I , V. 0! I. I Hon-I I» In; I? I- [Harm 51‘} $.NLlnuvl b.\ :H\I\.\\ul.| 9 - A t | y. I . I 9.! (lllII‘VMV‘U‘Iln v . lo‘Kll ll} iiillul‘fllflrfl. x I 3.1.1:} 0.. MW gimp- !! . \M‘ N -PP3% -- .12. I , a -01 pm"... - ( | o - i ‘ .|a I - .vn .‘ ‘ . . 1»ch. $.Etfil ! 51"?! Hun § Hi \. ‘ I..lm.\1fl§wnfln) ‘ 0 | no. I cl. 51.“ I‘l\|l ‘1“ E 0| .IJ‘ .I u ivlLl.L‘Pl\lo.|..o\.Au %? “\yl‘. n . l! ‘ . h. l . . o | i ‘. . . a. u .IlittclI‘ Eng ‘ufiwd ull' 1 \.l . ‘ ONIIWDMLVavJflbhmu 01.18 (:1er I. .IA‘ .r O In! . ‘I t . I In «I . - .. .1 O l 1.x \ > ‘ IHESES l mama? " macaw: Em“ University . l - A This is to certify that the dissertation entitled COMPOSITION AND STABILITY OF MECHANICALLY DEBONED CARP (CYPRINOUS CARPIO) WITH EMPHASIS ON LIPIDS AND TEXTURE DURING FROZEN STORAGE presented by Yathirajulu M. Naidu has been accepted towards fulfillment of the requirements for Doctoral Food Science & degree in Human Nutrition Major professor [yne October 25, 1983 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES 1—- BEIURNING MATERIAL§5 Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. 1W...— COMPOSITION AND STABILITY OF MECHANICALLY DEBONED CARP (CYPRINOUS CARPIO) WITH EMPHASIS 0N LIPIDS AND TEXTURE DURING FROZEN STORAGE By Yathirajulu M. Naidu 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 1983 u 259- ABSTRACT COMPOSITION AND STABILITY OF MECHANICALLY DEBONED CARP (CYPRINOUS CARPIO) WITH EMPHASIS ON LIPIDS AND TEXTURE DURING FROZEN STORAGE By Yathirajulu M. Naidu Composition and storage stability of mechanically deboned minced carp and hand filleted carp harvested from the Great Lakes were evaluated following freezing and frozen storage. Emphasis was on changes in lipids and their fatty acids. General stability characteristics were evaluated using TBA analyses, changes in various classes of lipids and fatty acids, shear values, water- holding capacity, protein solubility, and sensory evalu- ation. Suitability of one or more antioxidant formula- tions for increasing the shelf life of carp during long-term frozen storage was determined. Different packaging and storage environments to decrease lipid oxidations were evaluated. Seasonal variations in proxi- mate composition and detailed lipid composition of carp harvested from Lake Huron were determined. The yield of edible meat from the mechanical deboning process (40%) was greater than that from hand deboning (28.5%). Mechanically deboned flesh contained a lower percent protein and a higher percent lipid than did hand deboned flesh. This difference was mainly due to the Yathirajulu M. Naidu neutral lipid fraction. Phospholipids accounted for about l4% of the total lipids and were separated into six fractions of which phosphatidylethanolamine and phosphatidylcholine formed nearly 60% of the total phos- pholipids. Twenty-five different fatty acids from C14 to C22 with D to 6 double bonds were identified, of which C14’ C15’ C16:1' 518:1’ Cl8:2’ Cl8z3’ C20:1- 020:4. C20.5¢5 & w3, and C22:6 were the predominant fatty acids. BHA + BHT + ascorbic acid (with or without propyl- gallatel and Tenox 2 were identified as excellent antioxidant formulae for frozen storage of mechanically deboned carp, while Freezgard was found suitable for hand filleted carp. Little advantage of using vacuum and/or nitrogen to increase storage stability was apparent. Based on TBA numbers, retention of unsaturated fatty acids, taste panel evaluation for rancidity, water- holding capacity, shear values, and extractability of soluble proteins, it was concluded that the acceptable quality of mechanically deboned carp can be maintained for about 8 months during frozen storage while fillets with skin can be preserved for 8 months in comparison to 6 months for skinless fillets. The lowest lipid content of 6.45% was found in carp harvested in April and the highest in carp harvested during July (16.9%). An inverse relationship between Yathirajulu M. Naidu percent total protein and total lipid was observed due to seasonal differences. To my Dear Parents, Brothers and Sisters ii ACKNOWLEDGEMENTS The author wishes to express sincere appreciation and gratitude to his major professor, Dr. Lawrence E. Dawson, for providing guidance, assistantship support and marvelous friendship during the investigation and preparation of this dissertation. Thanks are extended to Mrs. Dawson. Appreciation is extended to Drs. A.M. Pearson, J.I. Gray, JiF. Price, C.M. Stine and A.M. Booren of the Department of Food Science and Human Nutrition and Drs. L.L. Bieber and H.A. Lillevic of the Department of Biochemistry for serving on the doctoral committee. Thanks are due to Dr. M.R. Bennink for providing the use of GLC facilities. To Drs. C.A. Arntzen, J. Duesing (Plant Research Laboratory) and K.K. Baker (Center for Electron Optics) for providing partial research assistant- ship support during graduate study at Michigan State University. Sincere appreciation and thanks are given to Rose Gartner, Norbottons, my parents, brothers and sisters. iii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . viii LIST OF FIGURES . . . . . . . . . . . . . . . . . . X INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE. Mechanical Deboning Composition of Fish Seasonal Variation. Fat in Fish Flesh . Protein of Fish Muscle and Texture. Lipid Oxidation and Stability . . Factors Affecting Lipid Oxidation Lipid Composition Light . . . . Ionizing Radiation. Peroxide. Enzymes . . Organic Iron and Trace Metal Catalysts. Mechanism of Lipid Oxidation. . . Role of Singlet Oxygen in Lipid Oxidation . . Evaluation of Lipid Autoxidation and Stability 2 N—l—J—l—lddd—ld—J—J—l mwuxthtcnososmmw—onoos U1 Percent Lipid Composition . . . 28 Oxygen Absorption . . . . . . . . . . . . . 28 Peroxide Formation. . . . . . . . . . 29 Decomposition of Peroxides. . 30 Predictive Methods for Evaluation of Lipid Oxi- dation and Stability. . . . . . . . . . . . . 31 Evaluation of Methods . . . . . 32 Sensory Evaluation of Lipid Oxidation . . . 32 Effects of Lipid Oxidation on Nutritive Values. 33 Antioxidation and Antioxidants. . . . . . . . . 35 Mechanisms of Antioxidation . . . . . . . . 35 Metal Sequestering Agents . . . . . . . . . 37 Synergism and Antioxidants. . . . . . . . . 33 Antioxidants. . . . . 39 Chemical Classification of Antioxidants . . 49 Most Commonly Used Food Antioxidants. . . . 4‘ Silicones . . . . . . . . . . . . . . . . . 45 iv Page Selection and Use of Antioxidants. . . . . . fig Extraction of Tissue Lipids. . . 49 Separation and Fractionation of Phospholipids. Gas Liquid Chromatography (GLC). . . . . . . . 5‘ METHODS AND MATERIALS. . . . . . . . . . . . . . . . 53 Processing and Treatment of Fish Samples . . . . 53 Fish Samples . . . . . . . . . . . . 53 Mechanically Deboned Carp. . . . . . . . . . 53 Hand Filleted Carp (with skin) . . . . . . . 54 Hand Filleted Carp (without skin). . . . . . 55 Antioxidants and Chelators Used. . . . . . . 55 Incorporation of Antioxidants into Mechanically Deboned Carp Flesh. . . . . . 55 Packaging of Mechanically Deboned Carp Flesh. . . 58 .Freezing and Storage of Mechanically Deboned Carp Flesh . . . . . 58 Incorporation of Antioxidants into Hand Filleted Carp (with and without skin). . . 58 Sampling of Carp for Seasonal Variation Analyses . . . . 50 Preparation of Samples for Further Analyses. . . 60 Materials for Chromatographic Analyses . . . . . 51 Chlorinated Solvents . . . . . . . 53 Petroleum Ether and Diethyl Ether. . . . . . 53 Analytical Method. . . . . . . . . . . 53 Moisture . . . . . . . . . . . . . . . . . . 53 Total Fat. . . . . . . . . . . . . . . . . . 54 Protein. . . . . .‘. . . . . . . . . . . . . 64 Ash. . . . . . . . . . . . 55 Extraction of Total Lipids . . . . . . 55 Separation of Neutral and Phospholipids. . . . . 67 Preparation of Silicic Acid Columns. . . . . 67 Application of Sample. . . . . . . . 57 Classification of Phospholipids. . . . . . . 59 Phosphorous Determination. . . . . . . . . . 70 Free Fatty Acid Separation . . . . . . . . . . . 71 2- Thiobarbituric Acid (TBA) Analysis . . . . . . 71 Gas Liquid Chromatography of Fatty Acids . . . . 73 Cholesterol Analysis . . . . . . . 74 Shear Values . . . . . . . . . . . . 75 Water Holding Capacity (WHC) . . . . . . . . . . 75 Protein Solubility . . . . . . . . . . . . . 75 Sensory Evaluation . . . . . . . . . . . . . . . 78 Statistical Analyses . . . . . . . . . . . . . . 78 RESULTS AND DISCUSSION. Characteristics of Mechanically Deboned Carp. Yie d . . . . . . . Proximate Composition Lipid Classes . . Phospholipid Composition. Fatty Acid Composition. Protein Fractions . . Shear Value and Water Holding Capacity. . Relative Stability of Mechanically Deboned Carp During Frozen Storage . . . Preliminary Studies on TBA Test . . . Effect of Antioxidants and Chelators on TBA Numbers of Mechanically Deboned Carp During Frozen Storage. . . . . . . . Effect of Storage Time and Treatments on Various Lipid Classes . . . . . . . . . Effect of Storage Time and Treatments on Various Phospholipid Fractions. . Effect of Storage Time and Treatments on Fatty Acid Fractions from Phospholipids Effect of Storage Time and Treatments on Extractability of Sarcoplasmic and Myofibrillar Proteins . Effect of Storage Time and Treatments on. the Shear Value of Fish Gels. . Effect of Storage Time and Antioxidant Treatments on Waterholding Capacity . Effect of Packaging Environment on Storage Stability of Mechanically Deboned Carp. . . Effect of Packaging Environment on Fatty Acid Fractions of Phospholipids. . . Frozen Storage Stability of Hand Filleted Carp. Evaluation of Fillets . Effect of Storage Time and Antioxidants on Ratio of Unsaturated to Saturated Fatty Acids from Phospholipids. . . . Effect of Storage Time and Treatment of Extractability of Sarcoplasmic and Myo- fibrillar Proteins. . . . Effect of Storage Time and Antioxidants on Shear Values of Fish Gels Prepared from Hand Filleted Carp. . . . Effect of Storage Time and Antioxidants on Water Holding Capacity of Fish Gels Prepared from Hand Filleted Carp. vi 97 106 113 115 119 121 123 126 128 131 133 139 142 144 146 Page Seasonal Variations. . . . . . . . . . . . . . . 149 Seasonal Proximate Composition of Carp Flesh. . . , , 149 Lipid CompositiOn of Carp FleSh: . . I : Z : ‘52 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . 159 APPENDIX......................‘54 REFERENCES . . . . . . . . . . . . . . . . . . . . . 153 LIST OF TABLES Table Page 1 Antioxidant treatments used for mechanically deboned carp flesh. . . . . . . . . . . . . . . 57 2 Antioxidant treatment used for hand filleted carp with and without skin. . . . . . . . . . . 59 3 Effect of method Of deboning on yield of minced carp. . . . . . . . 30 4 Effect of method of deboning on proximate composition Of minced carp. . . . . . . . . . . 31 5 Effect of method of deboning among the lipid classes in minced carp. . . . . . . . . . 34 6 Effect of method Of deboning on composition Of phospholipids in minced carp. . . . . . . . . . 85 7 Fatty acid composition Of phospholipid and neutral lipid fractions from minced carp. . . . 37 8 Effect of method of deboning on nitrogen levels of total and various extractable protein fractions from minced carp. . . . . . . . . . . 90 9 Mean TBA numbers for mechanically deboned minced carp stored at -18°C for ll months . . . 98 10 Effect Of time and antioxidant treatment on the phospholipid fatty acid fractions and ratio of saturate to unsaturated fatty acids in mechanically deboned carp during frozen storage . . . . . . . . . . . . . . . . . . . . 117 ll Effect Of time and antioxidant treatment on extractability of sarcoplasmic and myofibrillar protein nitrogen from mechanically deboned minced carp during frozen storage . . . . . . . 120 12 Effect of time and antioxidant treatment on shear values of fish gels prepared from mechan- ically deboned minced carp during frozen storage . . . . . . . . . . . . . . . . . . . . 122 viii Table Page 13 Effect Of time and antioxidant treatment on water holding capacity (percent liquid extractable) of fish gels prepared from mechanically deboned minced carp during frozen storage. . . . . . . . . . . . . . . 124 14 Effect of storage environment on TBA numbers from mechanically deboned minced carp during frozen storage. . . . . . . . . . . . . . . . 127 15 Effect of storage environment on various groups Of phospholipid fatty acids and ratio of unsaturated fatty acids to saturated fatty acids in mechanically deboned minced carp stored at -20°C for eleven months . . . . . . 129 16 Mean TBA numbers for mechanically deboned minced carp treated with various antioxidants and stored in presence Of various packaging environments at -18° C for 11 months . . . . 132 17 Effect Of time and antioxidant treatment on the ratio Of unsaturated to saturated phospholipid fatty acids in hand filleted carp during frozen storage. . . . . . 140 , 18 Effect of frozen storage and antioxidant 1 treatment on shear values of fish gels l prepared from hand filleted carp. . . . . . . 145 19 Effect of frozen storage and antioxidant treatment on waterholding capacity of fish gels prepared from hand filleted Carp with and without skin. . . . . . . . . . . . 147 20 Length and weight of carp samples during various months. . . . . . . . . . . . . . . 150 21 Seasonal variation in composition of carp flesh lipids. . . . . . . . . . . . . . . . . 154 22 Seasonal variation in phospholipid composi- tion of flesh lipids from carp harvested from Lake Huron. . . . . . . . . . . . . . . . . . 155 23 Seasonal variation in fatty acid composition of flesh lipids from carp harvested from Lake Huron . . . . . . . . . . . . . . . . . . . . 156 ix Figure Page 1 Mechanism of Auto Catalytic Autoxidation. . . . 20 2 Mechanism for Metal Catalysis Reaction in Lipid Oxidation . . . . . . . . . . . . . . . . . . . 22 3 Mechanism of Singlet Oxygen Formation . . . . . 24 4 Some Final Products Of Peroxide Degradation . . 27 5 Proposed Mechanism of Antioxidation . . . . . . 35 6 Stable Resonance Hybrids Formed by Phenolic Antioxidants..................42 7 Structure of Most Common Food Antioxidants. . . 43 8 A New Antioxidant in Use in Europe. . . . . . . 44 9 Shear Force Values and Panel Sensory Scores for Firmness Of Fish Gels Prepared from Hand and Mechanically Deboned Minced Carp. . . . . . . . 92 10 Waterholding Capacity and Panel Sensory Score for Juiciness Of Fish Gels from Hand and Mechanically Deboned Minced Carp. . . . . . . . 93 ll Absorption Spectrum from Malonaldehyde and 2- Thiobarbituric Acid (TBA) Complex from Minced Carp Tissue . . . . 95 12 Mean Rancid Odor and Flavor Scores for Fish Meat Loaf Prepared from Mechanically Deboned Minced Frozen Carp Stored up to 11 Months . . . 102 13 Effect of Treatment and Time on Total Phospho- lipids and Free Fatty Acids in Mechanically Deboned Minced Carp During Frozen Storage . . . 108 14 Effect of Treatment and Time on Total Trigly- LIST OF FIGURES cerides. and Mono and Diglycerides in Mechani- cally Deboned Minced Carp During Frozen Storage . . . . . . . . . . . . . . . . . . . . 109 Figure 15 16 17 18 19 20 21 Effect Of Treatment and Time on Total Choles- terol and "Other" Unidentifiable Fractions in Mechanically Deboned Minced Carp During Frozen Storage. . . . . . . . . . . . . . . . Effect of Antioxidant Treatments and Time on Various Phospholipid Fractions in Mechanically Deboned Minced Carp During 11 Months Of Frozen Storage. . . . . . . . . . . . . . . Effect of Antioxidants and Frozen Storage on TBA Numbers of Hand Filleted Carp with Skin. Effect Of Antioxidants and Frozen Storage on TBA Numbers of Hand Filleted Carp without Skin . . . . . . . . . Taste Panel Scores for Rancid Odor and Flavor of Antioxidant-treated Frozen Carp Fillets With and Without Skin. Effect Of Frozen Storage and Antioxidants on Solubility of Specific Protein Fractions from Hand Filleted Carp, With and Without Skin. Seasonal Variation in Proximate Composition Of Carp . xi Page 110 114 134 135 137 143 151 INTRODUCTION The commercial fishing industry in the Great Lakes has been declining because Of the decreased availability of fish species that are in high demand. One option to revitalize this commercial industry is to promote the use of underutilized fish species such as carp and suckers. The fresh water mullet (sucker) has proven to be an eConomical source of acceptable fish products (Baker gt_al., 1977; UGLRC Report, 1978; Lindsay, 1975; and Morris and Dawson, 1979). Carp was not as acceptable as mullet due to various problems such as its poor image, muddy flavor, and pesticide accumulation. Recent studies support the potential for new products from mechanically Processed carp (Price gt 11., 1981; Rippen, 1982). Acceptance of underutilized fish species can be 1'01’1uenced by a better understanding of the problems associated with various flesh characteristics, composition, and their changes during processing and storage for each sPecies. The use of mechanically deboned minced carp fliésh in restructured products might improve utilization FNTtential for carp. The intramuscular bone structure of CaYT)IS unsuitable for hand or mechanical filleting (IJawson 23.31.: 1978; Noble, 1974). This can be overcome by the use of mechanical deboning equipment. Successful utilization of mechanically deboned carp in varioUs restructured products might require different lengths of frozen storage period for the mince. During frozen storage, various chemical changes may occur, depending on flesh composition and type of fish species. This may affect flavor, texture, and other sensory qualities of such products. Babbitt (1972), King (1962), Miyauchi (1972), Lee and Toledo (1977), and Morris and Dawson (1979) observed that a rapid deterioration of mechanically deboned fish flesh occurs during frozen storage. This deterioration may be greater in mechanically deboned products which have been subjected to tissue destruction, exposure of lipids to heme and nonheme iron, incorporation of oxygen or to increased flesh contact with enzymes. For example, minced flesh with kidney tissue may result in poor texture because of the activity of alkaline protease, which can bring about slow proteolysis even in frozen storage. Volatile compounds associated with skin are incorporated into tissue mince during mechanical deboning, and can accelerate lipid oxidation and can add components which may increase the development of "off" Odors (Cole and Keay, 1976; Teeny and Miyauchi, 1972; Bremner, 1977; Patashnik _ti_l., 1973). Mechanical deboning results in mixing the high fat content red muscle, more susceptible to lipid degradation with the white muscle, thus imparting the deteriorative changes to the whole flesh (Mai and Kinsella, 1979). Texture changes also occur during frozen storage. They may be related to changes in pH, free fatty acid release and various reactions between proteins and lipids and resulting degradative products. These teXtural changes lead to altered functional characteristics of the mechanically deboned fish flesh during frozen storage. They are major concerns in storage of frozen fish mince (Iredale and York, 1977). Increased information regarding the advantages and disadvantages for storing both carp fillets and mechanically deboned minced Carp should be of value in developing or improving market opportunities. Different antioxidant systems or application levels vary in effectiveness for different species of fish during frozen storage. Teeny and Miyauchi (1972) found that BHA, BHT, and their combinations were effective in minimizing lipid oxidation in stored trout. Propyl gallate has been shown to be very effective for bullfish (Greig, 1967). Acetylated monoglyceride was used as a successful antioxidant for fresh fish (Stemmber, 1975) and Deng gt 11° (1977) showed that TBHQ was an effective antioxidant for mullet- and Morris and Dawson (1979) reported that Freezgard was effective for suckers. Hence, determination of an effective antioxidant system is an initial step for successful frozen storage Of carp. Specific objectives of this study were: 1. to study the yield and compositional charac- teristics of mechanically deboned carp with emphasis on lipids. to compare the effect of various antioxidants and combination of antioxidants for extending shelf life Of carp flesh during frozen storage. to evaluate the changes which take place in lipid composition of carp and to evaluate textural changes during frozen storage. to determine the differences between the quality and stability Of mechanically deboned flesh and fillets from carp during frozen storage. to determine the seasonal variation in flesh composition of carp harvested from Lake Huron. REVIEW OF LITERATURE During the past decade, there has been an increase in demand for fishery products throughout the world. This trend has accompanied a general decline in the availa- bility of traditionally established high value species of both marine and fresh water fish. In the United States annual per capita consumption of fish increased from approximately 11 pounds in 1960 to 13.7 pounds by 1970 (Miyauchi and Steinberg, 1971). Baker (1978) estimated that as much as 80 percent of the possible utilizable Species of fish from the Great Lakes fish were under- utilized. Many Of these species are unintentionally caught and are discarded either because of unestablished markets or because of unavailable processing and handling pro- cedures. Some species Of fish may be underutilized because of the presence Of intramuscular bones that pose problems for hand or mechanical filleting (Dawson gt 11., 1978; Noble, 1974). Odd shapes and sizes of fish have been identified as problems in mechanical handling of some fish in commercial processing (Bremner, 1977). Keay (1979) and Teeny and Miyauchi (1972) pointed out that small-sized species would be uneconomical to process by conventional methods. In addition to the above-mentioned problems leading to the underutilization of potentially available fish species, appearance, names, flesh color and flavor are other factors resulting in underharvesting or discarding Of these species harvested accidentally (Baker, 1978; King, 1978; Teeny and Miyauchi, 1972; Patashnik £3 31., 1974). Mechanical Deboning Since the introduction of mechanical deboners in 1940 in Japan, industrial production of restructured fish products has increased significantly (Lanier and Thomas, 1978). This process involves passing of headed, gutted, split, and washed fish between a pressure belt and a revolving perforated drum resulting in separation Of flesh that is squeezed through small perforations from bone, skin and scales which do not pass through perforations. Lanier and Thomas (1978) described in detail the design and operation of a fish deboner, and their use can pave the way for commercial utilization of such fish with intramuscular bones that are not suitable for hand or mechanical filleting. Within reasonable limits, most fish, irrespective of their sizes and shapes, can be effectively deboned. The effectiveness of deboning is very noticeable in case of predominantly boney species Of fish. Bone contents of 0.13 to 0.16 percent in mechanically deboned fish flesh were reported by Zapata (1978) and Apolinario (1975). ‘ ‘ The greatest advantage of mechanical deboning of underutilized fish species is that it allows the incorpo- ration Of the mince into various restructured products in various proportions. This leads to increases in the possibility for the development of new products with specified texture and flavor, and reduces the chances for undesirable products based on factors like species identity, flavor, texture, appearance, and color (Bligh and Regier, 1976; Hewson and Kemmish, 1976; Jaurequi, 1978; Moledina _t_;1., 1977). One major advantage attributed to the use of mechanical deboner is the increased yield of the edible meat. Flesh yield depends on the type of fish being deboned. Yields from 32 to 70 percent have been reported for various fish species. Reseck and Waters (1979), Kudo 33 31. (1973), and Noble (1974) reported a 70 percent yield for a small boney fish, whereas a yield of 25 to 30 percent was found for hand deboned products (Miyauchi and Steinberg, 1971). The left over fish frames from mechanical filleting can be mechanically deboned to recover edible meat using mechanical deboning (King and Carver, 1971; Moledina _t _l., 1977; Noble, 1973). Miyauchi t l. (1975) and Bremner (1977) have shown that variations in the mechanical deboner, such as belt pressure and size Of perforations of the drum, can influence the yield of edible flesh, bone, skin, and scale Content. The use of mechanically separated fish flesh in restructured fish products requires more attention to quality changes which occur during processing and storage. Lee and Toledo (1977), Babbitt (1972), King (1972) and Miyauchi (1972) indicated that fish flesh deteriorates rapidly after mechanical deboning, with major changes occurring in flavor and color. Some of the theories proposed for these accelerated adverse changes in mechani- cally deboned fish muscle include incorporation Of soluble constituents from skin and bone marrow and mixing of oxidation susceptible lipid fractions from flank muscles (Watts, 1954; Blackwood, 1974; Steinberg, 1975). Direct contact between fish flesh and iron parts of a mechanical deboner was reported to induce accelerated lipid oxidation (Castell 3331., 1966; Lee and Toledo, 1977). The aging of some fish from 1 to 4 days in ice can lead to an increase or decrease in acceptable fish flavor. Raja and Moorjani (1971) attributed such flavor improvement to the formation of inosine 5 monophosphate (IMP) by enzymatic deamination and phosphorylation of ADP. Mechanical deboning can directly influence flavor change in fish products. Such changes were associated with the inclusion Of soluble and volatile compounds extruded from skin during mechanical separation (Crawford _tggl., 1972) and inclusion of organel tissue and peritoneal membrane which have been reported as a basis for Off flavor in mechanically deboned meat (Dingle and Hines, 1975). Blood may impart a metallic flavor to minced fish product (Keay, 1979). A rupture of tissues and release Of cell contents can accelerate enzyme induced flavor changes. (Howgate, 1976). A blend of spices and smoke flavor ingredients can improve the flavor Of mechanically deboned carp (Patashnik _t _l., 1974), while a mix Of other acceptable fish mince or minced shrimp has been recommended for improving flavor (Babbitt gt_al., 1974). Mahohar gt 11. (1973) reported that polyphosphates may not effectively and consistently improve flavor. Composition of Fish The proximate composition of fish muscle varies widely due to species (Stansby and Olcott, 1963), nutrient availability (Cutting, 1969), season (Finne gt 11., 1980), or location (Deng gt 31., 1976). Composition may vary from 30 to 90 percent moisture, 6 to 28 percent protein, 0.1 to 65 percent fat, and 0.3 to 1.7 percent ash. Fish have been classified based on protein and fat content into five categories (Stansby and Olcott, 1963). These categories are (1) low fat, high protein fish; (2) medium fat, high protein fish; (3) high fat, low protein 10 fish; (4) low fat, low protein fish; and (5) low fat, very high protein fish. Respective examples are cod, Salmon, trout, clam, and tuna. Seasonal Variation Generally, migratory fish have highly significant differences in proximate composition among seasons. This also applies to most other fish with high fat content.‘ Changes during the year in availability of food, tempera- ture of water, diet or physical, chemical, and physiological changes brought about by reproductive cycle, play a sig- nificant role in proximate composition Of the species (Sidwell, 1976; Stansby and Lemon, 1941). In most species, principle constituents affected are moisture and lipid content followed by protein (Leu gt 31., 1981). With these variations, components of each constituent can undergo profound changes. For example, individual fatty acids or fractions of phospholipids of the total lipid, can vary greatly. Contents of moisture and total lipids shows an inverse linear relationship to each other (Venkataraman _£._l-’ 1968; Leu gt gt., 1981). Generally, it has been observed that monoenes are least affected by seasonal variation while the polyenes are affected the most. Venkataraman gt g1. (1968), Deng gt_gl. (1976) and Leu gt_gl. (1981) observed that the high lipid content coincides 11 with the prespawning period while low lipid content is found after spawning. Fat in Fish Flesh Stansby (1973) reported that "Next only to moisture, the fat content of fish varies more than any other chemical components. Although the range in fat content can vary, the content of individual species may range from 0.5 to 25 percent. A majority of fat in fish occurs in the form Of phospholipids and triglycerides. More of the fatty acids in phospholipids are generally polyunsaturated than those in triglycerides. In some fish species, most of the fat appears as phospholipids comprising basic cellular components. However, in fish with high fat content, the fat exists as fat depots at various locations throughout the body". In some fish, significant amounts of fatty acids may occur in unusual forms. For example, 20 percent of fatty acids occur as alkoxydiglycerides in sharks and there are known cases where they occur as wax esters (Stansby, 1973). Fat content may vary from very low percentages in edible flesh to high percentages in liver and ovaries. Most fish have, in addition to intracellular phospho- lipids, a store of triglycerides which are found in layers beneath the lateral line in skin along the dorsal and ventral areas (Liciardello gt_gl., 1980). 12 During any one season of the year, individuals of a species, even from the same catch, may vary considerably from other fish in fat content. Maximum triglyceride content has been reported during summer months and minimum content has been observed just before the end of winter (Deng gt gl., 1976). The fat content of muscle from different parts of a fish differs considerably; for example, a steak taken from the tail end Of the fish may contain 10 to 60 percent less fat compared to a similar steak taken from the area adjacent to the head, depending on species of fish. Belly flaps usually contain higher fat contents than remainder of fish (Leu gt_gt., 1981). The fat content of red or dark muscle of fish is higher than that of white or light muscle and the fat from red muscles is known to be more susceptible to oxidative rancidity (Lee and Toledo, 1977).. In general, more than 20 fatty acids are present in a species Of fish, varying in chain length from C14 to C24 and occasionally include C12 and C26' Twenty to thirty percent Of the fatty acids are saturated and a majority are C16 and C18 fatty acids. From thirty tO sixty percent of the fatty acids are monoenes. Polyenes of C16’ C18’ 020, C22 form the major portion of polyunsaturated fatty acids. 022:5, C22:6, C20:5 and C20:4 form major portions of polyunsaturated fatty acids in most fresh water fish 13 species (Stansby, 1973). Kinsella gt_gl, (1977) determined the fatty acid content of 18 species of fresh water fish. They reported a marked variation in fatty acid composition among various species. They a1So reported that at any given time, large fish contained a higher fat content than small fish within the sampled species and catch. Hayashi and Takagi (1977) reported that stress can influence the fatty acid content of fish. They reported that gill netted fish had a lower phospholipid to neutral lipid ratio than did trawl netted fish, with significant decreases in C20:5 w 3 and C22:6 w 3 acids. This was attributed to special physiological conditions caused by excessive stress. Protein Of Fish Muscle and Texture The protein content of fish may vary from 6 to 28 percent, depending on the species, season, and the stage of reproductive cycle (Stansby and Olcott, 1963). A major percentage of protein nitrogen is myofibrillar protein (58 to 73 percent), followed by the soluble sarcoplasmic protein (20 to 30 percent) and stromal protein (3 to 10 percent) (Moorjani gt_gl., 1962). Myofibrillar proteins contain 40 to 60 percent myosin and 20 to 40 percent tropomyosin (Dyer and Dingle, 1961). 14 Preservation of fish by freezing causes protein denaturation leading to a marked effect on the textural quality of the meat. These changes occur rather slowly in frozen fish but significantly affect the acceptability Of texture (Hao Chu and Sterling, 1970). Depending on the condition of the flesh and rate of freezing, ice crystals Of various sizes can form inside and outside the cells resulting in mechanical damage to muscle cells (Love, 1968; Kent, 1975). Increased salt concentrations due to a decrease in available water could result in protein dena- turation (Castell gt_gl., 1970; Sikorski gt_gl,, 1976). ++ and Cu++ ions have been shown to bring about protein Ca insolubilization by favoring protein lipid interactions (Pigott and Shenouda, 1975; Buttkus, 1971). Lipid hydrolysis can cause protein denaturation. Lipid oxidation, resulting in the formation of free radicals may also lead to protein deterioration. Dyer and Dingle (1961) reported a decrease in fish myofibrillar protein extraction with increased free fatty acid formation. The free fatty acids formed may bind polypeptide side chains and polar groups in intermolecular hydrophillic and hydro- phobic ionic linkages and decrease the protein solubility. LPolymerization of protein may lead to decreased solubility during lipid oxidation (Funes and Karel, 1981; Tappel, 1961). Availability of water may control the extent of polymerization (Schaich and Karel, 1975), thus 15 the method of freezing is important. Denatured proteins exhibit poor waterholding capacity which leads to lower product yields and brings about deteriorative effects on texture, flavor, color, and nutritional characteristics of fish products (Eskin t al., 1977). Lipid Oxidation and Stability The process of spontaneous oxidation on exposure to the air is by no means limited to lipids from foods. It is exhibited by various chemicals, such as hydrocarbons, aldehyde ethers, sulfhydryl compounds, phenols, amines, and sulfites (Labuza, 1971). Among the constituents of foods susceptible to autoxidation are the unsaturated fatty acids in their various forms or combinations and minor constitu- ents which influence aroma, flavor, color and vitamins (Lea, 1962). Factors Affecting Lipid Oxidation One characteristic feature of lipid autoxidation is that it can be influenced by factors other than the usual variations in concentration of reactants and temperature. Some of the most important factors affecting lipid oxida- tion are listed below. 16 Lipid Composition Lipid composition involves the nature and proportions of the unsaturated fatty acids present (Lea, 1962). Since double bonds are the active sites of oxidation, fatty acids vary in their reactivity depending on their degree of unsaturation (Privett, 1959). More specifically, autoxi- dation of a particular lipid will depend on its content of unsaturated fatty acids and their degree of unsaturation. Since the fatty acid composition of fish varies with season, diet, and the stage of reproductive cycle the fish is undergoing, these factors can indirectly play a marked role in autoxidation. um Ultraviolet and blue lights impart a significant influence on lipid autoxidation (Lea, 1962; Sherwin, 1968). Light falling in these wave length can accelerate the rate of lipid autoxidation. One Of the primary effects seems to be the acceleration of hydroperoxide decomposition in fats (Lundberg, 1962). IonizinggRadiation Alpha and beta particles and X-rays have definite effects on the autoxidation rate of lipids (Chipault, 1962). They can catalyze peroxide decomposition and formation of free radicals (Lundberg, 1962). 17 Peroxide The peroxides present in the food system and those formed as a result Of initial autoxidation of lipids play an important role in the further initiation and fate of autoxidation reactions (Lea, 1962; Sherwin, 1968). Enzymes Lipoxygenases (lipoxidases) influence the rate and course of fat oxidation (Lea, 1962; Tappel, 1962). Enzymes are also known to be involved in initiation Of fat autoxi- dation (Clegg gt gl., 1953). Organic Iron and Trace Metal Catalysts This classification includes hematin compounds such as hemoglobin and heavy metals such as Co, Cu, Fe, Mn, and Ni. Detailed reviewsinvolving such catalysts were reported by Ingold (1962), Lundberg (1962), and Tappel (1962). In addition to the faCtors mentioned above, considera- tion of the method of catch, physical state prior to pro- cessing, handling conditions and storing temperatures might influence physical/chemical changes in fish muscle. Mechanism of Lipid Oxidation Proposed mechanisms and theories of lipid oxidation have been reviewed by Privett (1959), Tarladgis (1961), Lundberg (1962), Keeney (1962), Ingold (1962), Labuza (1971), and Sherwin (1972). 18 According to Lundberg (1962) the oxidation of food lipids involves autoxidative reactions which are accom- panied by various secondary reactions having oxidative or nonoxidative character. Autoxidation is defined by the same author as the reaction of any material with molecular oxygen. The principal mechanisms involved in the primary autoxidation of fatty materials have been elucidated fairly well. Lundberg (1962) stated that, "A large number of secondary reactions, many of which are virtually unstudied are also involved in the oxidation deterioration of lipid materials". He also reviewed and discussed the general features and kinetics of oxidation of fats. "First, it is an autocatalytic reaction, i.e. a reaction whose rate increases with time due to formation of products which themselves catalyze the reaction. Second, the reaction primarily involves unsaturated acyl groups, and hydro- peroxide groups appearing in alpha position relative to a double bond. Depending on the amount of original unsatu- ration in the acyl group and other factors, the formation Of hydroperoxides may or may not involve the shift of a double bond. Third, among the common fatty acids and their derivatives, the rates Of autoxidation are greatly dependent on the degree of unsaturation of the fatty acids. Fourth, various extraneous influences may be present that affect the rate of oxidation. Some of these factors have been 19 presented earlier. Fifth, as with other chemical reactions, temperature has a marked effect on the rate of autoxidation. Sixth, although the major products of fat oxidation are hydroperoxides, certain secondary products not derived from peroxides are formed concurrently. Finally, hydroperoxides themselves do not contribute directly to any appreciable extent to the undesirable flavors and odors of autoxidized foods. Rather, the rancid flavor and Odors are due to a host of secondary substances derived through various reactions and further oxidation of peroxides and their degradation products". Following his kinetic studies on lipid oxidation, Lundberg (1962) presented a free radical chain mechanism for lipid autoxidation. A generalized form of this almost universally accepted theory of autoxidation is presented in Figure l. Privett (1959) presented an essentially similar mechanism. Figure 1 shows that an initiator (light, heat, heavy metal, enzymes) catalyzes the removal Of hydrogen atom from an unsaturated fatty acid (RH), produ- cing a free radical (R.). A molecule of oxygen combines with the free radical to form a peroxy radical (ROO.) which can remove a hydrogen from a new fatty acid (R'H) and thus result in propagation of this chain reaction. The effect of increasing temperature on the rate of catalytic autoxidation is somewhat greater than in most chemical reactions. Increasing temperature accelerates Haunt 20 Initiation: RH I . R. + H. or RH + 02 I . R. + H00 R. + 02 s R0000 Propggation: R00. + R'H +7 ROOH + R'. Decomposition ROOH > R0. + 0H R0. + RH > ROH + R. .OH + RH 4+ H20 + R. ROOH «+ ROO. + H. ROOH 4% R. + H00. Termination R. + H. ‘ e RR R. + H. 2, RH R0. + OH. 4+ ROOH R00. + H 4% ROOH R0. + R > ROR ROO. + R00. —> ? Note: I = Heat, metals, light, enzyme R. = Alkyl free radical RR = Polymer R00. = Peroxy radical ROOH = Hydroperoxide Figure 1.--Mechanism Of Auto Catalytic Autoxidation. 21 not only the chain propagation reactions, but also the peroxide decomposition, thereby making a greater Concen- tration of free radicals available for the initiation and further propogation of this reaction chain (Lundberg, 1962). Similar reactions were also illustrated by Sato and Herring (1973) and Dugan (1976). Various mechanisms have been proposed for the effects of trace metals on oxidation (Lundberg, 1962; Ingold, 1962). ‘ynuqa:*y The following mechanisms were reported by Lundberg (1962), first a "reduction activation", involving the oxidation Of a metal ion (M"+), as well as the production of hydroxyl ions and free radicals. A second mechanism involves reaction of metal ions, which may initiate reaction chains. A third mechanism involves reaction of molecular oxygen with metals to form complexes, which give rise to subsequent formation Of hydroperoxide (H00.) radicals. Yet another mechanism that has been found applicable in some cases is the formation of free radicals by direct reaction of metal ions with an olefinic substrate. A schematic view of some Of these mechanisms is shown in Figure 2. Other effects of trace metals on hydroperoxide decomposition and on termina- tion reactions have also been discussed by Ingold (1962). Mechanisms involved in the peroxidation of fatty materials by biological catalysts have been investigated and reviewed by Tappel (1961, 1962). In the latter report schemes are presented for better understanding of such mechanisms. 22 Reduction Activation M"+ + ROOH R0. + OH' + M("+1)+ Oxygen Activation .M"+O ‘V 2 [M("+1) 05] + RH + 04"“) + 05] R. + [M(n+1)+ [Hmll’+ 05H] Direct Reaction with Substrate 5- Mn+ + H00. n+ + M("+1)+ RH Figure 2.--Mechanism for Metal Catalysis Reaction in Lipid Oxidation. +H ‘mfiv 23 Frankel (1962) presented a rather extensive review on isolation methods for hydroperoxides, and on their purity determination, characterization, and decomposition. Keeney (1962) reviewed and presented illustrations of the secondary products from degradation of hydroperoxides. Several of the major possible pathways were considered. As previously indicated, lipid autoxidation is an extremely complex process. This complexity results from various simultaneous reactions occurring and their interacting reactants and products (Privett, 1959). Role Of Singlet Oxygen in Lipid Oxidation Singlet oxygen has been proposed as an intermediate reaction in photo-sensitized initiation which differs from free radical initiated autoxidation reactions. The presence Of hydroperoxides with nonconjugated double bonds in the primary reaction indicates participation of singlet oxygen in photosensitized initiation of autoxidation (Vianni, 1980). B-carotene (a singlet oxygen quencher) can inhibit photosensitized initiation of autoxidation, but butyl hydroxytoluene (BHT) (a free radical inhibitor) cannot inhibit this reaction showing that photosensitized oxidation is a different process (Van Santen, 1970). A general mechanism of singlet oxygen in the process of photosensitized initiation of lipid oxidation as prOposed by Rawls and Van Santen (1970) is represented in Figure 3. S + h 3s'* + 3O ROOH Excited form Singlet state Triplet state Sensitizer moat-o It- ROOH Free Radicals Figure 3.--Mechanism of Singlet Oxygen Formation. 25 Singlet oxygen reactions could be 1.SXK times faster than the triplet oxygen initiation, hence could be an important factor in lipid oxidation. Hydroperoxide formation has been mainly influenced by singlet oxygen in milk lipid oxidation in the presence of light, copper and xanthine oxidase (Aurand gt gl,, 1977). In light induced oxidation reactions, riboflavin acts as a sensitizer through the production of singlet oxygen (directly from its photosensitized triplet state). Singlet oxygen formation occurs by dismutation of the super oxide anion (05). In this system oxidation was prevented by using singlet oxygen quenchers showing the participation of single oxygen initiation. Cort (1974) reported that in the absence Of photosensitizers, singlet oxygen does not significantly influence lipid oxidation initiation. It is important to note that free radical terminators such as BHA or BHT are of little Use in stopping initiation of photosensitized lipid oxidation. Evaluation of Lipid Autoxidation and Stability Numerous objective methods have been developed for the evaluation of lipid autoxidation and stability (Erickson and Bowers, 1974). However, in any particular evaluation using an Objective method, correlation with a sensory evaluation of the food product is essential. It is important to recognize that when a direct method is used u‘ 26 for detection of lipid deterioration in a food product, the food should be prepared, packaged and stored Under the normal conditions that are planned for its future storage. The product should be examined periodically by use of proven sensory evaluation methods (Erickson and Bowers, 1974). Sensory or taste panel evaluations are subjective methods of evaluation and rely on subjective judgment of individuals. Nevertheless, proper training of personnel and use of appropriate statistical sampling and evaluation procedures can result in an Objectivity approaching that which is expected from chemical analyses (Erickson and Bowers, 1974). Use of a sensory panel may well be the only reliable option available. Erickson and Bowers (1974) classified the existing Objective methods for the evaluation of lipid autoxidation and stability into four groups. In the first group are those methods based on lipid composition, that is, the fatty acid composition, their relative prOportions, and unsaturation. The second group include those methods based on the absorption of oxygen, an indicator of progress of lipid oxidation. The third group comprises those methods based on the intermediate compounds formed, for example, peroxides as an index of progress of oxidation. The fourth group includes methods involving the measure- ment of one or more Of the final reaction products resulting from the peroxide decomposition (Figure 4). These authors 27 .cowuouacmmo muwxocwa to mpuauocm Pmcwu msom--.¢ mesmwu mcongouocc»: muwumeoc< mucouuuo mmcopmx meeaeee_< I mcmumm o mpogoop< o mmstcm _ a mPU e. < Beats ‘(k u _ m_eaez I mcmsxpom new: 28 pointed out that the oxidation process in a lipid system is a dynamic or continuous series of reactions; therefore, any individual determination is subjected to the relative dynamic parameters involved. Percent Lipid Composition These methods include the determination of the native proportion and unsaturation degree of the unsaturated fatty acids present in the food. Generally speaking, the higher the proportion and degree of unsaturation of the fatty acids, the more liable the lipid system is to oxida- tion (Dugan, 1955). Usually the techniques used to evaluate lipid oxidation involve thin-layer and gas chromatography. Such procedures are very attractive for pure lipid systems. However, problems may arise from the isolation of lipids from complex systems. A reduction in the surface for attack by oxygen, artifacts introduced by impure solvents, failure to exclude oxygen and incomplete extraction of fraction components involved, preclude or increase the difficulty of using such methods for processed foods. Oxygen Absorption Since oxygen is a reactant in lipid oxidation, the measurement of its uptake may constitute an Objective method for lipid oxidation assessment. Nevertheless, due to very low threshold values of many off flavor compounds which result from lipid oxidation, it is apparent that 29 such compounds could be formed without any measureable Oxygen uptake, a fact that limits accelerated oxygen absorption methods. Examples Of such methods are the active oxygen method--A.O.M. (A.O.C.S., 1974), an oxygen— absorption method proposed by Eckey (1946), the oxygen bomb method (Blackenship _t _l., 1973) and monometric oxygen absorption (Johnston and Frey, 1941; Lancaster .;E._l-’ 1956). In each of these procedures, and for any product, it is necessary to develop an absorption curve and correlate its value with gp appropriate sensory evaluation. One final caution, which must be considered for complex systems such as prepared foods, is the fact that the nonlipid constituents may also absorb oxygen (Erickson and Bowers, 1974). Peroxide Formation Peroxide formation methods are of very limited use in evaluating oxidation in prepared foods although suitable for pure fat systems. Peroxide formation is a direct result of oxygen absorption. However, peroxides are intermediates in lipid autoxidation. At any time, the amount of PGTOdeESpresent is a function of the rate of their formation and decomposition. If these rates are the same, significant development of Off-flavors occur in a relatively short time. Alternately, if the rate of decom- position is retarded, peroxides may accumulate without affecting flavor since they are essentially flavorless. 30 An example of a method in this group is the peroxide value determination (A.O.C.S., 1974), which is also used in the active oxygen method. Decomposition of Peroxides These methods involve the measurement of products formed during the decomposition of peroxides. Unfortunately, there is an array of such products (Figure 4) and some of them can cause Off-flavors at very low concentrations. Extensive time and money involved for accurate determina- tions of such low concentrations may limit their use as an index of lipid oxidation (Forss, 1973). Examples of methods in this group are the carbonyl test (Henick gt gl,, 1954; Dugan, 1955; Chang and Kummerow, 1955; McKerrigan, 1957; Lea and Swoboda, 1958), and the 2-thiobarbituric acid (2-TBA) test (Tarladgis gt_gl., 1960, 1962, 1964; Tarladgis and Watts, 1960; Pohle gt gt., 1964; Jacobson gt gl., 1964; Yu and Sinnhuber, 1964, 1967; Ho and Brown, 1966; and Marcuse and Johansson, 1973). Other methods, using ultraviolet and infrared spectrophotometry, polarography and gas chromatography have been proposed for measuring products from peroxide decomposition (Dugan, 1955; Scholz and Ptak, 1966; Sherwin, 1968; Jarvi gt gl., 1971). This application, however, is rather limited unless only one fractional component is evaluated and this requires a parallel sensory measurement for assessment of correlations. 31 Predictive Methods for Evaluation of Lipid Oxidation and Stability Several choices are available to an investigator in selecting a method for predicting lipid stability. How- ever, most procedures are not as sensitive in the early stages Of Off-flavor development as is sensory evaluation. Certain foods require extended periods of time for the development Of Off-flavors. Thus a practical test used under accelerated conditions of off-flavor development and its coupling with a chemical method are essential (Erickson and Bowers, 1974). This acceleration is achieved by intro- ducing one or more factors of autoxidation such as oxygen, temperature and photosensitization. It is important to avoid completely an artificial situation such as ultraviolet light as a factor of acceleration. Another important consideration is that, in accelerated situations, the actual kinetics and some of the reaction pathways are probably different (Moser _t _t., 1965; Erickson and Bowers, 1974). When elevated temperatures are involved in addition to unsaturated fatty acids, the saturated fatty acids may actively take part in reactionsivith oxygen (Erickson and Bowers, 1974). This method of prediction is useful only when evaluating pure Oils, fats or fried foods with low moisture and which are stored at room temperatures or refrigerated temperatures. This method is not applicable 32 for stored frozen foods (Erickson and Bowers, 1974). Evaluation of Methods Due to the variety of methods available for deter- mining rancidity and stability of fats and fatty foods, the choice of a method for a particular food may be difficult. This choice may be dictated by equipment availability, nature of the problem, financial considera- tion, time, significance, and confidence level desired (Erickson and Bowers, 1974). It is important to note that in spite of all the methods available and the development of new ones, no single ideal method applicable to all products and problems exist. Sensory Evaluation Of Lipid Oxidation Taste and smell are the only two human senses which have not been relegated to a second position in modern processing and development operations. The development of automated mechanical control, and sophisticated sensing equipment has not only displaced the other human senses, but has brought about new standards of quality and uniformity (Evans, 1955). Sensory panels may fall in three different classes: quality control, grading, and consumer preference panels. Reports are available dealing with each Of these and with statistical methods associated with them (Amerine gt gt., 1965; A.S.T.M., 1968a, 1968b, 1968c). Evans (1955) 33 reviewed and discussed the various methods of sensory evaluation of fats and Oils. He presented an extensive discussion of and recommendations for sample preparation and presentation, methodology testing, panel member selection, sample size, score sheets, interpretation of results, motivation of panelists, taster performance, and supplementary chemical tests. Fioriti £1.11- (1974) also discussed and compared various objective and sensory methods of measuring fat oxidation. Effects of Lipid Oxidation on Nutritive Values From various existing reports and reviews (Kaunitz, 1962; Kummerow, 1962; Klinger, 1974), there is some evidence of toxic effects from consumption of autoxidized and thermally abused fats and oils. Chang and Watts (1952) reported a decrease in unsaturated fatty acids, primarily, oleic acid, when fresh and cured pork, beef, lamb, and turkey were cooked. The losses were not considered important when ordinary cooking was done. Similar findings were reported by Phillips and Vail (1967). Labuza _t _l. (1971) reported co-oxidation of some vitamins as an undesirable effect Of lipid oxidation, and suggested that cross-linking oxidized lipids with proteins reduced their biological availability. Georgieff (1971) discussed the possibility that an excess of unstable free 34 radicals without sufficient inhibitors, such as vitamin E, may be linked to some type of cancer. Kummerow (1962) reviewed and discussed the possibilities Of carcinogenic activity Of heated fats. Johnson gt gt. (1957) and Nolen (1972) reported that thermally oxidized oil is absorbed at a lower rate than fresh Oils. Rats fed a diet containing 10 to 15 percent thermally oxidized Oils had a lower growth rate (Poling gt_gt., 1962; Witting t 1., 1957; Johnson _t gt., 1957; Bottino, 1962). Increased ratio of liver weight to body weight was reported as a consequence of consumption of thermally abused oils by rats (Johnson _t_ __l_., 1957; Poling 53331., 1962). Other results of consumption of thermally abused oils and fats by rats involve reduced fecal nitrogen (Nolen, 1972), loss of hair and discoloration (Bottino, 1962), and in some cases, death (Bottino, 1962; Witt'ing gt a_l_., 1957). Matsuo (1962) presented an extensive review Of the work concerning the feeding of rats with autoxidized and thermally polymerized fish Oils and agreed with the above authors. Poor growth was Observed as a result of feeding thermally abused corn Oil to chicken (Kummerow, 1962). Pyridoxine and riboflavin were found to protect rats against poor growth induced by consumption of commercially heat abused fats (Witting t 1., 1957). In an eleven- month experiment, rats fed hydrogenated soybean Oil which 35 had been used for 56 hours Of frying, showed no adverse effects including offsprings. When lipids are heated excessively, some toxic effects have been reported from feeding studies (Deul _t _l., 1951; Poling gt gl., 1962; Chang and Watts, 1962). Poling gt gt. (1962) warned that there is a potential toxicity when fats are abused excessively. Matsuo (1962) reported negative growth effect on rats from consumption of unheated autoxidized fish Oils. This author stressed the point that highly unsaturated fats and Oils, even when not heated, may be dangerous due to autoxidation alone. Antioxidation and Antioxidants Mechanism of Antioxidation Antioxidants are substances which react with free radicals formed during lipid autoxidation to give stable products and extend the shelf life Of the substance. Antioxidants, however, do not avoid or block autoxidation completely or permanently; they merely retard the chain reaction process; and eventually will be lost from the lipid (Stukey, 1962) or become oxidized (Weiss, 1970). Lundberg (1962a) reviewed the early work on antioxi- dation and presented schematically one of the most accepted mechanisms Of antioxidation (Figure 5). In any Of these mechanisms, antioxidants are proposed to act as hydrogen donors or free radical acceptors. This form Of R00. + AH2 AH. + AH. R00. + AH2 (ROOAHZ). + R00. AH2 + R00. AH. + R00. ROOH + AH. A + AHz (ROOAHZ). STABLE PRODUCTS AH. + ROOH ROOH + A Figure 5.—-Proposed Mechanism of Antioxidation. 37. action is called "primary" inhibition and an "auxiliary" inhibition involves removal or destruction of hydroper- oxides without the formation of chain—initiating free radicals. Stuckey (1962) referred to four possible mechanisms by which an inhibitor may function as a chain stopper for the free radical mechanisms of lipid oxidation. These are: hydrogen donation of antioxidant, electron donation by the antioxidant, addition of the lipid to the aromatic ring of the antioxidant and formation of a complex between the lipid and the aromatic ring of antioxidant. He presented evidence from the literature which strongly corroborates the first mechanism. However, evidence which favors the other three mechanisms was also discussed. Although antioxidants have been used successfully to retard lipid oxidation, the exact mechanisms Of their action have not been completely clarified. Metal Sequestering Agents Metal sequestering agents are called metal scavengers or metal chelating agents. They bind metals and thereby inactivate the participation Of trace metals such as copper and iron which are powerful pro-oxidants Of fats and oils (Lundberg, 1962b; Weiss, 1970). The most commonly used metal sequestrant is citric acid. Others are isopropyl citrate, stearyl citrate, ascorbic acid, monoglyceride citrate, and polyphosphates. 38 Synergism and Antioxidants The antioxidant effectiveness of a mixture of two substances is frequently greater than the sum of the inhibitory effects that are obtained when the same quantity of each antioxidant is used alone. This phenome- non of both substances helping one another is termed synergism (Lundberg, 1962b). In some cases, one of the two substances is far more effective than the other when used alone, and in such cases the more effective material is referred to as primary antioxidant, and the less effective material is referred to as the synergist. How- ever, synergism is frequently Observed with substances that have approximately the same order of effectiveness when used alone, and are similar in chemical structure, such as butylated hydroxyanisole and butylated hydroxy- toluene. Various explanations and mechanisms have been proposed for synergism among antioxidants. The acid synergists are also metal chelating agents and the action has been considered as the only explanation for such synergism (Lundberg, 1962a). (Mixed free radical acceptor synergism, however, Should receive another explanation.) Other existing theories of antioxidant- synergism were discussed by Lundberg (1962b) and Stuckey (1962). 39 Antioxidants According to Weiss (1970) antioxidants were developed primarily for use with pure fats. Presently their use is permitted in many countries for food use and for animal feeds (Lea, 1962). Stuckey (1962) affirmed that the development of antioxidants actually started during World War I with nonfood materials such as rubber, gasoline, and plastics. Sherwin (1972) stressed the point that antioxidant free radicals, formed when interferring in the autoxida- tion prOcess, cannot initiate or propagate the oxidation reactions. This same author stated that antioxidants do not function by competing with the substrate for oxygen and that they are not oxygen absorbers or adsorbers, but merely are free radical inhibitors. This seems to be the case, although Weiss (1970) referred to antioxidants as using up available oxygen in the shortenings to which they were added. Antioxidants, as other additives for use in human foods, are subjected to regulations under the Food, Drug, and Cosmetic Act; the Meat Inspection Act; the Poultry Inspection Act; and the U.S. Food and Drug Administration Food Additive Amendment. The antioxidants permitted are not likely to harm the consumer. The level of antioxidant usage in foods is also regulated (U.S.D.A., 1960). 40 Chemical Classification Of Antioxidants Antioxidants, other than those having metal-Seques- tering action, were classified by Stuckey (1962) into three groups: phenols, amines, and aminophenols. In general, they are structurally similar in that they contain unsaturated benzene rings plus either hydroxy or amino groups. Some are also effective polymerization inhibitors and others retard degradation of polymeric systems by ozone. Phenols are commonly used antioxidants when low color, low toxicity, or a combination of these properties is more important than extreme potency. MOst natural and synthetic food grade antioxidants belong to the phenolic class of compounds (Dugan, 1960). Antioxidants with amino or diamino groups attached to unsaturated benzene rings are usually extremely'potent, often quite toxic and generally form intense colors when oxidized or when they react with metals to form salts. They are usually quite stable to heat and caustic extraction. An example is diphenylamine which is widely used in rubber industry (Dugan, 1960; Sherwin, 1968). Aminophenol antioxidants have both amino and phenolic groups as potential sites of antioxidant activity. They are used in the prevention of gum formation in gaso- line (Lundberg, 1962a). 41 The unsaturated benzene rings found in the antioxi- dants are responsible for the stabilization of free radicals through the formation of stable resonance hybrids after hydrogen is donated to free radicals during oxida- tion (Figure 6). Most Commonly Used Food Antioxidants At present, the commonly used food antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl galate (PG), tocopherols, and hydroquinone. Structures of the first three are represented in Figure 7. Among the metal sequestering agents, citric acid is used most commonly (Sherwin, 1972). Due to the occurrence of synergism among these antioxidants and due to regulations regarding the amount that may be added to foods (U.S.D.A., 1960), they are used most commonly in mixtures rather than separately (Sherwin, 1972). The development Of new antioxidants has been continu— ously undertaken by many workers. Olcott (1974) reported a new antioxidant being widely used in Europe (Figure 8). He stated that the compound was excellent for fats and oils and was not absorbed in the gastrointestinal tract as most others are. The latter aspect is undoubtedly very promising since such a compound could be used without limitation and could possibly solve tough problems of 42 .mucmuwxowpc< owpocmze an umecom woven»: monocommm mpnmum--.m mcamwm J4Io_I .: QIQIQ ,1 Ge? :0 0T— , . o o .o . I 43 c / O/\0 I C3 ”8 PROPYLGALLATE Figure 7.--Structure Of Most Common Food Antioxidants. 44 2-3: Figure 8.--A New Antioxidant in Use in Europe. 45 lipid oxidation in many foods that are still unresolved. Silicones Silicone compounds are being used more and more for their antioxidant and their anti-foaming action (Weiss, 1970; Freeman gt gl., 1973). When added to frying fats and shortenings, they result in improved stability. The action Of such polymethyl siloxomes, more simply sili- cones, supress foaming, decrease oxygen contact, and supress build-up of foam promoting oxidation products in the fats or Oils being heated. The mechanisms for this supression have been presented and discussed by Freeman _t _l. (1973). Recommended usage levels for Silicones are in the range Of 0.5 to 3.0 ppm (Weiss, 1970; Freeman _gt._t., 1973). In addition to citric acid as a chelating agent, especially in high moisture and fatty foods, ascorbic acid has been utilized. Ascorbic acid will chelate metal ions; however, the mechanism Of antioxidant activity is more complex in high moisture systems (Labuza, 1971). An oxygen scavenger mechanism attributed to ascorbic acid was proposed by Cort (1974) as an explanation for increased stability of oil systems containing ascorbic acid. Ascorbic acid has been shown to have pro-oxidant activity in meat (Love and Pearson, 1971; Benedict gt gl., 1975). Citric acid and ascorbic acid have beneficial 46 synergistic effects with Type I antioxidants (Dugan, 1960). In addition to citric and ascorbic acid, poly- phosphates are also known to serve as metal Chelators. Selection and Use of Antioxidants The selection and use of antioxidants and/or mixtures should be done carefully and with consideration of the conditions surrounding the manufacture, handling, and use of the food product to be treated. According to Sherwin (1972), the major selection criteria to be considered include: antioxidant potency, solubility or dispersa- bility, discoloration tendencies, food pH, processing, antioxidant Odor and flavor, mode of application, and future trends in industry for that product. Some important points to remember in using antioxi- dants are the necessity of early addition, uniform and complete distribution of the antioxidants in the food being treated. It is important to observe these points due to the chain nature Of the antioxidation Of lipids, the unrestorability of oxidized fats and Oils, and the small amounts of antioxidants normally used (Lea, 1960; Sherwin, 1960). The reduction of oxygen incorporation and minimization of trace metals present are also important. The relative efficiencies of a series of antioxidants can be altered considerably by small changes in the 47 composition of substrate, or even in the temperature of oxidation (Lea, 1960). The difference in the activity of an antioxidant from one complex food to another can be very large (Lea, 1962). After the above considerations and the lack Of data regarding the use of antioxidants for a particular product, one may eventually decide upon experimentation to devise a new formulation or determine among various possible ones which product gives the best results. However, with a large number of antioxidants available, one may have too many combinations for a practical experiment even though it could give the best solution to the problems. This problem can be solved partially through elimination Of poor formulations. The four most commonly and widely used antioxidants and their charac- teristics were presented in several reports and reviews available in literature (DUtton gt gl,, 1948; Kraybill _t _t., 1954; Gearhart and Stuckey, 1955; Sims and Hilfman, 1956; Cooney gt gt., 1958; Lea, 1960, 1962; Dugan, 1960; Lundberg, 1961, 1962a, 1962b; Stuckey, 1962; Weiss, 1970; Jacobsen and Koehler, 1970; Mickelberry, 1970; Sherwin, 1972; Joint FAD/WHO Expert Committee, 1972; Bishov and Hekick, 1972; Klinger, 1974). The following factors are important when selecting an anti— oxidant for a particular food: 48 1. Trace metals such as Fe, Cu, Co, and Ni are expected to be present in foods and they‘ catalyze fatty acid oxidation. 2. Citric acid and ascorbic acid can chelate metals and show some synergistic effects on BHA and BHT. 3. BHA and BHT show similar mode of action; sometimes BHT may be slightly more potent than BHA. 4. BHA and BHT show good carry-through properties during baking, cooking, and frying. 5. BHA and BHT show good synergism when used in 1:1 proportions. 6. Propylgallate shows synergism with BHA and not BHT, does not have good carry-through property and may develop undesirable color when used in the absence of metal Chelators. Extraction of Tissue Lipids Qualitative extractions of tissue lipids can be achieved by using a combination of polar and nonpolar solvents. Nelson (1975) suggested hydrolysis before extraction to facilitate separation Of protein-bound lipids and allow free access to nonpolar solvents. Folch t gt. (1957) used a 2:1 chloroform-methanol mixture to extract tissue lipids. Bligh and Dyer (1959) used a 49 mixture of chloroform-methanol and water as lipid extracting solvents. A chloroform-methanol mixture has been suggested as a good lipid extracting solvent by others including Ostrander and Dugan (1961) and Radin (1969). Based on the effect of various combinations of solvents on extraction of total lipids, fatty acids, and triglycerides, Sheppard gt gt. (1974) found that digestion with 4N HCl followed by diethyl ether extraction or 2:1 chloroformzmethanol extraction were satisfactory methods for lipid extraction. Separation and Fractionation 9f Phospholipids Silicic acid column chromatography is the most popular and efficient method used to separate polar lipids from tissue lipids Of animal origin (Hanahan gt gt., 1957; O'Brien and Benson, 1964; Kata gt gt., 1966; Peng and Dugan, 1965; and Nelson, 1975). Usually neutral lipids, glycosphyngolipids and phospholipids can be separated by this procedure. The treating of Silicic acid with isopropanOl-KOH to separate free fatty acids has also been reported (McCarthy and Dutchie, 1962). Thin layer chromatography (TLC) has become a major procedure for lipid fractionation in the past two decades. It has advantages over silic acid column separation with respect to resolution, speed Of separation, and simplicity of procedure (Nelson, 1975). Silicic acid column SO chromatography can be very useful when large amounts of the fractions are required for further analyses. ‘Complex mixtures of lipids can be successfully and reliably separated using two-dimensional TLC on silica gel plates. Skipski t al. (1962) separated phospholipids and cere- brosides on silica gel plates using a mixture of chloro- form, methanol, acetic acid, and water as developing solvents. Parker and Peterson (1965) found a special washed silica gel H plate and a solvent mixture of chloroform, methanol, acetic acid and water to be good for microfractionation of phospholipids. The two dimensional TLC method of Rouser gt_gt. (1966) with chloroform, methanol, water, n-butanol acetic acid and aqueous ammonia for first dimension and chloroform, acetone, methanol acetic acid, and water for second dimension is a popular and successful method for phos- pholipid fractionation. Following fractionation of phospholipids, further estimations have been achieved by gravimetric, colorimetric, and titrimetric analyses. Colorimetric methods are generally accepted because of high reproducability of results. Common methods include those of Morrison (1964), Parker and Peterson (1965), and Rouser t 1. (1966). 51 Gas Liquid Chromatography (GLC) Purified total lipids or fractions belonging to various classes of lipids need to be converted to a volatile form before GLC analyses. This may be achieved by converting lipids directly into volatile forms or by hydrolysis of the lipids, and converting fatty acids separated into volatile forms (Metcalfe gt gt., 1966). Reagents and methods depend on the type of compound being analyzed. For conversion of fatty acids with less than eight carbons in the chain, BF3-butanol reagent is preferred, while fatty acids with more than eight carbons in the chain can be converted using BF3-methanol or BCl3-methanol (Metcalfe _t _t., 1966). Soponification before esterification often gives best results. Some of the common procedures in general acceptance and wide use are those methods of fatty acid methyl ester preparations reported by Morrison and Smith (1964), McGinnis and Dugan (1965), and Metcalfe _t _t. (1966). Since the introduction of GLC as a technique for separation Of Carboxylic acids (James and Martin, 1952), it has been successfully used as a major technique for separation Of lipids, alcohols, hydrocarbons, pesticides, and amino acids. Efficient and normal separation of a chemical mixture can be accomplished by use of a liquid phase similar in 52 chemical structure to that Of the mixture (Orr and Callen, 1958). They used a polyester type of liquid phase for successful separation of polyunsaturated fatty acids. Some widely used liquid phases are adipate and succinate polyesters of diethylene glycol. Silicone polymers Of type SE-30 especially for high temperature separation of natural triglycerides has been recommended by Kuksis (1965). Factors such as type and amount of liquid phase, temperature, gas flow rates, type of compound being analyzed and sample size have influenced efficiency of separation and resolution of chromatograms (Mehlen- backner, 1960; Seino gt gt., 1973). Qualitative identification of the various peaks on fatty acid GLC chromatograms can be accomplished by using retention value or by using the plot of log retention time of standard fatty acids against fatty acid chain length. This method described by James (1960) results in a linear straight line relationship for homologus series, thus is very useful for qualitative identification of fatty acids. METHODS AND MATERIALS Processing and Treatment of Fish Samples Fish Samples Carp (Cyprinous carpio) samples used in this study were obtained from a commercial fishing plant, Bay Port Fish Co., Bay Port, Michigan. All fish were harvested from Lake Huron. The fresh fish were transported to MSU Meat Laboratories, ice packed in cardboard boxes. Overall processing and treatments were completed within 36 to 48 hours of initial harvest. The fish used were 39 to 62 cm long and weighed from 2.50 to 4.10 kg each. Mechanically Deboned Carp About 500 kg of carp were washed thoroughly in cold running water, weighed, and manually deheaded, gutted and split dorsoventrally parallel to the backbone. The split halves were washed in cold running water, care being taken to remove peritoneal membranes, connective tissues in the abdominal cavity and kidney tissue, and immediately layered with crushed ice before deboning. A Bibun model S0513 deboner (Bibun Co., Fukuyama Kovoshima, Japan) was used to debone the fish. This is a moving belt type machine equipped with 3 mm hexogonal 53 54 perforations in the drum. Dressed fish were fed into the machine so that the flesh side was next to the drum perforations and the skin side was next to the belt. The meat was pressed through the drum perforations to the inside while bone, skin, scales, and some connective tissue pass outside the drum. The mince coming out of the inner surface of the drum was collected in rectangu- lar pans and stored at 2°C for further treatments. All treatments were completed within 6 to 8 hours on the same day. Hand Filleted Carp (with skin) A total weight of 400 kg carp used in this study was ice packed in waxed cardboard boxes and transported to the University Meat Laboratories. The fish were thoroughly washed in cold water (2°C) to remove slime and were weighed. After weighing, fish were manually deheaded, gutted, and split dorsoventrally so that two fillets and a vertebral column were separated. The fillets were washed thoroughly using running cold water, and care was exercised to remove peritoneal membranes, connective tissues in the abdominal cavity, kidney and other organ tissues. The washed fillets were weighed and stored in layers of crushed ice for further treatment. 55 Hand Filleted Carp(without skin) The skins were removed from one sample of the fillets by hand using a sharp knife, washed, weighed and stored in layers of crushed ice for further treat- ment. Antioxidants and Chelators Used Only legally permitted antioxidants were used, except for one treatment consisting of ethylene diamine tetra-acetic acid (EDTA). Following are the antioxidants and Chelators used in this study: 1. 2. Tenox 2® - Eastman Kodak Co., Rochester, N.Y. Tenox BHA‘D (Butylated hydroxy anisole) - Eastman Kodak Co., Rochester, N.Y. Tenox BHTG (Butylated hydroxy toluene) - Eastman Kodak Co., Rochester, N.Y. Propylgallate (PG) - Eastman Kodak Co., Roches- ter, N.Y. L+ ascorbic acid (AA) - Eastman Kodak Co., Rochester, N.Y. Citric acid (CA) - Eastman Kodak Co., Rochester, N.Y. EDTA - Mallinckrodt, St. Louis, MO. Freezgard (Formula FP-88E) - Stauffer Chemical Co., Westport, CT. 56 Tenox 2G is a commercial food grade antioxidant mixture. It consists of 20 percent BHA, 6 percent propyl- gallate, 4 percent citric acid, and 70 percent propylene glycol. Freezgard®’(Formula FP-88E) is a mixture of NaCl, Na tripolyphosphate and Na erythorbate with a suggested use of 0.18 percent based on flesh weight. All Of the above antioxidants were incorporated at the rate Of 0.01 percent fora single antioxidant or 0.02 percent fln‘a combination Of antioxidants (with no single antioxidant exceeding 0.01 percent) based on the total fat content, except for Freezgard®, which was used 0.18 percent based on flesh weight. The quality and quantity Of antioxidants used are indicated in Table l. Incorporation of Antioxidants into Mechanicaltnyeboned Carp Flesh The antioxidants which were not soluble in water were dissolved in propylene glycol to make a 10 percent solution before appropriate addition to the sample Of the deboned carp flesh as shown in Table l. Minced fish was mixed in a Hobart Kitchen Aid food mixer equipped with a paddle (The Hobart Mfg., Co., Troy, OH). The stainless steel mixer bowl was covered with a water repellant wax coated cardboard during four minutes Of mixing at medium speed to allow the addition of N2 during 57 .a:u.oz gnu—c _uueu so non-a a. go's: usaouoocu unouxo .ugm.oz and _oaou co comma “season as. nos—a» pp

PP< 59 F -- ooo. -- -- -- -- ooo. -- -- -- A< __< "muoz .ooECPBeoo_eoo .uoo Pogo“ co oomoo ucoucmom .oocoomo ooozm .omcoomo oppoowoocumzp op.onno.o “_nomp mo.onmo.o opnomp omcoeoo P~.on~o.m omnomm .Np.onsm.e .Pmnooo Pocoomopoeo Pooch oN.onNm.m Rmnoeo mp.onoh.m ownome A ”opoz .omooooo ooozm .omcoooo oppoowconomzp ¢.m whmu m.N ohmm mcmsgo 0.5 mommp m.o whomp owQVFowvsou m.n onmo_ o.a _Fnoo_ Fooomooo_ao.oeeemeea m.o_ F_noN~ o.op monoem oeocompxoooeeomoea o.o_ opnmmm o.op o_n~mm eo_o»eooe_am m.- mpnnmo m.NN thoom moosopooogpoPAowposomogo o.mm omnmmm _.mm “_Hoow ocwpogopzowuogomoso & Aoommwu m oop\osv & Roommwp m oop\mev «a: Fox mmopu owowposomozo mcwooooo we oozumz .ocoo ooocws ow movowpozomogo mo oowupmoosou co mcwcoomo mo oozpme we powwow .o mpoo» 87 Table 7. Fatty acid composition1 of phospholipid and neutral lipid fractions from minced carp. Method of Deboning Fatt acid: MD2 H03 Phospho- Neutral Phospho- Neutral lipids lipids lipids lipids 14:0 26 491 26 409 15:0 8 67 7 57 16:0 382 1719 378 1357 17:0 4 86 4 309 18:0 219 376 229 89 20:0 4 104 4 110 Saturates 643 2843 648 2406 14:1 2 131 2 108 15:1 -- 60 -- 49 16:1 152 3254 156 2790 17:1 12 230 12 200 18:1 220 3150 212 2587 Monoenes 376 6825 382 5734 18:2 47 664 53 549 18:3 + 20:1 72 468 73 397 20:2 4 175 4 152 20:3 10 38 10 33 20:4 + 22:0 191 239 190 201 20:5 u 6 236 96 229 79 20:59» 3 12 343 9 317 22:4 8 38 8 27 22:5 0 6 32 12 28 10 22:5 w 3 40 125 41 99 22:6 321 238 319 199 Polyenes 973 2436 974 2063 1Mean of duplicate samples. 2Mechanically deboned. 3Hand deboned. Note: Values are mg fatty acid/100 g tissues. 88 deboned and hand deboned minced carp lipids did not differ. However, the ratio of saturated fatty acids to that of unsaturated fatty acids of phospholipids in mechanically deboned minced carp was 1:2.11 compared to 1:2.09 from hand deboned minced carp. While this ratio was very similar between two treatments, neutral fatty acids accounted for about 1:3.26 and 1:3.24 in mechani- cally deboned and hand deboned minced carp respectively. The polyunsaturated fatty acids formed a much higher percentage of phospholipids (about 49 percent) compared with about 20 percent of neutral lipids, However, the total amount (mg fatty acid/100 9 tissue) of polyunsatu- rated fatty acids from neutral lipid would account for a substantially higher percentage of total polyunsaturated fatty acids in the whole system. Carp lipids did not contain detectable levels Of C15:l fatty acids in the phospholipid fraction. C14:0, muiCl6:0 were the major saturated fatty acids, Cl6:l and C18:l were major monoenes and Cl8:2, C18:3, c20;3, C20:4, C20:5 w 6, C20:5 w 3 and C22:6 were the major polyenes. The fatty acid composition of carp was similar to that reported by Mai and Kinsella (1979), except for minor concentration variations. This may be due to the variations in fatty acid composition of same species of fish from different areas, different seasons or different feed availability. 89 Protein Fractions The level of nitrogen in the various fractions of the proteins from mechanically deboned and hand deboned minced carp are presented in Table 8. Higher percentages of sarcoplasmic and nonprotein nitrogen were found in mechanically deboned than in hand deboned carp. Stromal protein nitrogen was found to be much higher in hand deboned than mechanically deboned carp. These high values of sarcoplasmic and nonprotein nitrogen may be due to the incorporation of more free amino acids and proteins associated with the skin bone marrow and intra tissue fluids released during mechanical deboning (Webb _t _t., 1976). The lower stromal protein nitrogen levels may be due to the screening effect of mechanical deboning resulting in separation of collagen and fibrous connective tissue as reported by Satterlée gt_gt, (1971) and Webb _t _t. (1976). To develop the desired textural property of comminu- ted products using muscle proteins, an understanding Of the solubility of individual proteins is important (Webb, 1974). However, the results based on protein fraction solubilities of fish muscle tissue are of no use in evaluating texture qualities (Webb, 1974; Webb _t gt., 1976). Basic information could be important in the development of comminuted products from mechanically deboned minced carp. For example, Carpenter and Saffle 9(1 .cooouu—z u x .z pogo» oo con-o cooogu.c accuse; - N .uoo.ooo.scoooo o sot» on + «ammo» oo—xz a: zoo: "coo: o.~no..o. o~n-n. o..«o.... sonoon .on~o.n~ o.«o.o oonom.o_ m.n_.. oo. noeooo~ oeox o._«mo..o Nonooo .o«-.o. oonhon .«oo.- oonoo. oonpo.o_ ”_«ooo oo. -noo.~ »_poo.e.ooex u o: a o: a o: a o: um o: z _.secom a e.eooceeo= z c.._.co.cexz z o.en..eooc.m z _eoe» oe.oeooo co oegoo: Aoanuou a oopsolv «oo.uooco ooouoco .osou sous—u not» moo.uuoco c-ouogo opaque-tuna uao.co> no. pouou oo apo>op cooocu_o oo oovoooue oo vogue: yo cocoon .o ops.» 91 (1964) found a positive correlation between salt soluble proteins and emulsifying capacity of red meat muscle tissue. In this study no significant differences were observed in the myofibrillar protein fraction (salt soluble proteins) between mechanically deboned and hand deboned minced carp tissue. Shear Value and Waterholdinngapacity The data presented in Figure 9 indicate that the fish gels prepared from mechanically deboned carp flesh had significantly lower shear values and firmness, as rated by a panel, compared to the fish gels prepared from hand deboned minced carp. The data on waterholding capacity, determined by the amount of liquid separated by centrifugation and panel scores for juiciness of fish gel prepared from mechanically deboned and hand deboned minced carp are presented in Figure 10. A high percentage Of liquid separated from carp gel denotes a low water- holding capacity and vice versa. Although the water holding capacity Of fish gel made from mechanically deboned minced carp was lower than from hand deboned carp (i.e., higher percent Of extractable liquid), it was not significantly different from that of the gels prepared from hand deboned minced fish gel. A signifi- cant difference in juiciness was identified by the taste panel evaluation scores (2.86:0.4 and 3.8:0.5). 92 2.2 2.0 109 A MD. if E 3"1.3 E ,2 2 u; == ==: 5 g E E L E E g m .. IE 1.7 gig Egg g; u: 3- E -"'-'-' "’ to E E _. a 1.6 '_-'-'___= 5 Lie" .3 = E a. II a: ..=-='=: s: 1.5 ;- g E E == _-== SIGNIFICANT AT P 0.05 MD - MECHANICALLY DEBONED. HD = HAND DEBONED Fl'90re 9.--Shear force values and panel sensory scores for firmness of fish gels prepared from hand and mechanically deboned minced carp. 93 20 ‘ 5.0 ND 16 g NO 9.0 5 HD o E t E l s 212 =-.-_= MD' 3.0 > 2 E E B _J = = > :9 E E a. " on = E gm E 8 E E 2.0 tag .2. :=: g _. .. X E -=- a: a “J = = c: U N E E I D. > A '51.: g 1.0 g 0 = = 0.0 ' SIGNIFICANT AT P 0.05 MD = MECHANICALLY DEBONED. HD = HAND DEBONED Figure 10.--Waterholding capacity and panel sensory score for juci- ness of fish gels from hand and mechanically deboned minced carp. 94 Lipids, their degradation products and their reac- tions with protein could be limiting factors affecting frozen storage life of fish fillets (Castell et 31,, 1966; Anderson and Steinberg, l964; King 35 al., l962; Hanson and Olley, 1965; Takama, l974). The results from this study show that there are some additive effects when mechanically deboned minced fish is stored in the frozen state. Some adverse effects observed due partly to incorporation of high fat content from mechanically deboned flesh, were loss of solubility of some protein fractions, loss of waterholding capacity, and decrease in firmness of fish gels. To what extent these properties would effect the subsequent comminuted fish products were not determined, since product development from the frozen minced fish was not one of the objectives. In a similar study on the effect of mechanical deboning on fish flesh, Webb _3 _l. (1976) concluded that a more firm product was obtained from hand deboned fish, probably due to the high soluble fraction in it. The shearing action during mechanical deboning could produce a stress upon myofibrillar proteins which may result in denatura- tion of fibrills and thereby low textural qualities of mechanically deboned fish. 95 Relative Stability of Mechanically Deboned Carp During Frozen Storage During the mixing of antioxidants with mechanically deboned minced carp, the product temperature increased to l3:l°C. This temperature increase could be reduced by processing the fish in a controlled temperature room as in commercial seafood processing plants. Careful mixing was done to assure a homogenous distribution of antioxi- dants. Preliminary Studies on TBA Test A series of preliminary tests were conducted to determine the best conditions and procedures for deter- mining TBA numbers. Preliminary trials indicated that a 35 minute heating period, followed by a l0 minute cooling period in the procedure suggested by Tarladgis gt 31. (l960) resulted in a more constant color formation in blanks than the l5 hour holding period at room tempera- ture for color development as suggested by Tarladgis _t _l. (l964). Absorption spectrum of the pigments formed from TBA reaction in fish sample distillates are shown in Figure ll. A wave length of 538 nm was found to be most sensitive to the malonaldehyde-Z-TBA complex pigment formation from minced carp flesh lipids. Distillation trials, using l,l,3,3-tetra ethoxypropane (TEP) to determine average recovery and determination of 96 0.12“ 0J0! DAN 0J5! ABSORBANCE 0.02 0.00 LEO‘fiDWDWWSiIJSlDSZOBOS‘IOSSOSfiD' HAVE LENGTH (71m) Figure ll. Absorption spectrum from malonaldehyde and 2-Thiobarbituric acid (TBA) complex from minced carp tissue. 97 factor K for converting absorbance values to TBA numbers, was conducted according to Tarladgis gt al. (1960). A recovery of 68.4 to 71.3 percent was achieved in accord- ance with results reported by Tarladgis £3 31. (l960). A conversion factor of 7.8 was used to express absorbance readings as TBA numbers throughout this study. Effect of Antioxidants and Chelators on TBA Numbers of Mechanically Deboned Carp During Frozen Storage TBA values were determined and used as numerical indicators to assess the degree of lipid oxidation in mechanically deboned minced carp subjected to various antioxidant treatments. Initial TBA number for mechani- cally deboned minced carp was 0.86:0.14. TBA numbers determined at various storage time intervals through eleven months of frozen storage (lBOC) for all the product treatments are listed in Table 9. TBA numbers increased slightly during the first three months of frozen storage, increased more rapidly during the 4 to 8 month period and then increased rather slowly from 8 to ll months of storage. This trend is somewhat similar to the general pattern of TBA values as reported by Deng gt_§l, (1977) for changes occurring in frozen mullet fillets. A similar trend with time of frozen storage was reported by Morris and Dawson (1979) in sucker flesh (except that no leveling off trend was 98 .mucosuomcu meoEo Amo.ox: monocomevu azuupuvcmwm mumu.uc_ asapou o =_ mgouuop unmgomm.o "uuoz .apasom m ooopxmuagov—ocopoe as no cummogaxo cowuo:¢5couuu o soc» muuoup—aog N we com: — u~¢.e uoo.e apo.~ umm.. am~._ .mm.o oma.o as~ xoeoh ump.a oom.m umo.o unm.~ n~0._ umm.o umo.F nwucam~uuca u.n.. van." acm.~ umo.. a.n.. .co._ a8.— ue + << + Axe + «In u_o.e u.o.n mnm.~ oo..~ no... .mn.. 084 on + <0 + pzm + «zm u~m.m u-.c omm.n umm.~ nPA._ amm._ un..~ << + an + «In un..m uma.. u_o.o u_~.m noo._ moo.— ~.~._ <0 + an + «In u.o.o oun.m ump.. ucm.n a_¢._ .om~._ -_.. << + Axe + <19 afio.. upm.m u~m.¢ afim.n nom._ .c~.. ao~.. «u + sz + <=m amn.m a_s.m noo.m one.” ae~._ amm._ ~m_._ hzm a ——< . “pm <~m “am can <~e muueesuem am“ No. 3.0 mmm <85 mdeua,o< -.. m.n .m... a.” .ne.. ”NM ~o.~ cqn N..~ own “demoed: eemoeu.z can mcu omogoum Augucoe. nopcoa concoum .mgucoe cm>o—m so» uoo~- an umcoum acou caucme coconut x__eupco;ume c. muwuc xuuou uoaacauom cu muwuo xuuou woo-cauamc: yo o.uoc uco mu.ua xuuou v,a_—o;nmozq we masocm maovcc> co u:oE:oc.>co omocoum we uuouuu .m— o_nu» 130 those packaged in nitrogen. The higher retention was due to less degradation of polyenes, since the variation due to packaging treatment among saturates and monoenes was minimum. However, there were no statistically identifiable advantages due to packaging treatment at end of eleven months of storage. From 4 to 9 months of storage, nitrogen packaged samples had a higher ratio of unsaturated to saturated fatty acids, with the ratio at 8 and 9 months being significantly different from that of 4 months. Using mechanically deboned chicken and turkey meat, Jantawat (1978) observed that vacuum and nitrogen packaging treatments resulted in significantly higher retention of polyenoic fatty acids during four months of storage. How- ever, in this study it was observed that only the nitrogen package showed some advantage in improved retention of polyenoic fatty acids of carp tissue over a period of 8 to 9 months. A possible explanation of nitrogen back flush having advantages over vacuum packaging would be the fact that the limited oxygen left after vacuum packaging is further diluted by nitrogen addition and hence decreases availability of oxygen for lipid oxidation. A general trend shows a decrease in oxygen due to vacuum and/or N back flush resulted in unsaturated fatty acid retention and decreased TBA numbers. The effect of storage environment and antioxidant treatments on the TBA numbers at the end of 4, 8, and 11 131 months of storage of mechanically deboned minced carp are listed in Table 16. No significant differences were found among samples packaged in air, vacuum, and nitrogen. The TBA numbers for samples packaged using a nitrogen back- flush were slightly lower than all others. This difference, among packaging treatments, became slight after eleven months of storage. The packages did retain the vacuum and nitrogen tensions at the end of eleven months, however, no effort was made to measure the exact amount of vacuum or nitrogen retained. Deng _t__l, (1977) reported that vacuum packaging in combination with antioxidants improved storage stability of mullet as indicated by TBA numbers and peroxide values. In this study vacuum packaging did not improve the antioxidant effect, while a nitrogen back flush in combination with antioxidants did result in low TBA numbers. This may be due to the differences in vacuum levels used (the vacuum levels used in above author's experiment are not reported) and the differences in type and quantity of lipids, between carp and mullet. Frozen Storage Stability of Hand Filleted Catg Carp fillets with and without skin treated with Tenox 20, FreezgardD, BHA + BHT + PG + citric acid, and BHA + BH + PG + ascorbic acid were frozen and stored at -20°C to monitor rancidity development. 132 .opanm mxxmuxgau_aco_az as we nommocaxo mcovuoc.5couou o scum .gmapm goon :ooocu*z n z meovuuuF—aoc N no com: .s==u~> n > .L—< u c_<~ — < z > < z > ~< _— m o mucouvxovuc< Augucosv «ewe uoaco»m mucmmmcn c_ umcoum cco macaw—no.uco uneven) gu_x umummcu acme vouc_s vmcooou x__uu.cogume to» .mzucoe p. to» uom_- an mucmscoc_>cu m:.auxuua maopca> 8o .mcmneac . I e g 1 g’ 1 o O :2 / = 4. _, ' o II 55 c: / 1.1.1 ac: .- z 0 = z 0 <2 2, I . m . .— /./ O 0.0 2 3 4 6 8 9 11 STORAGE TIME (MONTHS) Figure 18.--Effect of antioxidants and frozen storage on TBA numbers of hand filleted carp without skin. 136 differences occurring at 6, 8, and 11 months of storage. TBA values were 2.64, 5.01, 7.41 for fillets with skin and 4.18, 6.41, 8.24, for fillets without skin (6, 8, and 11 months of storage, respectively). Polyphenol antioxidant treatments on fillets with skin were ineffective because of the lower exposed muscle surface compared to skinless fillets. A slightly lower TBA number in comparison to control (but not necessarily statistically significant) were observed for skinless fillets treated with Tenox 20 and BHA + BHT + PG in presence of either ascorbic or citric acids. FreezgardG treated fillets with and without skin resulted in lowest TBA numbers among the treatments. Slight differences in TBA numbers were observed for products similarly treated between fillets with and without skin, but no significant differences were found. Taste panel evaluations of fish loaves prepared from antioxidant treated carp fillets stored for O, 6, 8, and 11 months at -20°C are illustrated in Figure 19.. Lowest scores were recorded for Freezgard® treated samples. As explained earlier, a hedonic scale of 0 to 5 with 0 = no detectable rancidity and 5 - very rancid was used. All fillets with skin stored for 8 months were acceptable by the panel in comparison to only 6 months storage for skinless fillets (from comments on panel scoring forms). Based on an acceptable score of 2.0 or lower, phenolic 137 VV+9d+LHH+VH8 J L V3¥Ud+lH8+VH8 8” x0N31 908V9X3383 F LLET WITHOUT SKIN WOULNOJ l 1 I l I l 1 1 4 v, 1 l I 1 VV+9d+LH8+VH8 I L I V3+9d+LH8+VH8 93 xo~31 T I I I 9G8V92338d FILLET WITH SKIN 108LN03 3' M N H ( GIDNVB A83As§ 'ALIGIDNVU BTSVLDBLSO ONsO ) 80AVTJ 3 8000 013NV8 803 838038 TBNVd (1 6 811 61811 £18 0.0 SflflflfiEiiflflflHS) SNJWGEIIHJUHS) dant-treated frozen carp lOXl Figure 19.-Taste panel score for rancid odor and flavor of ant fillets with and without skin. 138 antioxidants preserved acceptability for 6 months compared to 8 months for FreezgardO treated fillets. Mullet fillets dipped for one minute in TBHQ, sodium EDTA, and ascorbic acid, singularly or in various combina- tions, resulted in a stable product with little rancid flavor (Deng gt gt., 1977). They also showed that skinless fillets stored frozen for 9 months resulted in higher TBA numbers than fillets with skin. A similar trend in the control fillets was observed in this study, but not for antioxidant treated fillets with or without skin. This may be because of the increased exposure of muscle surface which facilitate penetration of antioxi- dants into the skinless fillets. Results of this study also indicate that the phenolic antioxidants which are not soluble in aqueous solutions, are less effective for fish stored in the form of fillets. Those antioxidants, which resulted in lower TBA numbers in mechanically deboned minced carp, resulted in comparatively higher TBA numbers than fillets treated with Freezgasz. The increased variation in TBA numbers from phenolic anti- oxidant treated fillets compared to mechanically deboned minced carp (presented earlier) may be the result of nonuniform penetration of antioxidants into fillets. Sodium erythorbate has been shown to extend shelf life of frozen Argentine hake fillets (Licciardello t 1., 1980), while ascorbic acid was found to be an 139 effective antioxidant in extending the shelf life of frozen chub (Greig, 1967). In the above studies correla- tion coefficients r= -0.767 andY=-0.72 were reported between panel acceptance scores and TBA numbers. While a correlation coefficient between TBA numbers and sensory evaluation values of -0.74 (p < 0.05) was calculated in the present study, it was not possible to accurately determine the exact level between acceptable and unac- ceptable rancidity level based on TBA numbers. Hence, the arbitrary sensory score of 2.0 was used as an index level for acceptance which translates to acceptable frozen storage quality of 8 months for fillets with skin and 6 months for fillets without skin. Effect of Storage Time and Antioxidants on Ratio of Unsaturated to Saturated Fatty Acids from Phospholipids Variations in ratio of total unsaturated to saturated phospholipid fatty acids in carp fillets at the end of 3, 6, 8, and 11 months of storage and treated with various antioxidants with and without skin are listed in Table 17. Control samples of fillets with and without skin had no significant differences in their saturation ratios. However, irrespective of treatment, fillets without skin tended to retain a higher ratio of unsaturated to saturated phospholipid fatty acids. No significant differences in ratio between fillets with and without skin were found in 140 .mo.ova an can—cu a cw monocoee_u acoueu.cmwm mueomwcnma C .mupa_—o;qmogn Ea mu*uu huuuu vouocauom _ouOu on vmuocauomca pouou ovum; mo m:_c>_ me.— mm._ «0.. po.~ ~<._ mm.— —~.— mo._ <9 + an + »:m + z : z_meoz .- om: z_xm p=o=h_z hu44_m 2.xm =h_z hm44_m i.om com o .2 u _E _ u+oa+§m+z m: z_meo<< <<44_: cm: 43 z_xm hzozp_3 hu44_u z_xm =h_z hm44_u .om cam 144 carp. Effect of Storage Time and Antioxidants on Shear Va1ues of Fish Ge1s Prepared from Hand Fi11eted Carp Mean shear va1ues (kg force/g) of fish ge1s prepared from hand fi11eted carp with and without skin and treated with various antioxidants are 1isted in Tab1e 18. Shear va1ues decreased with storage time with maximum decrease for contro1s. Significant1y higher shear va1ues were found for samp1es of fi11ets with skin than without skin when treated with Freezgardg, about 10, 13, and 30 percent for the storage periods of 3, 8, and 11 months, respecti- ve1y. Lower shear va1ues were found during the first 3 months storage. A maximum of about 6.8 percent increase in shear va1ues was recorded for fi11ets treated with other antioxidant treatments. Fi11ets stored without skin and treated with Freez- garcfS had significant1y higher shear va1ues as compared to contro1 after 11 months of storage. A11 samp1es of fi11ets without skin from a11 treatments had 1ower shear va1uesthan the counterparts from fi11ets with skin. During the first 8 months of storage, no significant differences due to treatments were observed. 145 Tab1e 18. Effect of frozen storage and antioxidant treatment on shear va1ues of fish ge1s prepared from hand fi11eted carp. Fi11ets with Skin Fi11ets without Skin Treatment Storage (months) Storage (months) 3 8 11 3 8 11 Contro1 1.79 1.47 1.12 1.68 1.32 0.96 Freezgard‘3 1.98* 1.67* 1.48* 1.79 1.43 1.30* Tenox i” 1.76 1.55 1.20 1.62 1.34 0.98 BHA+BHT+PG+AA 1.76 1.57 1.16 1.65 1.30 1.00 BHA+BHT+PG+CA 1.78 1.56 1.14 1.60 1.32 0.89 1 Va1ues are mean of 2 determinations (kg force/g samp1e. * Denotes significant difference in a co1umn (p<0.05). 146 Effect of Storage Time and Antioxidants on Water Honing Capacity of Fish GeTS Prepared from Hand Fi11eted Carp Percent extracted 1iquid from fish ge1s during centrifugation were used as a measure of waterhoning capacity. An increased percentage of extracted 1iquid indicates a Tower water honing capacity. These percen- tages for the antioxidant treated samp1es from f111ets with and without skin at the end of 3, 8, and 11 months of storage are 1isted in Tab1e 19. Freezgard‘D treated samp1es resu1ted in significant1y higher waterho1ding capacity (1ess extractab1e 1iquids) except for fi11ets without skin stored for 11 months. The fi11ets treated with inso1ub1e pheno1ic antioxidants had sTight1y higher or simi1ar waterhoning capacities re1ative to those of the contro1 samp1es. In genera1, the fi11ets without skin resu1ted in 1ower waterho1ding capacities than the fi11ets with skin throughout frozen storage. Texture ana1yses in this study, inc1uding protein so1ubi1ity, shear force and waterho1ding capacities, indicated that an improved qua1ity of frozen fish can be maintained by storing the carp as fi11ets, rather than as mechanica11y deboned minced carp (not significant1y). An indirect re1ationship between 1ipid oxidation measurements and texture re1ated measurements was observed. For examp1e, there was an increase in TBA numbers and decrease in unsaturated to saturated fatty acid ratio of 147 Tab1e 19. Effect of frozen storage and antioxidant treatment on water ho1ding capacity of fish ’ ge1s prepared from carp fi11ets with and without skin. Fi11ets with Skin Fi11ets without Skin Antioxidant Treatment Storage (months) Storage (months) 3 8 11 3 8 11 (% extractab1e 1iquid) ' Contro1 17.8 20.0 23.1 18.4 21.7 23. FreezgardE 16.4* 18.3* 20.8* 17.2* 19.7* 22. Tenox 29 17.5 19.5 23.0 13.4 21.2 23. BHA+BHT+PG+AA 17.2 19.0 22.6 18.0 20.9 22. 0010010000 BHA+BHT+PG+CA 17.3 19.2 22.1 18.1 21.2 22. 1Va1ues are mean of 2 determinations (% extractab1e 1iquid). *Denotes significant difference in a co1umn (p<0.05). 148 phospho1ipids with increase in storage time. These factors appear to be re1ated to a decrease in so1ubi1ity of protein fractions, waterho1ding capacity, and shear va1ues. Cheng gt al. (1979a) reported that waterho1ding capacity, shear va1ues, and protein so1ubi1ity va1ues decreased with an increase in frozen storage time. The decrease in soTubi1ity of myofibri11ar (sa1t so1ub1e) fractions from carp tissue was higher than that of sarco- p1asmic fraction. However, sarcop1asmic fraction so1u- bi1ity in mechanica11y deboned minced carp decreased rapid1y during frozen storage, compared to that of fi11ets. This may be due to increased exposure of sarc0p1asmic protein due to mechanica1 breaking of tissue during the deboning process. This damage is minimum during hand fi11eting. Those antioxidant treatments which resu1ted in 10w TBA numbers, or affected a retention of a high ratio of unsaturated to saturated phospho1ipids, a1so resu1ted in retentions of high amounts of so1ub1e protein fractions, high waterhoning capacity, and high shear va1ues. This shows an indirect re1ationship between 1ipid oxidation changes and texture re1ated parameters. Lee and To1edo (1976) and Cheng gt al. (1979a) discussed the va1ue of protein so1ubi11ty, water- ho1ding capacity, and shear va1ues of frozen fish in predicting genera1 textura1 qua1ity of fina1 product. 149 Seasona1 Variations The carp used in this study were harvested from the Saginaw Bay area of Lake Huron. Carp harvested around the 15th of each month from June 1980 through May 1981 were ana1yzed for proximate composition and 11P1d composition. During this study, due to adverse weather conditions and/or no fish harvest, samp1ing was not done from January through March1981. The mean 1engths and weights of carp samp1ed during different months are 1isted in Tab1e 20. Mean 1ength for the samp1e was 67 t 3.0 cm and weight was 3.88 i 0.25 kg. Seasona1 Proximate Composition of Carp F1esh Variations in proximate composition of carp f1esh during the period of study are summarized in Figure 21. Average va1ues of ash content varied from 0.86 to 1.06 percent, fat content from 7.49 to 16.8 percent, protein content from 12.1 to 17.65 percent and moisture from 69.25 to 75.43 percent. An inverse re1ationship was found between protein content and 1ipid content with the maximum protein content of about 16.92 and 17.65 percent for the carp harvested in October and December, respective1y, whi1e the 1owest tota1 protein content (about 12.1 percent) was found in carp harvested in Ju1y. Lowest tota1 1ipid content was observed for carp harvested in October and Apri1 with 7.60 and 6.45 percent, respective1y. In the 150 Tab1e 20. Length and weight of carp samp1es during various months. Month of Harvest Length Height (cm) (k9) June 1980 69 t 4.1 3.65 i 0.31 Ju1y 1980 66 i 3.3 3.71 i 0.42 August 1980 70 i 5.0 3.81 i 0.20 October 1980 68 i 1.5 4.17 i 0.33 November 1980 70 i 3.0 4.30 i 0.25 December 1980 69 i 6.1 4.00 i 0.16 Apri1 1981 60 i 6.5 3.58 i 0.17 May 1981 65 i 5.1 3.71 i 0.14 Note: Mean i SD for n = 4 to 7. 151 .ggmu mo cowpvmoasou mmePxoca cw cowpmvcm> chommmm .FN meamvd >oz puo mum m=< Jaw 22w 1 m6 o4 :w< m w % S m w #5. NH m. m... 3 m S 23.—baa ”H an /\ $55 8: mm 152 month of_Ju1y the carp f1esh contained the highest percen- tage fat accounting for 16.85 percent of the tota1 f1esh. Highest and 1owest moisture 1eve1s of f1esh were observed during the months of June and Apri1 accounting for about 69.25 and 75.43 percent, respective1y. Genera11y, no significant differences in tota1 ash content were observed due to seasona1 variation. In aquatic anima1s, factors such as avai1abi1ity of food, temperature of water, differences in diet and reproductive cyc1e stage are important factors that affect proximate composition of a species (Sidewe11, 1976; Stansby and Lemon, 1941; Leu gt al., 1981). The 10w 1eve1s of tota11ipidsobserved in Apri1 might be the resu1t of post-spawning stage of carp. This minimum tota1 1ipid content was reported by Hardy and Keay (1972) and Stansby and Lemon (1941). Tota1 1ipid content of carp, and most fish in genera1, is dependent on size and age of the fish. By se1ecting fish with a sma11 range in 1ength and weight, variations due to differences in size can be minimized. Using this se1ection method, fish of about the same age were obtained and possib1e composition variations due to age were minimized. Lipid Composition of Carp F1esh Five c1asses of 1ipids consisting of phospho1ipids, mono, and dig1ycerides, trig1ycerides, free fatty acids, and tota1 choTestero1 were separated by TLC from f1esh 153 1ipids of carp samp1ed during various months. The distri- bution of c1asses of 1ipids during various months of ana1yses is shown in Tab1e 21, The variations in tota1 phosphoTipids, free fatty acids, and tota1 choTestero1 were sma11 when expressed as percentages based on tota1 1ipid content. However, 1arge variations in phospho-h 1ipids expressed as mg/100 g wet tissue were observed with 1owest va1ue in fish harvested in Apri1 (883 mg/ 100 of tissue). The ratio of phospho1ipids to other c1asses of 1ipids were simi1ar for each month. Signifi- cant variations in tota1 mono and dig1ycerides and trig1ycerides from carp tissue were observed. A significant1y higher trig1yceride content was observed in carp harvested during the months of September through December than from other months. This may be due to variations in energy metabo1ism before the spawning season and a1so may be due to decreasing water temperatures. A simi1ar resu1t was imp1ied by Hatanabe and Acman(1977) regarding oysters. Since phospho1ipids are important in functions other than energy transformation,comparativer 1esser changes in phospho1ipid fractions can be expected than for nonpo1ar or neutra1 1ipid fractions which p1ay an important roTe in energy metabo1ism (Stewart 33 11., 1972). Simi1ar exp1anations can be attributed to the data on variation in phosphoTipid c1asses (Tab1e 22). 154 .mo.OVa cmzo_ A—ucauwu_cm_m. .mmmmzucmcon ov_m:* m? uvq__ pouou mo amoucougua .mamm_u o oo_\cowuuocc u_a_p we com: ”muoz Akm.nv age “No... Nam Aqm.omv _ONm Ae~.o_v Noe, Ao~.m_v cam. ».z Ame.mv NON Aw.... ne~ Am~.nev ~omn Aeo.av mom Aoo.m.v Nam _.La< Aam.nv MAN A_m.nv N.— A_n.n~. “smm .Am..mv ovm Am..e_v mm.F 2.85.0.9 ”Nm.nv m.” Ac..n. a.” Am_.msv mama .Awm..v ~m. “so.m_v men. .masmsoz Am..m. .m~ A~_.m. on~ “88.nhv monm .Aom..v am. .om.¢_v m~o_ Lunouuo A~8.n. omN Acn.mv m- Aon.n~v _Nom .ANn... can A8..m.v mm,— Lmasmuaam Rom.nv 5.. A¢~.m. co. .Ao~.nc. wens flam.n_. «em. .w..mpv -m_ ..=m=< Amo.nv mum Amn.mv .mw .Aom._ov «aka Aon.e.. .m- A.M.“.v ooNN »_=a A_o.nv mpm Awm.nv a“. .Aom.mmv meow A~m.n_v ooa. Amm.o.v m~m~ mesa pocoumopogu _ouop pacemaom .—~ m_nmp 155 Fou_mo=_,»a...;amoza . H. ocmE~.o:osuopxu.uugamoga 1 ma c.g_po.ucau 1 u :.pa»eoa:¢:am a 2m .=._o;u.»u.uagamoga . u. .m:o_uoc_scoumu m mo some mamm_u aoofixoe use mm:_o>~ oo.oo~ amo~ oo.oo~ 0mm 00.00“ me- oo.oo~ -m_ oo.oo— moo” oo.oo~ mo—~ oo.oo~ ~mw~ 00.00“ mmum oo.oo~ mwmm pouch flafllwflfllflflflmflwflmmflmiflflmflgW 2.5. mm.~ ~m~ me.~ mm mm.@ mm sm.w m- mm.~ mm o~.m mm mm.“ sea om.o om~ nv.n on" u e~.~ meg mo.~ no cw.w mo~ NH.w o- cm.m mm ~o.w no" wo.m om” NM.“ mmfi mo.n cod Ha m~.o~ mow n~.- mm mm.~_ cm~ mo.~_ m¢~ mm.- m- m~.o~ aw" _v.o~ mom om.o~ mam m~.o~ wmm ma ~o.n~ oww mo.m_ ~o~ mv.m~ va om.- m- mo.- o- mm.- ~v~ c~.- ovm mm.- 9mm om.- «MN zm mm.- mmo vw.- _ow mm.o~ mmw oo._~ mow oo.o~ efiw No.- _mm mm.- mow mo.m~ on cm.~m mmm ma o~.mn "an o~.mm mmw mm.~m mmm N~.~m omv mm.~m mmm m~.vm ofiv mm.cm nmo m~.mm mko ~m.cm mom um E _... moo—\me Emacs—BE a: a 0335 <._ k. a coin... a... I“. m 093.: f. a 8:95 <1. w a 8:9: 5 a. a 8:9. <0 n a 819.. 133: uogamoga xoz __La< .uoo .>oz .uuo .uamm umaoa< span mend 1011110011... n1” 9 .8" .5 .I u. . ..1..1. 1n n14. 1 1.1.1.1; 11¢ 1 A filial-1.— w 3'11"”. 11.1 a“ «fig 10:... 11%|. l ’81"”fl1l1l..1.. 4 Cal 1 I. |.l 13W... .cocax ago; soc. umumm>ccz acou soc. muwa.P cmmpe go comuwmoaeou v_a__ocnmoga c_ co_um_ca> _acOmmmm11.- mquh 166 .Ammamm_u m ooH\msv mucucvsgmumu N do come mcm won—u> "muoz “mom” mmmm New“ @qmm New“ “mks ~_o~fi mmom~ ommeg 4oz .uuo .uamm umzma< apaw mean uuau :5sz 8.3 :5... “3333.2 33 =5... 3.3.: :3: .6 5.5.3858 Bum x33 5 5.53;: .2533 11.3 “.55 157 Fatty acids of tota1 1ipids from carp f1esh are 1isted in Tab1e 23. Twenty-five different fatty acids with carbon chain 1engths of 14 to 22 were identified. The average fatty acid composition was made up of 26 percent saturated fatty acids, 38 percent monoenes, and 36 percent poTyenes. The major saturated fatty acid was C16:0 (16 percent of tota1 Tipid), fo11owed by C18:0 (4 percent) of tota1 fatty acid content. Major monoenes were C16:1 (12 percent) and C18:1 (23 percent) of tota1 1ipid fatty acids. Major po1yenes were C18:2, C18:3, C20:4, 020:5 w 6 and C22:6 which accounted for more than 26 percent of tota1 1ipid fatty acids. No significant differences were found in tota1 saturated fatty acids among seasons. Tota1 monoenes showed a decreasing trend from June to September foTTowed by an increasing trend unti1 December. Hhen each major monoenoic acid C16:1 and C18:1 were considered, this decreasing trend was noticeab1e. Tota1 po1yenoic acids showed an increasing trend from June through October f011owed by a dec1ine in November and then an upward trend. Ota and Yamada (1974) reported a decrease in C16:1, C18:1, C18:2, C18:3, acids in winter and an increase in 020:4, C20:5, and C22:6 acids in f1esh 1ipids of Masu Sa1mon. They a1so noticed that these changes varied wide1y. This change in fatty acids from summer 158 to winter may be due to changes in specific fatty acids as identified in this study. These changes in this study may have been affected by ages of fish from 4 to 6 years, and it is known that wider variations due to season occur in young fish than 01d fish, other factors being equa1. Fish from different habitats are known to show variations in composition due to seasona1 changes. Deng gt al. (1976) reported that the unsaturated fatty acid content increased from summer to winter in mu11et from gu1f coast areas whi1e the unsaturated fatty acids in the same species from another area a few hundred mi1es away (0akhi11) was significant1y different. These authors recommend a wide and extensive sampTing for such a study. Tota1, po1ar, and nonpo1ar Tipids of shortneck c1am musc1e have been reported to show a decrease in amount with decrease in water temperature (Ueda, 1974). A simiTar trend was observed in carp f1esh 1ipids in the present study. SUMMARY AND CONCLUSIONS Composition and storage stabiTity of mechanica11y deboned minced carp and hand fi11eted carp harvested from the Great Lakes were eva1uated during 1ong-term frozen storage. Emphasis was p1aced on changes in Tipids and their fatty acids. Stabi1ity characteristics were eva1uated using TBA ana1ysis as an index of 1ipid oxida- tion. Changes in various c1asses of 1ipids and fatty acids, shear va1ues, waterho1ding capacity and protein so1ubi1ity were determined. Suitabi1ity of one or more antioxidant formu1ations for increasing the she1f 1ife of carp during 1ong-term frozen storage was determined. Possibiiity of varying storage environments as added advantages to decrease 1ipid oxidation was eva1uated. Seasona1 variation in proximate composition and detai1ed 1ipid composition of carp harvested from Lake Huron was determined. A summary of research and recommendations based on this study are as fo11ows: 1. A higher usab1e meat yie1d of near1y 40 percent can be achieved by mechanica11y deboning carp as compared to 29 percent for hand deboning and mincing. This increased yie1d is especia11y advantageous when the commercia11y harvested carp f1esh is to be incorporated 159 160 into restructured products. Hechanica1 deboning resu1ted in higher fat content of the product and a 1ower protein percentage of minced f1esh when compared to hand deboned and minced f1esh. An increase in tota1 fat was the resu1t of an increase in various classes of 1ipids inc1uding phosphoTipids, mono- and dig1ycerides, trig1ycerides,‘ free fatty acids and tota1 cho1ester01. The hand deboned f1esh contained a 1ower percent of neutra1 1ipids compared to mechanica11y deboned f1esh; this was the resu1t of se1ective remova1 of depot fat during the hand deboning process. A higher 1eve1 of free fatty acids was found in hand deboned f1esh compared to mechanica11y deboned f1esh. This may be the resu1t of product temperature increasing during processing and an increased processing time during hand deboning. A comparison of carp f1esh after hand deboning and mechanica1 deboning did not show substantia1 differences between various c1asses of phospho1ipids which inc1uded phosphatidy1— cho1ine, phosphatidyTserine, sp1ingomye1in, phospha- tidy1ethan01amine, phosphatidyTinosito1, and cardioTipin. Twenty-five different fatty acids were identified inc1u- ding fatty acids in the range of C14 to C22 and O to 6 doub1e bonds. In both neutra1 and po1ar 1ipid fractions from mechanica11y deboned and hand deboned samp1es, no 519- nificant differences in presence or absence of specific fatty acids were observed. Major phospho1ipid fatty acids were 161 C14:0, C16:0, C16:1, C18:1, C18:2, C18:3, C20:1, 020:4, C20:5 w 6, C20:5 w 3, and C22:6. Mechanica11y deboned f1esh contained 1ower amounts of extractab1e sarcop1asmic, myofibri11ar, and stroma1 protein nitrogen than hand deboned f1esh. Mechanica11y deboned carp had 1ower shear va1ues and was rated Tess firm by a taste pane1 in comparison to hand deboned carp. Mechanica1 deboned f1esh a1so had 1ower waterhoning capacity than hand deboned f1esh. 2. This conc1usion was based on the resu1ts from TBA ana1yses, changes in various 1ipid c1asses, retention of unsaturated fatty acids of phosphoTipids, waterho1ding capacity, shear va1ues, extractabiIity of protein, and taste pane1 data to identify rancidity. Tenox 2® , BHA + BHT + PG + AA and BHA + BHT + AA were satisfactory antioxidant formu1ations for 1ong—term frozen storage of mechanica11y deboned minced carp. These antioxidants maintained satisfactory keeping qua1ity of mechanica11y deboned minced carp for at 1east 8 months. 3. Air, nitrogen backf1ush and vacuum were used as different environments to store mechanica11y deboned minced carp. Nitrogen backf1ush treated samp1es had s1ight1y 1ower TBA va1ues than vacuum and air packaged samp1es. The cost invo1ved in bu1k storage in presence of nitrogen may 1imit this advantage, especia11y for carp which is a 10w va1ue fish. 162 4. StabiTity of hand fi11eted carp during frozen storage was eva1uated. Fi11ets with skin were more stab1e than fi11ets without skin. Treatment of fi11ets with nonaqueous soTubTe antioxidants resu1ted in 10w penetration and poor distribution of the antioxidant. Freezgard'D was an exce11ent antioxidant for carp fi11ets during frozen storage. It was predicted from this study that she1f 1ife of 6 and 8 months for carp fi11ets with and without skin can be achieved in frozen storage using Freezgardg. Overa11 qua1ity of deboned minced f1esh from frozen stored fi11ets may be superior to that of products from mechanica11y deboned minced frozen stored carp. This improved qua1ity may not be advantageous from the point of view of increase in space and energy invo1ved in frozen storage of fi11ets. 5. Seasona1 variations in proximate composition and detai1ed tota1 1ipid fatty acid profiTes were determined. Highest tota1 fat content was observed during the months of May and Ju1y whi1e a 1ower range in tota1 1ipid percentages was observed during October through Apri1. One practica1 imp1ication of this observation may be that the carp harvested during the months of May through Ju1y wi11 require an additiona1 process 1ike washing of the mince to reduce tota1 1ipid content for further storage or processing. NonpoTar 1ipids contributed to the vari- ation in tota1 1ipids. Ratio between type of fatty acids 163 and qua1ity of fatty acids remained about the same irre- spective of seasona1 variation. Variations were found in tota1 saturated fatty acids (major ones were 14:0, 16:0, and 18:0), tota1 monoenes (major ones were 16:2 and 18:1) and tota1 poTyenes (major ones were 18:3, 20:4, 20:5 w 6 and 22:6). APPENDIX 164 22 9ms:zz EMS:ZZ‘: 9:0: +o:zz £28t§ ‘30: ‘— I:st—=: 1:95: 0:9t ‘ asmdsaa 3 1 9 50 40 30 TIME(MIN) Typical GLC chromatogram of fatty acis from total lipids in carp f1esh A1. 164 28 9mS=ZZ ems:zz-=: 9:0: +o:zz £:8t+ I30z1— flat-g: “931* 039! " asnoasaa 3 1 g 50 40 30 TIMEUVIIN) Typical GLC chromatogram of fatty acis from total lipids in carp flesh A1 165 GDLESTEROL CHOLESTANE RESPONSE GLC TIME (MIN) A2. Typical GLC chromatogram of total cholesterol found in carp flesh lipids 166 EWHEMTHIJOFIuNCHXTY Please read the fallouing before you start the evaluation. nuxamelxnngInquunndtn»:wfluheonhytheluflcn31hsnzi.RNIZD Motthepmdmtmmttheaccepubilityofmmitulf.co dhnmganianytuaulnu‘uxnnMItheizodmn:andxanktmelxumxnslxuudon the:nurfid‘UHRnimr!raneklodourcrdy. (I) ‘maaare1ghn51FUUR1qnced funniest saunas.insnse‘uunazandzumk 'uInufiarluncnatnsni Hidia‘wutiomllinecrithelturiaxnzn lhurnmdud behavrupnanntflx;the.um;ne.lx>rh131yomrlnuu0udth11sdpcfiflcurnde and:-IdpcnwaUH-beuann'UHndngcm’u¢>-qnes. (II) Alsoymareprwidedwithfaxmplesinmncupscoveradwith alunim foils, please snell fliese.a1eatatinemdnnkfliemanm ash!)the1nnn'pnxmdmn2asanxwe. GOHKEQGHfiKQCGE W Sawfle