.o No.o “ oo.o no.0 “ mo._ No.o N mm._ mo.o N v~.c oo.o n om.m oo.o N mF.F mo.o N mm._ energy—ocamoza —ouo» xmxcap oo.o m~.o mm.o mm.o mm.~ m¢.¢ mm.o om.o mcrEw_o:mcuw_xu_uozgmoza u u No.0 No.o ¢_.o _m.o mo.o mo.o mcwcmm_>uwuocamoga ~_.o mv.o mo.o om.~ mn.~ mo.“ om.o o_.~ mcwpogupxuwuazamoga op.o oo.o mo.o N~.o mm.o mm.o Np.o NN.o cw_wxsomcwnm No.o No.0 u u u u u » mc__onu_xuwuocamocaomx4 ooo.o N ~e.o .o.o H mm.o mo.o “ m_.~ _o.o w mm.~ om.o u Np.o mo.o n mm.mp No.0 H mm.o mo.o u mp.~ mmu_awpo;amoca peach “new m\oev macocqmoca uwaP—oznmoca :mxuwcu teas ugm_s Xena beats xeao “cows xcco ugo_4 mmapu u.a_Focamoga c_xm mcom uzo: azqz .HuaJu au..-..ll.|¢1..4-nvi..uvjdainldvuuawnu-id.qua-ualulkanuadl-vainli.uauunqgrii.1u.--..u-..44n....iu -Nhnw -4fl...4....hw...u-. wuqdiuuni c-n- « .._.i.- mmzmmwu sexes» uco coxuwzu ace» momma—u u_QWPocamozq new mu_a_Pocamoca pouch .m m_nc» 59 from 40 percent (dark chicken skin) to 60 percent (light HDCM) of the total phospholipid contents. The next major component found in almost all the tissues is phosphatidylethanolamine. The only ex- ception is the phospholipids of skin from dark meat of chicken where the quantities of its Sphingolipid and phosphatidylethanolamine are almost equal. Light and dark tissue phospholipids within and among bird species showed similar trends for both compositions and rela- tive quantities of the major phospholipid classes. It is evident from table 3, that the total phospholipid contents of both MPCM and MPTM were intermediate between those of bone and skin tissue or most resemble those from bone tissue, when compared among the three. Quantitation of phospholipid classes also support these results. Except for Sphingolipids from dark MPCM, the quanti- ties of phosphatidylcholine, phosphatidylethanolamine and sphingolipid of both MPCM and MPTM were found to be somewhat similar to those of bone tissues rather than the hand deboned or skin tissues. Cholesterol Total cholesterol values for all tissues are given in Table 4. An example of a chromatogram for total cholesterol is given in Appendix C . Mean values are given in mg per loo 9 fat fbr tissue correlation pur- poses and in mg per g tissues to compare with previously reported results by other researchers. Total cholesterol contents for light HDCM, dark HDCM, light chicken skin and dark chicken skin are 42.8, 70.4, 75.03, and 91.01 mg/lOO g tissues respectively. These values were fOund to be lower than those reported by Mickelberry et al. (l964, l966) who reported 60 .ume coconut ucmxu .umme nommouoca xppmuwcocumzn .mcowumcwELmumu c soc» m:o_umw>wu vcoucoum can wcomza co._ “ mo.- NM.o a oo.mm m~.o n mm.mm _N.o “ om.m~ ew.o H o~.mm co.~ “ mm._e _e.e n mm.ap_ mm.o a -.om mama?“ a oo_\Pogoumo_o;u as eo.o “ a~._ No.o a mm.P No.o n mm.m mN.o “ o~.NF mm.o N m~._. mm.o n oe.o_ Fm.o “ mfi.m No.9 n me.“ one m\Fogmpmm_o;u me xoxcsh m_._ n .o.mm em._ a mo.m~ NN.¢ “ oo.~e_ 05.. a mN._o_ om.m n oa.o~ mm.p a om.~v No.e “ oo.o__ N..N a om.m~ mamm_u m oo_\Foaaumo_ogu me mo.o “ om.~ no.0 n mo.~ m~.o a o_.o_ q..o “ om.m_ km.o a ow._~ Ne.o n om.~P ep.o H mm.m ¢_.o a oe.m be» m\_oca~mm_onu me cmxuwzu Jean “new; Jena u;m_4 xcao u;m_4 xeao p;m_4 83 «$2 ...... lllllrleislTillillellulll mwamm_u xmxcau new cmxu_:u wo acmucou mFoLmummpogu _uuob .e m_noh 61 the values of 57.7, 82.6 and l09 mg/l00 g for chicken breast, thigh and skin tissues. Sweeney and Weihrauch (1976) however, clearly in- dicated that dietary fats and age of the bird influence cholesterol content of the bird tissues. In addition to these biological factors, methods for determinations also affect final cholesterol values. Thus, large variations can be expected from different laboratories. These explanations are also true for turkey tissues. Cholesterol contents of turkey tissue found in this study were: 4l.8, 55.8, 53 and 72 mg/lOO g for light meat, dark meat, light skin and dark skin while those obtained by Neudoerffer and Lea (l968) were: 84 mg/l00 g for breast meat and HG mg/lOO g for dark meat. Light tissue lipids from both turkey and chicken contained higher levels of cholesterol than dark tissue lipids, when reported as mg cholesterol per g fat. However, when the values were calculated on tissue weight basis, dark tissues containedhigher levels of chol- esterol than their corresponding light ones. Differences in total lipid contents between light and dark tissues are responsible for these results. As shown in Appendix 0, dark tissue, in general had about twice as much lipid as did white tissues. From data shown in Table 4, it is apparent that sources of cholesterol in MPPM come from their three component tissue choles- terols. Cholesterol contents (mg/g fat) of both MPCM and MPTM are lower than those found in their corresponding HDM and bone tissues but higher than those of skins. For MPCM, the values obtained are closer to those of skin rather than hand deboned meat or bone tis- sues. Thus from this result alone it seems appropriate to conclude that a greater amount of fat from skin enters into MPCM during the 62 machine deboning processes. For turkey, cholesterol content per g of MPTM fats more closely resembled those from bone fats when com- pared among the three types of tissue. So, at this point, the con- clusion is that bone lipid cholesterol appears to have a significant contribution to cholesterol content of MPTM. Free cholesterol contents of MPCM and MPTM are reported in Table 5. An example of a chromatogram for free cholesterol is given in Appendix E. It is apparent that most of the cholesterol in both chicken and turkey tissues are in the form of free cholesterol. This finding coroborates the work of Mickelberry et al. (l964) and Moerck and Ball (l974). For MPCM only about 6-8% of their total cholesterol was in the form of cholesterol esters. Higher levels of ester forms are found in MPTM. For this particular meat species, approximately l2-l3% of the total cholesterol was cholesterol esters in their tissue lipids. Correlations An attempt was made to correlate quantities of cholesterol, phospholipid and fatty acid distribution profile found in each type of mechanically processed meat with those found in their composite tissues. Various proportions of meat lipidzbone lipidzskin lipid which were likely to be representative models fbr MPPM lipids were set up. Calculations were made in accordance with each set of the hypothesized pr0portion to obtain the final quantities of cholesterol, phospholipids (total and fractions) and fatty acid compositions in mechanically processed meat lipids. It was found that a model of meat fatzbone fatzskin fat of l:3:6 seemed to be most appropriate 63 .mcowumcwecmumu e acct cowumw>mv cgmucmvm ace mcmwzm NP.N H Rm.~oF N¢.P H m~.oN _¢.¢ H mm.e_F mm.o H N~.ow H=HHHH m ooF\FocmHHmFo;u as mo.o H so.m NF.o H mo.m F~.o H m~.m No.o H me.“ Hey m\FoLmHHmPo;u as sea: ow.¢ H oo.mo_ mm.m H mm.mm No.e H oo.o._ NP.~ H om.mn H=HHHH m oo_\_ocmHHmpo;u as o~.o H mh.e ¢N.o H mo.m ep.o H mm.m ¢F.o H oe.m Hat m\FocmHHmpo;u me zuaz gene HemHH Heme Hzmws maxp new: Pogmummpozu muck PoeaHHH_o;u .HHOH mpmos xoxgau new cmxuwgu ummmmuoca »__muwcmcums mo ucmucoo m_ocmumm_ocu mmgu .m mpame 64 for MPCM fats based on fat contributed by each tissue. For turkey, a slightly different pr0portion was concluded. A value of 1:4:5 meat fatzbone fatzskin fat was hypothesized as MPTM lipid model. Comparisons of calculated and analyzed total cholesterol and total phospholipid contentscrflipids from both meat Species are shown in Table 6. Table 6. Comparisons of analyzed values and calculated values of total cholesterol and total phospholipid contents from mechanically processed chicken and turkey meats Total Cholesterol Tbtal Phospholipids (mg/9 fat) (mg/9 fat) Meat Types Calculated Analyzed Calculated Analyzed Values Values Values Values MPCM Light 5.56 5.40 2.55 2.18 Dark 5.40 5.23 1.20 0.98 MPTM 7.40 7.65 1.88 1.87 Light 5.60 5.75 1.28 1.13 Dark From previous findings and discussions, conclusions were drawn as f01lows: 1) There was little difference among the fatty acid components of neutral lipid from various tissue samples. 2) Phospholipid fatty acid components of MPPM resembles the fatty acids of bone or hand deboned meat phOSpholipids more than those from skin phospholipids. 65 3) Tbtal phospholipid contents and quantity of each phospho- lipid class in MPPM are most similar to those from bone tissues. 4) Cholesterol contents of MPPM resembles the cholesterol con- tents of skin tissues or bone tissues more closely than that from muscle tissues. In contrast to the hypothesized models, the above conclusion indicated that, except for neutral lipid fatty acids, bone tissue lipids seem to have a predominant effect on MPPM lipid composition. However, as evident from data shown in Table 6. total cholesterol and total phospholipid contents of both MPCM and MPTM lipids are in good agreement with their corresponding hypothesized values. For phos- pholipid classes and phospholipid fatty acids of MPCM and MPTM, al- though the actual findings seem to be contrary to the hypothesized models, they are explainable. Although it was hypothesized that 50-60% of fats in MPPM are from skin fats, skin fats contain the lowest levels of phospholipids. Thus, even if 5-6 times as much skin fat as meat fat did enter into the machine processed meat fats, the proportion of skin phospholipids in the resulting MPPM phospho- lipids were still lower than those from either the hand deboned meat or bone tissue. As a result, the MPPM phospholipid fatty acids and their phospholipid classes resemble most the bone and hand deboned tissue phospholipids or bone phospholipids instead of skin phospho- lipids. According to the suggested models fatty acid composition of MPPM neutral lipids should resemble most those found in their correspond— ing skin neutral lipids. However, as already mentioned in the 66 previous discussion little differences were shown among neutral lipid fatty acids from various tissues within each bird species. Thus, Specific conclusions can not be drawn with confidence for this par- ticular lipid component. 67 Part II. Stabilitygof MPPM A. Effects of Inert Gas and Vacuum Packaging on Storage Stability of MechanicalLy Processed Poultry Meats Mechanically processed chicken and turkey meats (MPCM and MPTM) were individually mixed under inert atmospheres, packed along with either N2 or CD2 gases or under vacuum and frozen at -18°C, immedi- ately after packaging or after holding for 72 hrs. at 4°C. Samples were stored up to 4 months at -18°C. For each evaluation period, frozen samples were randomly selected, partially thawed at room tem- perature and prepared for chemical evaluations. This experiment was designed to study storage stability of mechanically processed poultry meat (MPPM), under different types of microenvironmental atmOSpheres. Along with the above factor, the effect of prefreezing hold time on subsequent storage stability of MPPM was observed. The specific purpose of this study was to evaluate the influences of packaging methods and storage conditions on oxidative rancidity development in MPPM, in order to develop practical approaches to aleviate stability problems . Changes in Fatty Acid Composition Mean values for fatty acid unsaturation ratios of both MPCM and MPTM are presented in Table 7. Statistical analyses of these data are shown in Tables 7and 11. Fatty acid distribution profiles for MPCM and MPTM phospholipids before and after 4 months storage at -l8°C are shown in Appendices F and G. Earlier studies by many researchers revealed that the neutral lipid fraction of various kinds of meat including poultry, oxidized very slowly compared to phospholipids (El Gharbawi and Dugan, 1963; 68 Table 7. Mean unsaturation ratios1 and their Tukey separations2 f0r MPCM and MPTM packed under C02 or N2 or vacuum and stored at -18°C fOr 4 months. Storage time (mo.) Meat Type Treatment 0 2 3 4 Unsaturation Ratio MPCM Immediate freezing _ c02 2.15a 1.68a 0.88a 0.73a N2 2.05:b 1.69: 0.84: 0.69: Vacuum 2.18 2.06 1.04 1.18 72 hrs. prefreezing hold time a a a a 002 1.85 1.25 0.91 0.52 Mg 1.78: 1.41:a 0.74: 0.75:a Vacuum 1.80 1.63 0.92 0.93 MPTM Immediate freezing C02 1.53: 1.05: 0.80: 0.54: Mg 1.56a 1.18a 0.81b 0.85b Vacuum 1.60 1.14 0.95 0.93 72 hrs. prefreezing hold time C02 1.55: 0.91:b 0.87: 0.37: N2 1.56a 0.97a 0.88b 0.74b vacuum 1.60 0.82 0.65 0.71 1Mean of 2 replicates, expressed as polyunsaturated, C 18:3 to 22:6/palmitic acid. 2Comparison among packaging treatments of each storage interval and freezing treatment. Like letters among treatments within a column denote no significant difference (P = 0.05). 69 Keskinel et al., 1964; Lee and Dawson, 1973 and Moerck and Ball, 1974). Special attention is thus spent only on the changes in fatty acid composition of phospholipid fractions. It was evident from the fatty acid composition of meat from all treatments after storage that hex- aenoic, pentaenoics, tetraenoics and trienoics were the major sub- strates of oxidative deterioration in both species of meat. Hence, the progress of autoxidation of polyunsaturated 18:3 to 22:6 carbon fatty acids were selected as the indices to follow autoxidative de- terioration of MPPM phospholipids. Using palmitic acid as the stable component, the unsaturation ratios were calculated from total per- centage area of polyunsaturated 18:3 to 22:6 carbon fatty acids over percentage area of palmitic acid. Changes in the unsaturation ratios for both MPCM and MPTM are graphically presented in Figures 1 and 2. Three way analysis of variance for this particular test showed a significant treatment effect for all 3 main factors, both in MPCM and MPTM. Significant interactions were also observed in MPCM be- tween freezing-storage and gases-storage and fOr all types of 2 and 3 way interactions for MPTM. Total lipid content of mechanically processed meat samples for this study were: 17.29% of wet tissue for MPCM and 16.98% of wet tissue of MPTM. It is apparent from the data that unsaturation ratio f0r all samples decreased as storage time increased. For MPCM, there appeared to be an induction period between 0 and 2 months of storage Since the unsaturation ratios of most samples declined slowly during this time. This induction period was then followed by an accelerated rate of autoxidation which continued to about 3 months, and slowed after the third month. These autoxidative trends were 70 .oow um newcpo; .mg: Nu Lmuwm so mucwspmmcu gmuwm aFmpmwumeew gmcuww .oomF- um umgoum ncm Esaum> Lo Ncu Lo Nz Luvs: omxuoa 29;: Ho mcwawpocqmoza :_ muwum xuumm uwpmgaummcsxpoa mHNN op mHmF u 50 cowumcwxo .P mgamwu 62>; m2: moéopm Q a N w av v a N p n. u d d u q u q - O «Cu 0 «CU H I o z I NZ/. 0 ‘ . 1 1 P W > \‘ m n— H” w“ . m... N 4 o a w “u L (l I a o $540... an 04m: "oz—Nummu 3.50925: 71 .u°¢ pm mcwu_o; .mcg Nu Lmumm co mucmEummgu cmumm xpmpmvumeew cmzpwo .Uompn pm umgoum use Ezsum> Lo Nz co moo smug: cmxuma that yo mcwawpogamoga c? mcwum xuumm nmumcapmmczxpoa mumm ow muwp u yo cowumuwxo .N mcamwm ads: 22: wage; v n u w o c a a p a H H . . . H . H .u «Cu . N Ou \’ .u ‘ n m / Hz/ m /. ‘I‘ U l l J F w. \z .- mm N a U m o o « $30: an ad: 02.5%: 35822. L « 72 not observed in MDTM phosphospholipids. No apparent induction periods for the autoxidation which resulted in the decrease in unsaturation ratios were found in MDTM samples. Decreases in unsaturation ratios were rather steady for most of the storage period. A slightly lower . decreasing rate was observed in some treatments, during later storage periods. Samples packed under vacuum had higher numbers of unsaturated molecules (C l8:3 - C 22:6) in their phospholipids when compared with those found in samples packed in N2 or C02, at the end of stor- age period. The only exception was the vacuum packed MDTM samples which were held 72 hrs at 4°C before freezing. For these samples, unsaturation ratios were almost equal to those of the samples packed under N2. These observations are supported by statistical analyses. Tukey multiple comparisons of the means for MDCM samples indicate that at the 5% level, the average mean values for vacuum treated sample was different from the average means of the other two treatments, at the fourth month of storage. These results are true for both freez- ing treatment groups of MDCM. For MDTM the advantage of vacuum stor- age was shown only in the samples which were frozen without delay. In most of the samples, C02 packaging was found to be the least ad- vantageous when compared among three types of packing, at the fourth month of s torage . [Visual observations of the meat packages during frozen storage revealed water losses from the surface of meat samples which were stored under N2 or C02. Gas packed products in general, have some space left between surface of the meat and the film, whereas in vacuum packed products, the surface of the meat adhered thoroughly 73 to the surface of the packages. Losses of water from the surface of meat packed under N2 or C02 could occur as a result of exchanges in microclimatic water vapor between meat and voids between surface of meat and packaging film. Correlation of desiccation or "freezer burn" with oxidative rancidity and discoloration of meat has been observed earlier by Ramsbottom (1947) and Steinberg et al. (1949). Watts (1954) stated that a lowering of the meat pH can occur as a result of C02 packing. Oxidation of reduced myoglobin and hemoglobin pigments to their oxidized form could be accelerated by this acid pH. The ferric heme pigments have been reported to belactive biocatalysts in initiating lipid oxidation in meats (Tarladgis, 1961 and Watts, 1961). This could explain why vacuum packed samples are better than those packed in N2 and C02 with respect to oxidation of unsaturated fatty acid molecules, and why the unsaturation ratio of phospholipids from samples packed under C02 are the lowest among the three, at the end of the storage period. Holding meat samples at 4°C for 72 hrs. prior to freezing re- sulted in a significant increase in the development of lipid oxida- tion. As evident from the data (Table 1) and statistical analyses, unsaturation ratios of both MPCM and MPTM phospholipids from most treatments are lower in samples which had a delaying time between processing and freezing (followed by frozen storage), when compared with another group which was frozen immediately after packaging. As previously pointed out in Part I of this study, autoxidation of highly unsaturated fatty acids found in the phospholipid fraction of MPPM might have already been initiated by the machine deboning process. Subsequent holding of the meat below freezing temperature hence, 74 could result in a favorable condition for further autoxidative deter- ioration of these fatty acids. Vacuum packaging or packaging of the meat under inert atmosphere during these holding periods might par- tially minimize some of the oxidation reactions. However, consider- ing other types of prooxidants which are naturally present in the meat as well as atmospheric 02 which was incorporated into the meat tis- sues during the deboning process, it is obvious that oxidative degra- dation of polyunsaturated fatty acids of meat samples stored under. vacuum or inert gasses was not eliminated completely. 2-Thiobarbituric Acid (TBA) Tests In order to determine if the changes in polyunsaturated fatty acids were attributable to oxidation, TBA tests were used for numer- ically assessing the degree of lipid oxidation in MPPM samples during frozen storage. The mean TBA numbers are reported in Table£3, and statistical analyses are summarized in Tables 8 and 11 . Figure 3 and 4 show graphic presentation of the mean TBA numbers for MDCM and MDTM respectively. Analyses of variance showed significant effects for all of the main treatments. Significant interactions were also found in freezing- storage and packing-storage of MDCM and for all 2 and 3 way interac- tions of MDTM. Small differences in TBA numbers were found among variously treated MPCM samples between 0 and 2 months of storage. Significant increases in TBA numbers of these samples were evident at the end of 4 months of storage. As previously shown in the changes in unsatura- tion ratios, there appeared to be induction periods of about 2 months 75 Table 8. Mean TBA numbers1 and their Tukey separations2 for MPCM and MPTM packed under CD2 or N2 or vacuum and Stored at ~18°C for 4 months. Storage time (mo.) Meat Treatment Type O 2 3 4 TBA numbers MPCM Immediate freezing a a 002 0.86a 1.18a 4.40 4.24 N2 1.0231 0.83: 1.58?) 1.81: Vacuum 0.85 0.92 1.5 1.24 72 hrs. prefreezing hold time C02 0.83: 2.47: 4 5 a 5.09‘2‘J N2 1.23a 1.32b 1.4 b 2.330 Vacuum 1.23 1.20 1.71 2.19 MPTM Immediate freezing 002 4.083 6.9'a 8.553a 9.23:,l N2 3.97: 6.0 7.4 5.85b Vacuum 3.68 5.63 6.56 6.39 72 hrs. prefreezing hold time (:02 5.37:b 8.2133 9.5 aa 7.68: N2 4.60b 6.39 6.4 b 8.54 Vacuum _ 4.26 5.27b 4.44 7.45a 1Mean of 2 replicates within 8 determinations expressed as mg. TBA reactive substance per 1000 9 meat. 2Comparison among packaging treatments of each storage interval and freezing treatment. Like letters among treatments within a column denote no significant difference (P = 0.05). 76 .uae um mcwupo; .mg: mm cmpmm co mucmEummcp Lmumm xpmumwcoeem gmcpwm .Uam_- um umcoum use Eaaum> co Nz Lo moo smug: nmxuma 20¢: mo mg¢n§=c go Nz co moo Lune: cmxumg spa: we mg¢nE=g > o u“ .- N 0U l o L -— .- Nz \ n. a «00 250: an Bu: . 62.5mm“. E5822. .. .v wczmwm 0 aaewnN v91 0% 78 during which the rate of autoxidation was quite slow. High initial TBA numbers were observed in all treatments of MPTM. Unlike MPCM lipids, there was no obvious induction period of autoxidation shown in any treatment of MDTM. Rapid increases in TBA numbers were found in most of the treatments during freezing and subsequent storage. At the end of 4 months storage, significant effects of frozen storage on TBA changes were shown in all treatments of MPTM. Tukey comparisons of mean TBA numbers for MPCM revealed that samples stored under CO2 develop significantly higher TBA numbers than those stored under vacuum or in N2, at the end of the third and fourth month of storage. This significant difference was shown as early as during the second month of storage for MPCM samples which were held at 4°C for 72 hrs. before freezing. However, there was no significant advantage of vacuum packing over N2 packing at the end of storage for this particular meat. TBA nunbers of MPTM stored under C02 markedly exceeded those of the meat samples stored under vacuum or N2. These results were especially obvious for MPTM which was held at refrigerator temperature before freezing. Signif- icant differences between TBA numbers of samples packed under C02 and those stored under vacuum and N2 were observed as early as at the end of the second month of storage. The benefit of vacuum packaging over N2 and C02 packaging was shown in both groups of MPTM samples at the end of the third month. However, these advantages were not significant at the end of the storage period. For samples which were frozen with and without delay, a highly significant difference in TBA numbers was observed between these two groups. In both MPCM and MPTM, mean TBA numbers of all samples 79 frozen immediately after packing were lower than those found in sam- ples which were held 72 hrs. at 4°C before freezing. This result was obviously shown in MPCM at the end of 4 months of storage, and at the third month for MPTM. Correlation of Unsaturation Ratios and TBA Numbers In order to determine if a relationship existed between phos- pholipid fatty acid oxidation and TBA numbers, the mean unsaturation ratios were correlated with the mean TBA numbers across storage time. The results are presented in Table 11. High correlation coefficients were found for most samples packed under C02. The only exception is that of MPTM which was held 72 hrs. at 4°C before freezing, where low correlation (r = 0.61) was observed. All samples packed under N2 showed good correlation coef- ficients between oxidation of polyunsaturated (18:3 - 22:6) fatty acid in their phospholipids and the development of TBA reactive sub- stances (r = 0.82 - 0.92), during frozen storage. The only vacuum packed sample which failed to show a good correlation between these 2 tests was vacuum packed MPTM samples which were held 72 hrs. at 4°C before freezing (r = 0.48). Low correlations were, in general, found between those groups of samples in which either the TBA numbers or unsaturation ratio fluctuated highly during frozen storage. For example, C02 packed MPTM sample which was held 72 hrs. before freezing storage developed TBA numbers as high as 9.58 mg TBA reactive substance per 1000 g of meat at the third month of storage and then dropped to 7.68 mg TBA reactive substance per 1000 g of meat at the end of the fourth 80 Table 9. Correlation coefficients between unsaturation ratios and TBA numbers Correlation Sample Treatment Coefficient MPCM Immediate freezing C02 ”0.97 Mg , -0.92 Vacuum -0.95 72 hrs. prefreezing hold time C02 -0.97 N2 -0.82 Vacuum -0.89 MPTM Immediate freezing C02 -0.98 N2 -0.87 Vacuum -0.99 72 hrs. prefreezing hold time C02 -0.61 N2 -0.9l Vacuum -0.48 81 month. Although there were few treatments which failed to exhibit high correlation coefficients between the two tests, correlation coef- ficients obtained from the majority of the treated MPTM and MPCM samples led to the conclusion that there is a relationship between oxidation of polyunsaturated fatty acids found in phospholipid fraction of MPCM and MPTM and the developmentssof TBA reactive substances in the meat sample during frozen storage. These relationships have been previously reported in lipids from MPCM, by Moerck and Ball (1974). Total Phospholipid Phosphorus The mean total phospholipid phosphorus values for MPCM and MPTM samples are presented in Table 10, and are graphically illus- trated in Figures 5 and 6. Statistical analyses of these data are shown in Tables 10 and 11. As evident from the data and graphic presentation, a consider- able decrease in the total phospholipid contents were apparent in samples from all treatments as frozen storage time of the meat pro- gressed. Analyses of variance also indicated significant effects of storage on changes in phospholipids from both MPCM and MPTM samples. A Slight decline in the phospholipid contents of samples from all treatments was evident during the first 2 months of storage, then a high loss was shown in samples from most treatments between 2 and 3 months to 4 months of storage. At the end of the storage period, MPCM samples packed under N2 or CD2 Showed slightly higher phospholipid losses than those samples packed under vacuum. This trend was observed in both freezing groups 82 Table 10. Mean §otal phoSpholipid phosphorus] and their Tukey separa- tions for MPCM and MPTM packed under CD2 or N2 or vacuum and stored at -18°C for 4 months. Storage Time (mo.) Meat Type Treatment 0 2 3 4 Phospholipid P (mg/g fat) MPCM Immediate freezing 002 1.23 1.07 0.87 0.53 N2 1.13 1.05 0.79 0.55 Vacuum 1.13 0.96 0.73 0.65 72 hrs. prefreezing hold time 602 1.01 1 00 0.77 0.43 N2 0.99 0 96 1.05 0.49 Vacuum 0.97 l 07 0.90 0.96 MPTM Immediate freezing 002 1.08“ 0.90“ 0.6 0.30“ N2 1.10“ 1.05“ 0.8 “ 0.4;“ Vacuum 1.12“ 0.99“ 0.95“ 0.8 72 hrs prefreezing hold time 002 0.94“ 1.0; 0.53a 0.19a N2 0.94“ 0.8 0.70“ 0.35“4 Vacuum 1 .00“ 0.94“ 0.74“ 0.52“ 1 2Comparison among packaging treatments at each storage interval and freezing treatments. Like letters amont treatments within a column denote no significant difference (P = 0.05). Mean of 2 replicates with 4 determinations. 83 Table 11. Analyses of variance of the unsaturation ratios, TBA num- bers and phospholipid phosphorus Mean Square Source of Variation d.f. Unsaturation TBA Phospholipid Ratio Number Phosphorus MPCM Storage 3 0.91: 10.04: 0.48“ Freezing l 0.67a 2.46a 0.002 Packing 2 0.26a 13.59a 0.02a Freezing-storage 3 0.07 0.44 0.056 Freezing-packing 2 0.02a 0.15a 0.06a Storage-packing 6 0.05 2.43 0.06b Freezing-storage-packing 6 0.01 0.11 0.03 MPTM Storage 3 1.84: 98.60: 1.64: Freezing l 0.16a 5.01a 0.40a Packing 2 0.05a 65.40a 0.22a Freezing-storage 3 0.03a 5.87a 0.04a Freezing-packing 2 0.03a 3.60a 0.03a Storage-packing 6 0.04a 5.74a 0.11a Freezing-storage-packing 6 0.01 9.45 0.02 “Significant at 0.01% level. bSignificant at 0.05% level. 84 .oae um mcvao; .mg: mm cwumm so mucmsummcu memm xpmumwcwssw cmsuwm .uomp- um tocopm new E:=um> to «z co «co Love: cmxuma zuaz mo wagonamoga nwnwpogamogq pouch .m mesmwm «as; us: 3:05 n N p .u N N — nu . _ d . q _ 0 .d H" a nu 3 .4... u" o .8 m I . H L m « 9‘ nu 9‘ 4‘ .d u" 0 > H w . m 4 m. .vmmmmmwuunuau 4‘ J p co > I a . 1 III/111111: w o I I. n. m ( $1301 NR 04m... L 02_wa¢n_ 2.50925: 85 . .uaq um meet—o; .mc; Nu Levee co mucweHmmcu empem aFmpmHuwsew cmgpwm gem—i um umeoum can Eazuc> co Nz co «co cone: umxuwa zen: mo macocgmoca uwawpocamoga quop .o meamwu . nos: 22: 34:55 a a p o n u p o . _ . . . q o .8 o .8 m 0 NZ . / m I O 1 NZ 1 m 4‘ .I .1 > M O I H a m l 4 I u .«NHHHHUVMNwm. smllllllll.AVHHHUVIAHHLUWIIIIIIIII mm AV .4 pa: m M meOI NB 04m: 02.wamm wh<_0m<<<<. 11‘ n 1 86 of MPCM. However these differences are not statistically significant. Unlike MPCM, significant differences among three types of packings were Shown in total phospholipid contents of MPTM samples. Losses in phospholipid content were lowest in samples packed under vacuum and highest in those packed along with C02. These results are true for both groups of samples which were frozen with and without delay. The effect of holding the meat at refrigerator temperatures be- fore freezing on subsequent loss of total phospholipids are not sig- nificantly shown in MPCM. However, highly significant effects of this treatment were apparent in MPTM. Significant interactions between freezing-storage and freezing-packing were observed in phospholipid phosphorus from both types of meat. Lipolysis of phospholipids during frozen storage has been ob- served in meat from various animals. McMurray and Magee (1972) ex- plained that the enzyme phospholipase which occurred in mammalian tissue can cause a release of fatty acids from phospholipids. Forma- tion of free fatty acids and water soluble decomposed phospholipids were reported to occur as a result of the hydrolysis of phospholipids, (Awad et al., 1968 and Keller and King, 1973). Other reactions which might also involve and cause breakdown of phospholipids include lipid oxidation, lipid protein copolymerization and lipid browning reactions. All meat samples were allowed to thaw for only one hour at room temperature prior to each analysis. This short thawing period was selected so that the meat would be soft enough f0r convenient prepar— ation for each chemical analysis. With this treatment, a negligible amount of drip loss was found in the meat packages at each thawing. Thus, losses of total phospholipids from both MPCM and MPTM upon 87 frozen storage apparently were not from drip loss. Besides, all of the variously treated samples were frozen and thawed under similar con- ditions. Hence, loss of phospholipids may be attributed as a result of storage rather than freezing and thawing pgr_§g, Davidkova and Khan (1967) and Awad et al. (1968) reported decreases in phospholipid concentration with formation of free fatty acids, during frozen stor- age of chicken muscle and cod muscle. They explained that the drop in phospholipid concentration could be accounted for by the enzymatic hydrolysis of phospholipids. Significant differences in phospholipid losses among various treatments of MPTM indicated that enzymatic hydrolysis may not be the only cause for phospholipid degradation noted in this meat type. Close agreements between TBA numbers and phospholipid content of MPTM samples indicated thata breakdown of phospholipids occurred as a re- sult of oxidative degradation in addition to hydrolytic degradation. There was no significant effect of packing and freezing on total phos- pholipid changes at the end of 4 months storage for MPCM. This re- sult led to the conclusion that the drop in phospholipid concentra- tion in this meat during frozen storage may be mainly accounted for by the enzymatic hydrolysis reaction. The oxidation reaction has only a minor influence on phOSpholipid losses f0und in MPCM. This result seems reasonable since oxidative deterioration of MPTM samples, as indicated by changes of TBA numbers and unsaturation ratio, exceeded by far those of MPCM samples. 88 8. Effects of Air at Various Levels on Storage Stability of Mechanically Processed Poultry Meats In this study lipid oxidation in MPCM and MPTM during frozen storage, was followed over a wide range of air levels. Air pressures of 0, 5, 15 and 30 in. of Hg were assigned to MPPM and MPPM lipid extract samples, and the samples were stored at -18°C up to 3 months. At each test period, the samples were thawed at room temperature for 1 hr. and prepared for chemical evaluations. Changes in quantities of polyunsaturated fatty acids (C 18:3 - C 22:6) and 2-thiobarbituric acid tests were used for numerically assessing the degrees of lipid oxidation. Degradation of phospholipids as a result of frozen stor- age of the meat and their lipid extracts under these air tensions was also quantitated. This experiment was designed to determine maximum limit of air which may exist in the meat packages with- out causing development of oxidative rancidity in the meat samples. Along with air, storage stability of meat lipids in absence of all other meat components was also studied. Changes in FattygAcid Compositions The mean phospholipid unsa uration ratios (C 18:3 - 22:6/ C 16:0) are reported in Table‘12. Their statistical analyses are presented in Table 12 and 15. Fatty acid compositions of MPCM and MPTM phospholipids before and after 3 months storage are presented in Appendices H and 1. Changes in the unsaturation ratios from sam- ples from all treatments of MPCM and MPTM are graphically shown in Fig- ures 7 and 8. Significant differences in unsaturation ratios were found in MPCM as a result of storage, air tension, and extraction of meat 89 Table 12. Mean unsaturation ratios1 and their Tukey separations2 for MPCM, MPTM and their lipid extract samples, packed at 0, 5, 15 and 30 in. of air and stored at ~18°C for 3 months. Storage Time (mo.) Meat Type Treatment 0 2 3 Unsaturation Ratio MPCM Stored as meat 0" air pressure 1.9 1.4 a 0.9 5" air pressure 1.9 0.6 0.8 15" air pressure 1.9 0.8 a 0.3 30" air pressure 1.9 0.5 0.3 Stored as lipid extract 0" air pressure 1.79: 1.06a 0.97: 5" air pressure 1.79a 1.07: 0.8 15" air pressure 1.79a 0.81 0.6 30" air pressure 1.79 0.6 0.5 MPTM Stored as meat 0" air pressure 1.43: 0.70: 0.68: 5" air pressure 1:435 1.00a 0.5 15" air pressure 1.43a 0.60 0.2 30" air pressure 1.43 0 59a 0.31 Stored as lipid extract 0" air pressure 1.4 0.69a 0.80: 5" air pressure 1.4 0.57“ 0.71ba 15" air pressure 1.4 0.4gg 0.5?b 30" air pressure 1.42a 0.4 0.3 O 1Mean of 2 replicates, expressed as polyunsaturated C l8:3 - 22:6 fatty acids/palmitic acids. 2Comparison among packaging treatments of each storage interval and extraction treatment. Like letters among treatments within a column denote no significant difference (P = 0.05). 90 damp- pm 2ch m 3 a: 2:on use .me Ho .cw om ucm mH .m .0 pm umxuma mm_asmm Humcuxm cHaHH 208: can sum: 40 mowpmg cowumgzummca .m mcamwm ads: m2: uoéem a N w o N 9 Ha H H H H . H H O on o a. / On 0/ o n or o o a We / N m o m o m o o V o o w o o a a p N 8 m nu o o h0<¢hxm DE... .392 . 91 damp- an «.555 m 3 a: 3..on use he mo .cH cm can m_ .m .0 pm umxuma mmpasmw uumcuxm nHaHH zen: use Zhez Ho mowumg,=o_umcaumm=: 0‘) Has: 22: 3:05 o h0<¢th OE: .m mtamwd n u H c H H H a mp6 Ono n . N no G W on . m 0 N w o 1H“. O :92 -Lo 92 lipids. There was no significant effect of lipid extraction on changes in unsaturation ratio of MPTM. Significane of 2 and 3 way interactions for all treatment combinations were fOund in both MPCM and MPTM phospholipid unsaturation ratios. It is evident from the data and statistical analyses that a marked decrease in unsaturation ratios was found in samples from all treatments at the end of 3 months of storage. No apparent induction period nor any specific or typical trend of lipid autoxidation were observed in treatments examined. The short storage period (3 months) or the missing data (no test at 1 month interval) might have affected these results. Significant differences in unsaturation ratios were found among means of different air level treated samples. For MPCM, at the end of 3 months storage, the quantities of unsaturated molecules (C 18:3 - 22:6) left in the phospholipids were comparable between samples stored at 0 and at 5 in. of air and between those stored at 15 and 30 in. of air. There was a significant difference in the mean values of un- saturation ratios between these 2 pairs of samples. Similar results were observed in MPCM lipid extracts and in MPTM samples. Similar results, as previously mentioned for these 3 groups of samples was observed in MPTM lipid extract. The only exception was that there were significant differences between unsaturation ratios of samples stored at 15 and 30 in. of air for MPTM lipid extract samples. When MPCM samples were stored as lipid extracts, significantly higher quantities of polyunsaturated fatty acids (C 18:3 - 22:6) were found in their phospholipids when compared to those f0und in their corresponding samples which were stored as meats. Higher unsaturation 93 ratios were also observed in MPTM lipid extract samples. However, these differences were not statistically significant. From the results obtained, it appears that 5 in. of air was comparable to vacuum storage with respect to oxidation of poly- unsaturated fatty acids in MPPM phospholipids. Non-significant dif- ferences in the mean unsaturation ratios found between samples stored at 15 and 30 in. of air also led to the conclusion that air at 15 in. had the same effect as air at 30 in. on oxidation of MPPM phospholipid fatty acids. Lipid extract samples were expected to develop autoxidative deterioration more slowly and to a lesser degree than their correspond- ing meat samples upon frozen storage. Extraction of muscle lipids with chloroform-methanol and purification of the crude extracts with aqueous solution should provide meat fats which were free from most other water soluble components including the meat pigments and non-heme iron which have been reported to be present as trace components in MPPM. Thus, the autoxidation reactions which occur in isolated lipids should be entirely different from those which occur when these lipids are in the meat samples. 0n the other hand, when lipids are in the meat tissues, some of them might bind with proteins and be present in the form of lipoprotein complexes. Protein and water in meat tissues together might partially protect the lipids from being reached by 02 molecules which surround the meat blocks. Lipid extract samples, in contrast, were exposed directly to 02 molecules. This factor might have some compensation effect on losses of prooxidant substances found in lipid extract samples. As a result, a rapid development of oxidation was also f0und in the lipid extract samples. Hence, at the end of 3 94 months storage, there were no significant differences in fatty acid oxidation of MPTM and MPTM lipid extract samples, or only low level Significant differences in mean unsaturation ratios found between MPCM and MPCM lipid extract samples. 2-Thiobarbituric Acid (TBA)Tests TBA absorption values obtained for variously treated MPCM and MPTM samples are presented in Table 13 and graphically Shown in Figures 9 and 10. Analysis of variance and Tukey mean separations for these data are presented in Tables 13 and 15, respectively. Analysis of variance for TBA absorption values Showed significant effects for storage, air tensions and lipid extraction treatments for both MPCM and MPTM. Significance of all 2 and 3 way interactions was also observed in TBA absorption values from these 2 types of meats. TBA absorption values of all samples increased as a result of fro- zen storage. The induction period for the development of TBA reactive substances seems to occur between 0 to 2 months of storage for MPCM sam- ples stored at 0 and 5 in. of air. MPCM samples stored at 15 and 30 in. of air Showed a rapid increase in TBA absorption values between 0 and l to 2 months of storage and then slowed down. Similar trends of this rise and decline were observed in MPCM lipid extract samples. The TBA absorption values, however, behaved differently for MPTM samples. Rapid increases were observed in all treatments of MPTM early in the storage. There was no apparent oxidation induction period in any treatment of MPPM. After a 2 month frozen storage period, TBA absorption values of MPTM samples increased to approximately 1.5 to 1.6 nm and then,with subsequent storage periods, some of these values dr0pped. These increases and declines in TBA absorption values however, were 95 Table 13. Mean TBA absorption values1 and their Tukey separations2 for MPCM, MPTM and their lipid extract samples packed at 0, 5, 15 and 30 in. of air and stored at -18°C for 3 months. Storage Time (mo.) Meat Treatment Type 0 1 2 3 TBA Absorption Value (nm) MPCM Stored as meat 0" air pressure 0.089: 0.072: 0.111a 0.235a 5" air pressure 0.089a 0.070a 0.17 0.304: 15" air pressure 0.089a 0.076b 0.762b“ 0.885 “ 30" air pressure 0.089 0.454 1.106 1.173“ Stored as lipid extract 0" air pressure 0.059“ 0.116“ 0.185: 0.293: 5" air pressure 0.059“ 0.132: 0.421 “ 0.454 “ 15" air pressure 0.059“ 0.270b“ 0.700“ 0.63 30" air pressure 0.059a 0.444 0.727b 0.625 MPTM Stored as meat 0" air pressure 0.426“ 0.600: 1.000“ 1.150“ 5" air pressure 0.426: 0.819 a 1.25 1.31 15" air pressure 0.425 146$c 1.571 1.39 30" air pressure 0.426a 1.33 1.492b 1.38 Stored as lipid extract 0" air pressure 0.365“ 0.330: 0.214“ 0.400“ 5" air pressure 0.355“ 0.394 0.30 a 0.460“ 15" air pressure 0.365“ 0.48 0.60 0.710““ 30" air pressure 0.365“ 0.72 0.780b 0.939b 1Mean of 2 replicates with 6 determinations. 2Comparison among packaging treatments of each storage interval and extraction treatment. 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