.V. ....... Va. x.. w .819. “L fixatifln: ...h~.i .b . .,‘ JAM D '( .uou ..a.u.-u:'?i.c IIIIIIIII III H 293 00577I1476 LIBRARY Michigan State University I II II II III IIII II This is to certify that the thesis entitled ANTIOXIDANT ROLE OF NITRITE IN MEAT SYSTEMS presented by Linda Anne Freybler has been accepted towards fulfillment of the requirements for MaSterS degree in FOOd SCience . I Iméw Major proamr (/ Date 5/‘I/59 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES :- RETURNING MATERIALS: Place in book drop to remove this checkout from your "record. FHVES will be charged if book is returned after the date stamped below. V~¢N¢w¢fitfi .n. .. ’.,‘ "s i 1? .~~"‘Iq JAN I 9m .’|' l; I 1, 22“.- A u. 1... l‘:' "i ..| f g If: fipn , w «7 ’ 'zuL' Q I I 3 l, bk.) w J‘ ,t g, _. ANTIOXIDANT ROLE OF NITRITE IN MEAT SYSTEMS BY Linda Anne Freybler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1989 Sb’flifil ABSTRACT ANTIOXIDANT ROLE OF NITRITE IN MEAT SYSTEMS BY Linda Anne Freybler Stabilization of unsaturated lipids and heme pigments in meats by nitrite was investigated. Microsomes, mitochondria, and phospholipids from nitrite-treated pork I were less susceptible to metmyoglobin/hydrogen peroxide- initiated peroxidation than those from pork without nitrite. Reaction of pork phospholipids and polyunsaturated fatty acid ethyl esters with dinitrogen trioxide increased their stability to peroxidative changes. Infrared analyses and the ability of the reacted lipids to nitrosate secondary amines indicated that dinitrogen trioxide may react with unsaturated lipids to form nitro-nitroso derivatives, thus stabilizing the lipids toward peroxidative changes. Nitrite stabilized the heme pigments and prevented the release of nonheme iron in the presence of hydrogen peroxide and/or heat. Nitric oxide myoglobin also prevented lipid oxidation in water-extracted pork systems. Addition of nitrite to. pork before and after cooking increased its cxidative stability. To my husband, Timothy P. Freybler, and my parents, , Florence A. and Eugene G. Schab ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation and thanks to Dr. J. Ian Gray, my major professor, for his guidance and involvement in this study and for his assistance and patience in preparing this thesis. I would like to express sincere appreciation to Dr. A.M. Pearson, Dr. J. F. Price, Dr. D. M. Smith, and Dr. A. Thulin for serving on the guidance committee. Special thanks is expressed to Dr. A. M. Booren for his assistance in obtaining the meat used in this study and to Dr. A. Asghar for his assistance and guidance in conducting the laboratory experiments. I especially want to thank Joan and Terry Preybler for the use of their computer equipment to publish this thesis and for all the free dinners. I am deeply grateful to my parents, Eugene and Florence Schab; my brother, Joe Schab; and my brother and sister-in-law, Paul and Brenda Schab for their continuous support and understanding throughout my academic career. Finally, I would like to express my deepest gratitude to my husband, Timothy, for his confidence in me and my abilities, his moral support, and his continual love. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE REVIEW Classification of lipids Oxidation of meat lipids Mechanism of lipid oxidation Initiators/catalysts of lipid oxidation Reduction of particle size Role of sodium chloride Inhibition of lipid oxidation Phosphates Metal chelators other than phosphates Ascorbate Maillard reaction products Synthetic antioxidants Natural antioxidants Nitrite Color stabilization Cured flavor Antimicrobial properties Inhibition of lipid oxidation Mechanism of nitrite as an antioxidant (1) Stabilization of heme pigments (2) ”Chelation” of trace metal ions (3) Formation of nitrite-derived antioxidants (4) Stabilization of lipids EXPERIMENTAL Materials Reagents Source of meat Methods Phase I: Stabilization of meat lipids with nitrite Isolation of lipids (neutral and polar) from pork iv Page vii viii Fatty acid analysis Isolation of microsomes and mitochondria from meat Measurement of subcellular membrane protein Reaction of unsaturated lipids with dinitrogen trioxide Preparation of liposome system for peroxidation assay Measurement of metmyoglobin/hydrogen peroxide initiated-peroxidation in membranes and lipids N-Nitrosation of morpholine by lipids reacted with nitrite or dinitrogen trioxide Infrared spectrophotometric analysis of lipids Phase II: Stabilization of meat pigments with nitrite Preparation of nitric oxide myoglobin Release of iron from nitric oxide myoglobin Preparation of water-extracted muscle fibers Effect of prooxidants, antioxidants and heating time on lipid oxidation and free iron content of water-extracted muscle fibers Heat treatments Effect of the addition of nitrite before and after cooking on lipid oxidation and the free iron content of pork loin Analysis of lipid oxidation by the TBA procedure Determination of free iron content Statistical treatment RESULTS AND DISCUSSION Phase I Effect of nitrite on the peroxidative stability of microsomes and mitochondria Comparision of the peroxidative stability of lipids from cured and uncured pork Stabilization of polyunsaturated lipids toward peroxidation N-Nitrosation of morpholine by lipids reacted with nitrite or dinitrogen trioxide Structure determination of lipids from cured and uncured pork and lipids reacted with dinitrogen trioxide 36 38 39 4O 4O 41 42 43 43 43 44 44 45 46 46 47 48 49 49 49 52 58 61 63 SU LI Phase II Release of nonheme iron from metmyoglobin and nitric oxide myoglobin in the presence of hydrogen peroxide Effect of prooxidants, antioxidants and heat treatment on the oxidative stability nonheme iron content of water-extracted muscle Effect of nitrite addition on lipid oxidation and nonheme iron content of pork loin SUMMARY AND CONCLUSION LIST OF REFERENCES vi 66 66 69 77 82 85 LIST OF TABLES Table Page 1 Unsaturated fatty acid content of 5 phospholipids in some muscle foods 2 Neutral lipid fatty acid composition 54 of cured and uncured pork 3 Phospholipid fatty acid composition 56 of cured and uncured pork 4 Formation of N-nitrosomorpholine on 62 heating morpholine with phospholipids form pork and lipids reacted with dinitrogen trioxide 5 Additional IR absorption bands found in 64 phospholipids from cured pork and lipids reacted with dinitrogen trioxide 6 Effect of hydrogen peroxide, metmyoglobin, 7O nitric oxide myoglobin, ferrous and ferric iron on the oxidative stability of lipids in water-extracted muscle 7 Effect of hydrogen peroxide, metmyoglobin, 71 nitric oxide myoglobin, ferrous iron and ferric iron on the nonheme iron content of water-extracted muscle after storage for 72 hours at 4‘0 8 Analysis of variance (ANOVA) of TBA values 77 from eight treatments of water-extracted muscle fibers 9 Effect of nitrite (200mg/kg) when added 78 prior to heating and after heating, on lipid oxidation of pork lion stored at 4°C. 10 Effect of nitrite (200mg/kg) added prior 79 to and after heating on the nonheme iron content of pork loin Vii e LIST OF FIGURES Figure 1 2 Overall mechanism of lipid oxidation Proposed mechanism for the reaction of nitrite with polyunsaturated lipids Metmyoglobin/hydrogen peroxide-initiated peroxidation in membranes isolated from cured and uncured pork Metmyoglobin/hydrogen peroxide-initiated peroxidation of neutral lipids from cured and uncured pork Metmyoglobin/hydrogen peroxide-initiated peroxidation of phospholipids from cured and uncured pork ‘ Metmyoglobin/hydrogen peroxide-initiated peroxidation of linoleic acid reacted with dinitrogen trioxide Metmyoglobin/hydrogen peroxide—initiated peroxidation of polyunsaturated fatty acids reacted with dinitrogen trioxide Effect of hydrogen peroxide on the release of nonheme iron from metmyoglobin and nitric oxide myoglobin viii Page 33 51 53 55 59 6O 68 INTRODUCTION Lipid oxidation is a complex process involving the reaction of unsaturated lipids with oxygen and the subsequent formation of lipid hydroperoxides. These hydroperoxides can degrade to form a variety of compounds including alkanals, hydroxyalkenals, ketones, and alkanes which can cause undesirable changes in the flavor of meat (Love, 1987). The development of off-flavors in meats as a result of oxidation can occur in two situations. One occurs during refrigerated storage of cooked meat while the other occurs during frozen storage of raw meat (Reineccius, 1979). Nitrite functions as an antioxidant in cured meat. Sato and Hegarty (1971) reported that nitrite added at the level of 2000 mg/kg meat completely eliminated oxidation in cooked beef, while 50 mg/kg meat significantly inhibited oxidation development. In addition, Morrissey and Tichivangana (1985) found that levels as low as 20 mg nitrite/kg meat significantly reduced lipid oxidation in fish, chicken, pork, and beef. The mechanism by which nitrite prevents or inhibits WOF is not fully understood. A number of mechanisms have been proposed for the antioxidant action of nitrite including stabilization of the iron porphyrins, 2 "chelation" of trace metals, formation of certain compounds possessing antioxidant properties and stabilization of meat lipids against oxidation (Igene et al., 1985; Morrissey and Tichivangana, 1985). Although all of these mechanisms may have a role in the antioxidant properties of nitrite, although the stabilization of the iron porphyrins and the meat lipids will be the focus of this thesis. This study will be conducted in two phases to meet the following objectives: 1. Demonstrate that nitrite stabilizes meat lipids, thus preventing their rapid oxidation during the storage of meat. 2. Demonstrate that nitrite stabilizes the heme pigments, thus preventing the release of nonheme iron into the meat system. LITERATURE REVIEW Chlmdfnzniancnfnunds Animal fats provide both desirable and undesirable qualities to meats and meat products. Lipids are not only involved in the desirable flavor and aroma of meats, but they also increase tenderness and juiciness (Pearson, 1966). In addition, lipids are involved in the development of rancid off-flavors during the storage of meat products. Therefore, it is important to understand the classification and structure of meat lipids to ascertain their involvement in the quality characteristics of meat. Most of the lipid in meats is present in the form of triacylglycerols. These are esters of glycerol to which are attached three fatty acid moieties. Triacylglycerols differ greatly in their composition due to the presence of fatty acids which vary in their degree of unsaturation. Because of this, triacylglycerols differ in their susceptibility to autoxidation. Generally, as the degree of unsaturation of the fatty acids increases, their susceptibility to oxidation increases (Labuza, 1971). Although triacylglycerols are present in greater amounts in meat products, phospholipids oxidize much more rapidly and are believed to be more important than triacylglycerols in the initial oxidation of meat lipids ’ 4 (Gray and Pearson, 1987). A phospholipid consists of a glycerol molecule with fatty acids esterified at two positions and a phosphoric acid molecule connected to a nitrogen-containing alcohol at the third position. Phospholipids are mainly associated with the cell membranes, especially the mitochondria, and contain a high percentage of polyunsaturated fatty acids (Labuza, 1971; Asghar et al., 1988). Any process that destroys membrane integrity causes the phospholipids to be exposed to oxygen, enzymes, heme pigments, and transition metal ions which can catalyze their oxidation (Pearson et al., 1977; Asghar et al., 1988). The total phospholipid content of muscle tissues varies I among species and from muscle to muscle in the same animal. (Dugan, 1987). Phospholipids in muscle foods are more susceptible than triacylglycerols to oxidation due to their higher degree of unsaturation (Giam and Dugan, 1965; O'Keefe et al., 1968). Triacylglycerols contain mainly C1,, C16, and C1, fatty acids that are either saturated or contain one or two double bonds. In contrast, phospholipids contain not only the 01,. C1, and C13 fatty acids but also considerable amounts of the more unsaturated C ,0 and C 2, fatty acids (Igene et al., 1981). As the degree of unsaturation increases, the susceptibility of the fatty acids to oxidation also increases. Chicken is most susceptible to lipid oxidation due to its high amount of polyunsaturated fatty acids followed by pork, beef, and lamb (Table l). 5 I Table l. Unsaturated fatty acid content of phospholipids in some muscle foods.‘ Content (%) Fatty acid Lamb Beef Pork Chicken C18:1 19.51 33.44 12.78 20.25 C18:2 18.49 10.52 35.08 14.20 C18:3 0.43 1.66 0.33 0.90 C20:2 0.34 0.69 ---------- 020:3 0.62 2.77 1.31 1.30 C20:4 13.20 8.51 9.51 11.60 C20:5 ----- 0.76 1.31 1.55 C22:4 ----- 0.88 0.98 2.10 C22:5 ----- 0.92 2.30 5.75 C22:6 ---------- 2.30 5.75 ‘Adapted from Melton (1983). Oxidation of Meat Lipids Lipid oxidation is a complex process involving the reaction of unsaturated lipids with oxygen and the subsequent formation of lipid hydroperoxides. Hydroperoxides are transient in nature and can degrade to form a variety of compounds including alkanals, hydroxyalkenals, alkenals, ketones, and alkanes (Love, 1987). These compounds can cause undesirable changes in the flavor of meat and some can react with protein, vitamins, and pigments to cause changes in the nutritive value and color of meat (Tappel, 1962; Love and Pearson, 1971). There are two situations in meats where lipids are involved in the development. One situation occurs during refrigerated storage of cooked meat, while the other involves the frozen storage of raw meat (Reineccius, 1979). Although low a temperature storage can delay the rapid development of rancidity (Igene et al., 1979, 1980), lipid oxidation can still occur at refrigeration and freezer temperatures (Love and Pearson, 1971). Cooked meat is very susceptible to lipid oxidation. The term "warmed-over flavor” (WOF) was first used by Timms and Watts (1958) to describe the rapid development of oxidized flavors in cooked meat during refrigerated storage. Phospholipids have been identified as being major contributors to WOF (Igene and Pearson, 1979). WOF can also develop in raw meat that is ground, flaked, or restructured. These processes disrupt the integrity of the muscle membranes and result in the exposure of the phospholipids to oxygen (Sato and Hegarty, 1971; Gray and Pearson, 1987). Mechanism of Lipid Oxidation Lipid oxidation occurs through a free radical chain mechanism that involves the following stages (Farmer, 1943): Initiation RH + 02‘---> R. + OH‘ (1) Propagation R. + Oz«—---> R00. (2) R00. + RH ----> ROOH + R. (3) Termination R. + a. ----> RR (4) R. + R00. ----> 3003 (5) R00. + R00. ----> noon + 02 (6) In the initiation step, a free radical (R.) is formed when a labile hydrogen is abstracted from a site on the fatty acid (RH). The propagation stage involves the reaction of the free radical with oxygen to yield a peroxy radical (ROO.). Subsequently, hydrogen is abstracted from another fatty acid molecule and the products formed are a hydroperoxide (ROOH) and another free radical (R.). This free radical is capable of perpetuating the chain reaction. Alternatively, the termination steps involve the formation of nonradical products which stop the reaction. After the hydroperoxides are formed, they will decompose and produce more free radicals that can participate in the chain reactions (Figure 1). The hydroperoxides can also decompose to form compounds that are responsible for off-flavors and off-odors (Gaddis et al., 1961; Horvat et al., 1969). These compounds include ketones, aldehydes, alcohols, hydrocarbons, acids, and epoxides. The hydroperoxides are also involved in reactions that cause the oxidation of pigments, flavor components, and vitamins (Dugan, 1976). Unsaturated Fatty Acid Ir ‘ '—* Free Ra lcals Oxidation of Pigments. \ Flavors and Vitamins , + Oxygen -_ Hydrope‘rcxides Breakdown Products (including rancid 1r off-flavor compounds) Polymerization lnsolubllization such as ketones, (Dark Color) of Proteins aldehydes. alcohols. hydrocarbons. acids. and epoxidee Figure 1. Overall mechanism of lipid oxidation (Labuza, 1971). '9 Initiators/Catalysts of Lipid Oxidation Many forms of iron are found in meats and have been described as an insoluble fraction, ferritin, hemoglobin, myoglobin, and low molecular weight or free iron (Hazell, 1982). The amounts and ratios of these various forms of iron differ between species of animals and between muscles in the same animal. For instance, myoglobin is the predominant form of iron in beef and lamb, whereas in pork, the insoluble iron and the myoglobin are found in the greatest amounts. The insoluble fraction is the predominant form of iron in chicken (Hazell, 1982). The total amount of the various forms of iron in meat changes upon heating. Usually, the amount of nonheme or free iron increases with a subsequent decrease in the amount of heme iron (Igene et al., 1979; Schricker et al., 1982). Both heme and nonheme iron have been implicated as initiators/catalysts of lipid oxidation. Myoglobin has been cited in many studies as playing an important role in the initiation of lipid oxidation in uncooked red meat (Greene, 1969; Rhee and Ziprin, 1987). Recently, Kanner and Harel (1985) and Harel and Kanner (1985a, b) have suggested that the interaction of hydrogen peroxide with metmyoglobin generates "activated" metmyoglobin which is capable of initiating lipid oxidation. Rhee et a1. (1987) suggested that "activated" metmyoglobin was more important than nonheme iron as an initiator of lipid oxidation in raw meat. 10' This was based on the observation that the optimum amount of hydrogen peroxide needed for the catalyzing activity of metmyoglobin was far below the hydrogen peroxide level needed to cause the greatest release of nonheme iron from the metmyoglobin. In addition, this amount of hydrogen peroxide is readily available in postmortem skeletal muscles of red meat animals. Hydrogen peroxide is obtained from the autoxidation of oxymyoglobin and oxyhemoglobin, leading to the formation of metmyoglobin, methemoglobin and superoxide anion which dismutates to form hydrogen peroxide (Misra and Fridovich, 1972). Therefore, it appears that metmyoglobin, when activated by hydrogen peroxide, is an important catalyst of lipid oxidation in raw meats. Kanner and Harel (1985) and Harel and Kanner (1985a; b) proposed a mechanism by which hydrogen peroxide- activated-metmyoglobin initiates membranal lipid peroxidation. Initially, oxymyoglobin autoxidizes to form metmyoglobin and hydrogen peroxide. This activates metmyoglobin to form the porphyrin cation radical (EV-Fe“-O) which initiates lipid peroxidation by a two-electron reduction of the initiator as shown below: I”-Fe“-O + RH ----> P-Fe“-O + R. + H’ R. + Ozi---> R00. R00. + RH ----> ROOH + R. P-Fe"-o + soon ----> P-Fe3’ + R00. + 'on R00. + RH ----> ROOH + R‘ lI Nonheme iron has been shown to be the major catalyst of lipid oxidation in cooked meat. Sato and Hegarty (1971) demonstrated that nonheme iron rather than heme iron was responsible for the rapid oxidation of cooked meat during storage leading to the development of WOF. They reported enhanced oxidation of the lipid components when iron (ferrous chloride or ferric chloride) was added to water-extracted muscle fibers. However, there was little oxidation in samples to which heme compounds were added. u Furthermore, the ferrous form of iron was found to have a greater prooxidant activity than the ferric form. Studies by Love and Pearson (1974) confirmed that nonheme iron is the major prooxidant in cooked meat. They reported that levels as low as 1 mg/kg of nonheme iron caused increased lipid oxidation in cooked water-extracted muscle fibers as compared to the control samples. Tichivangana and Morrissey (1985) using a similar model system also showed that ferrous iron was an effective catalyst of lipid oxidation. It should be noted that the studies of Sato and Hegarty (1971), Love and Pearson (1974), and Tichivangana and Morrissey (1985) were carried out on water-extracted muscle fibers. Rhee et a1. (1987) and Asghar et al. (1988) suggested that the water extraction process not only removes the pigments but also may have removed hydrogen peroxide from the system. Hydrogen peroxide, as stated earlier, will 12 enhance the prooxidant activity of metmyoglobin as seen in the raw system. Igene et a1. (1979) demonstrated that heating releases iron from the heme compounds. This was subsequently confirmed by Schricker et a1. (1982), Schricker and Miller (1983), and Chen et al. (1984). At high concentrations, hydrogen peroxide in also capable of destroying heme compounds, thereby releasing the iron, and thus increasing the prooxidant activity of the system (Igene et al., 1979) The level of hydrogen peroxide required to destroy the heme pigments is higher than the amount used by Rhee et a1. (1987). Transition metal ions can promote lipid oxidation by facilitating initiation or by promoting hydroperoxide decomposition. The following mechanism has been proposed for hydroperoxide decomposition by iron and other transition metal ions (Ingold, 1962): Fe“ + ROOH ----> Fe3+ + R0. + on‘ (7) Fe“ + noon ----> Fe” + R00. + n’ (8) Reaction (7) involves the interaction of ferrous ion with the hydroperoxide and results in the formation of a very reactive alkoxy radical (RO.) which can participate in the propagation reactions of lipid oxidation. This reaction proceeds rapidly. Reaction (8) involves the ferric ion and it is a relatively slow reaction. It produces peroxy 13 radicals (ROO.) which are less reactive than the alkoxy radicals. The rate of both reactions is greatly affected by chelation (Aust and Svingen, 1982). Iron is also involved in the initiation of lipid oxidation. As stated earlier, the initiation of oxidation in lipids that are free of hydroperoxides can occur by abstracting a labile hydrogen from an unsaturated fatty acid to form an alkyl radical. A number of initiators for this reaction have been suggested. These include the hydroxyl radical (RO.), perferryl or ferryl radical, and the di-iron- oxygen bridged radical (Asghar et al., 1988). Investigators who studied enzymic lipid peroxidation in liver microsomes believe that the extremely reactive hydroxyl radical (RO.) is formed in tissues either by the Haber-Weiss reaction or by the superoxide-driven Fenton reaction. However, most investigators hold the view that the Fenton reaction is responsible for hydroxyl radical formation (Fong et al., 1976, Gutteridge, 1984). Several investigators have proposed that perferryl iron promoted the initiation of lipid oxidation in both NADPH- and superoxide-dependent systems (Svingen et al., 1979; Tien and Aust, 1982). An electron from NADPH or the superoxide anion reduces ferric ion to ferrous ion which can complex with dioxygen to produce the perferryl or ferryl radicals. These radicals are assumed to be able to abstract hydrogen from lipids and thereby initiate lipid oxidation. Minotti and Aust (1987) proposed that a ferrous- I 14 dioxygen-ferric complex is responsible for initiating lipid oxidation. This requires the presence of both ferrous and ferric ions. This complex can be formed either from ferric ions in the presence of a reducing agent such as NADPH, superoxide anion, or ascorbate, or from ferrous ions in the presence of an oxidizing agent like hydrogen peroxide. The concentrations of iron, reducing agents and oxidizing agents are critical to this mechanism. Reduction of Particle Size Particle size of meat is reduced by a number of processes including grinding, chopping, flaking, and emulsification. Sato and Hegarty (1971) reported that chopping fresh meat and exposing it to air increases the rate of lipid oxidation. Any process which reduces the particle size of meat disrupts the integrity of the membranes exposing the lipids to oxygen, thereby accelerating lipid oxidation (Pearson et al., 1983). The membrane-bound lipids, which are high in phospholipids, are very susceptible to lipid oxidation as a result of their high content of polyunsaturated fatty acids (Igene et al., 1980). Role of Sodium Chloride Sodium chloride or salt can initiate color and flavor changes in meat, however, the mechanism is poorly understood (Pearson et al., 1977). Sodium chloride has been - 15 shown to function as both an antioxidant and a prooxidant depending on the meat system studied. Chang and Watts (1950) demonstrated that the extent of oxidation in the presence of sodium chloride was dependent on the moisture content of the meat. Mabrouk and Dugan (1960) demonstrated that increasing the level of dissolved sodium chloride decreases the amount of oxidation occurring in aqueous emulsions of methyl linoleate. The inhibition resulted from a decrease in the dissolved oxygen in the emulsions as the I salt content increased. Some investigators postulated that the prooxidant effect of salt may be due to the presence of trace metal ion impurities in the salt. However, Olson and Rust (1973) reported that there were no differences in taste panel scores between samples cured with purified low metal salt and those cured with unpurified salt. Salih (1986) also reported that fine flake salt (a highly purified salt) had a significant (p<0.05) prooxidant effect on turkey meat. Therefore, the mechanisms involved in the effect of salt on autoxidation are not fully understood. However, it has been shown that low levels of sodium chloride accelerate lipid oxidation and cause color deterioration. Inhibition of Lipid Oxidation There are a number of compounds that can be used to inhibit lipid oxidation in meat products. These include phosphates, ascorbate, Maillard reaction products, and both synthetic and naturally occurring antioxidants. These 16 antioxidants vary in their mechanisms and in their effectiveness. Antioxidants are often used in combination which gives even greater protection from lipid oxidation due to the synergistic effects involved. Phosphates Timms and Watts (1958) reported that pyro-, tripoly- and hexametaphosphates protected cooked meats against lipid oxidation. However, orthophosphate was not effective in preventing oxidation. They proposed that the mechanism by which phosphates function relates to their ability to chelate heavy metal ions. This prevents the prooxidant effect of the metal ions in meat systems, especially ferrous iron which is a major catalyst of lipid oxidation in cooked meats. Sato and Hegarty (1971) verified the antioxidant activity of phosphates in their studies with cooked ground beef. Metal Chelators other than Phosphates Other metal chelators (sequestrants) that have been studied in meat systems include ethylenediamine tetraacetic acid (EDTA) and citric acid. Igene et a1. (1979) reported that EDTA chelated most of the free iron in cooked meat and therefore reduced the extent of lipid oxidation. However, EDTA has not been approved for commercial use in meat products. Benedict et a1. (1970) showed that citric acid had a limited effect in decreasing 17- lipid oxidation in ground beef. MacDonald et al. (1980a) reported that 1000 mg/kg citric acid reduced TBA values in refrigerated hams but it was not as effective as 50 mg/kg nitrite. Ascorbate Ascorbate can either catalyze or inhibit lipid oxidation depending on the amount that is added to meats. At low levels (up to 100 mg/kg). ascorbic acid catalyzes lipid oxidation in meat systems (Timms and Watts, 1958; Sato and Hegarty, 1971) . Ascorbate is a reducing compound and is capable of rapidly reducing ferric ions to ferrous ions producing a "redox cycle", and thus stimulating lipid oxidation (Kanner et al., 1986). In contrast, higher levels (greater than 1000 mg/kg) of ascorbic acid prevent lipid oxidation and WOF development. Sato and Hegarty (1971) stated that ascorbic acid may function either by acting as an oxygen scavenger or by changing the balance of the ferrous and ferric forms of iron. Furthermore, phosphate and ascorbate together act synergistically to prevent lipid oxidation (Timms and Watts, 1958; Sato and Hegarty, 1971). Therefore, phosphate and ascorbic acid are typically used in combination in meat products. Maillard Reaction Products The Maillard reaction, which involves both amino and carbonyl compounds, can produce products capable of 18' inhibiting lipid oxidation. These compounds can be produced during the retorting of beef and usually develop in overcooked meats. Zipser and Watts (1961) reported that overcooked beef contained components which prevented lipid oxidation. In addition, Sato et a1. (1973) extracted low molecular weight, water-soluble compounds from retorted beef and these compounds possessed antioxidant properties. Huang and Green (1978) showed that the liquid produced from the high temperature cooking of meat retarded warmed-over-flavor in both raw and cooked beef. Melanoidins and premelanoidins resulting from heating various sugar-amine mixtures are the compounds responsible for the antioxidant properties (Bailey, 1988). These compounds include maltol (3-hydroxy- 2-methy1-4H-pyran-4-one) and reductic acid (2, 3-dehydroxy- 2-cyclopentene-1-one) (Sato et al., 1973; Bailey et al., 1987). Synthetic Antioxidants There are two types of antioxidants that can be added to meat products to prevent lipid oxidation. These are the naturally occurring and the synthetic antioxidants. The synthetic antioxidants include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tertiary butylhydroquinone (TBHQ). Addition of antioxidants is regulated by law in the United States. A number of studies have evaluated the synthetic antioxidants either alone or in combination with other antioxidants. 19‘ Chastain et al. (1982) investigated the effectiveness of BHA and TBHQ, alone and in combination in preventing lipid oxidation in restructured beef and pork steaks. The samples treated with the antioxidants had improved flavor scores, lower TBA numbers and less discoloration than the control samples. In addition, BHA was better in preventing discoloration while TBHQ was more effective in decreasing off-flavor development. Chen et a1. (1984) also studied the effect of various antioxidants coated on or mixed with salt. Tenox 4, which is a mixture of BHA, citric acid, and PG effectively inhibited lipid oxidation. In addition, a combination of ERA and BHT mixed with the salt completely inhibited lipid oxidation. natural Antioxidants Alpha-tocopherol is a natural antioxidant used in the processing of meat. One method of incorporating alpha- tocopherol into a meat product is by adding it to the animal's feed. This increases the tissue level of the antioxidant. This is more effectively accomplished in nonruminant animals such as pigs and chickens than in ruminants. However, Brekke et al. (1975) found this to be an ineffective process, even in the nonruminants. Lin (1988) studied the effect of alpha-tocopherol- supplementation of broiler diets and reported that alpha-tocopherol stabilized membranal lipids against 20— oxidation. A number of spices such as sage, paprika, and rosemary, possess excellent antioxidant activity ( Loliger, 1983). However, the only spice that is used commercially as an antioxidant is rosemary. Chang et a1. (1977) found that 0.02% rosemary extract was as effective as Tenox VI ( a mixture of BHA, BHT, propyl gallate, and citric acid) in animal fat and superior to Tenox VI in vegetable oils. In addition, Barbut et a1. (1985) found that the antioxidant effect of 20 mg/kg rosemary oleoresin was comparable to that of a commercial BHA, BHT, citric acid mix. Rosmarinus officinalis L. and its extract contain over forty-five compounds possessing some antioxidant activity (Loliger, 1983). Carnosol and rosmanol are two such compounds having high antioxidantactivity (Inatani et al., 1983). In addition, rosmarinic acid and carnosic acid have been identified as antioxidants (Loliger, 1983). Rosmaridiphenol and rosmariquinone have also been isolated from rosemary and were more effective than BHA in preventing oxidation in lard by Houlihan et al. (1984, 1985). These compounds are thought to be capable of terminating free radical reactions and quenching reactive oxygen species. Plant proteins also have been examined as possible antioxidants. Rhee and Ziprin (1981) studied the effectiveness of oilseed proteins in gravies and sauces for preventing oxidation in cooked meats. They found that glandless cottonseed, peanut, or soy protein, when used in a 21 - gravy at a 3% level, decreased lipid oxidation in meat patties covered with gravy. Ziprin et a1. (1981) also studied the antioxidant activity of various defatted flours, protein concentrates, and protein isolates that were obtained from glandless cottonseed, peanuts, and soybeans. All of these decreased lipid oxidation in cooked ground beef patties. More details about these and other natural antioxidants can be found in the review by Rhee (1987). Nitrite Nitrate was first used in the curing of meat as an accidental contaminant (Lawrie, 1966). Since nitrate was found to stabilize cured meat color, it was considered a desirable curing adjunct. Although sodium nitrate had been used in the curing of meat for many years, sodium nitrite was not allowed for use in the curing process until 1925 after studies by Lewis and Vose (1926) and Kerr et a1. (1926) revealed that it could be substituted for sodium nitrate. Sodium nitrite quickly replaced sodium nitrate as a curing adjunct because it provided increased rates of color fixation and decreased the quantity of additive required (Moulton and Lewis, 1940). Nitrite provides a number of functions in the curing of meat products. It can be used to (1) stabilize the color, (2) contribute to cured meat flavor, (3) inhibit the outgrowth of Clostridium botulinum spores, and (4) 22 inhibit lipid oxidation (Kramlich et al., 1973). Color Stabilization The mechanism by which nitrite stabilizes the color of cured meats is as follows (Kramlich et al., 1973): Nitrite ------- > NO + H20 N0 + Mb ------- > NOMMb NOMMb ------- > NOMb NOMb + Heat + Smoke ------ > NO-Hemochromogen Mb . myoglobin, NOMMb - nitric oxide metmyoglobin Nitrite in meat products breaks down to form nitric oxide which then reacts with myoglobin to form nitric oxide metmyoglobin. This can be reduced to nitric oxide myoglobin. During the curing process, heat and smoke are added which are necessary to form nitrosohemochromogen, the typical cured meat pigment (Kramlich et al., 1973). Hustad et a1. (1973) reported that as little as 25-50 mg/kg nitrite produced the characteristic cured color. However, under commercial conditions, a level up to 75 mg/kg is needed to provide a satisfactory cured color (Rubin, 1977). Cured Flavor Nitrite is necessary for the development of the characteristic cured flavor (Cho and Bratzler, 1970). However, the chemical changes responsible for the unique 23 flavor are not entirely understood. Several authors believe that nitrite affects the flavor by inhibiting oxidation (Sato and Hegarty, 1971; Love and Pearson, 1976; Fooladi et al., 1979; Igene et al., 1979). However, Cho and Bratzler (1970) have suggested that nitrite reacts with tissue components to produce the characteristic cured flavor. Many researchers have attempted to identify the volatile compounds produced during the cooking of cured meat (Lillard and Ayres, 1969; Mottram and Rhodes, 1974). Gray et a1. (1981) summarized these studies and concluded that most of the studies demonstrated that the presence of carbonyl compounds in the volatile fraction from uncured meats contributed to the difference in aroma between cured and uncured meat. However, no one compound or class of compounds has been identified as being responsible for the cured meat flavor. Antimicrobial Properties Nitrite also inhibits the growth of food poisoning microorganisms. Tarr (1941, 1944) reported that 200 mg/kg nitrite at pH 6 inhibited the growth of many kinds of bacteria including C. botulinum and C. sporogenes. The effect of nitrite in controlling C. botulinum growth and toxin formation is enhanced with increasing nitrite concentrations (Foster and Duncan, 1974). To provide complete inhibition of clostridial growth, 100 or 200 mg/kg nitrite should be added and the pH of meat should be lower 24 than 6.2 (Graver, 1974). More information on the antimicrobial effects of nitrite can be found in a review by Sofos et a1. (1979). Inhibition of Lipid Oxidation Nitrite functions as an antioxidant in cured meat. Sato and Hegarty (1971) reported that nitrite added at the level of 2000 mg/kg meat completely eliminated WOF in cooked beef. Even 50 mg/kg of nitrite inhibited WOF development in cooked beef. Fooladi et a1. (1979) studied the effectiveness of nitrite in preventing WOF in cooked beef, pork and chicken. Nitrite added at the level of 156 mg/kg inhibited WOF development. In fact, there was a two-fold reduction in TBA numbers for cured beef and cured chicken samples, while cured pork samples had a five-fold reduction in TBA values relative to uncured samples. MacDonald et al. (1980b) investigated the effect of various levels of nitrite (50, 200, and 500 mg/kg) on lipid oxidation in cooked hams. They demonstrated a significant reduction (p<0.05) in TBA numbers for all the hams. In addition, the 200 mg/kg and 500 mg/kg nitrite concentrations were equally effective in reducing lipid oxidation. Nitrite at the lowest level tested (50 mg/kg) was more effective than BHT and citric acid in preventing lipid oxidation. Morrissey and Tichivangana (1985) also tested the effect of various levels of nitrite on lipid ‘25 oxidation. They found that levels as low as 20 mg/kg significantly reduced lipid oxidation in fish, chicken, pork and beef products. Mechanism of Nitrite as an.Antioxidant The mechanism by which nitrite prevents or inhibits WOF is not fully understood. There have been a number of mechanisms proposed for the antioxidant action of nitrite. The following mechanisms, have been proposed by Igene et al. (1985) and Morrissey and Tichivangana (1985): 1. Nitrite forms stable complexes with the iron porphyrins in meat systems thereby inhibiting heme-catalyzed lipid oxidation. 2. Nitrite may "chelate" trace metals preventing their catalysis of lipid oxidation. 3. Nitrite may form certain compounds which possess antioxidant properties. 4. Nitrite may stabilize meat lipids against lipid oxidation. All of these mechanisms may play an important part in the antioxidant properties of nitrite. Each of them will be discussed in more detail below. (1) Stabilization of the Heme Pigments Zipser et al. (1964) first proposed that nitrite can form stable complexes with the iron porphyrins in heated meat systems,thus preventing heme-catalyzed lipid oxidation. Nitrite is capable of converting meat pigments to nitric oxide hemochromogen when it is added to meat before heating. 26 ' This increases the stability of the cured meat to lipid oxidation during storage. Chen et al. (1984) reported that the amount of nonheme iron in uncured samples increased with increased heating. However, samples containing nitrite showed no increase in nonheme iron content after heating. These investigators attributed this to the stabilization of the porphyrin ring by nitrite which prevented the release of nonheme iron. Morrissey and Tichivangana (1985) also investigated the effect of nitrite on the stability of heme pigments in an aqueous extract of beef muscle. After heating, extracts with no added nitrite contained higher amounts of free iron and corresponding decreases in heme iron contents relative to the unheated extracts. However, extracts containing nitrite did not show any changes in the amount of free or heme iron resulting from heating. The authors hypothesized that nitrite reacts with myoglobin to form nitric oxide myoglobin. Upon heating, the stable complex, nitric oxide hemochromogen, is formed, thus preventing the release of free iron. (2) 'Chelation' of Trace Mbtal Ions The second proposed mechanism by which nitrite inhibits lipid oxidation is that it may "chelate" trace metals. MacDonald et al. (1980a) proposed this mechanism to explain the inhibitory effect of nitrite on the activity of ferrous ions. They used a model system of an emulsion of 27 linoleic acid, phosphate buffer and Tween 20 to examine the effect of ferrous iron and nitrite on lipid oxidation. Results indicated that ferrous iron was a strong prooxidant of lipid oxidation. When low amounts of nitrite (<25 mg/kg) were added to the system, the amount of oxidation of linoleic acid was greatly reduced. In addition, a 1:1 weight ratio of nitrite to ferrous iron provided the greatest prooxidant effect. Morrissey and Tichivanagana (1985) also proposed that nitrite forms inactive "chelates" or complexes with nonheme iron, copper, and cobalt, thus reducing the catalytic activity of these ions and subsequent lipid oxidation. These investigators used water-extracted muscle fibers to which they added the various prooxidants (ferrous iron, copper, and cobalt) and differing levels of nitrite. TBA data revealed that ferrous iron had the greatest catalytic activity of all the prooxidants. Nitrite, at concentrations as low as 20 mg/kg, caused a significant (p<0.01) reduction in TBA values while 50 mg/kg nitrite caused a highly significant (p<0.001) inhibition of lipid oxidation in all systems tested. Apte and Morrissey (1987) further studied the complexing of nitrite with metal ions by adding nitrite to pork and fish systems either before heating, after heating, 24 hours after heating or 48 hours after heating. Their results indicated that nitrite was an effective antioxidant when added to meat systems before heating and even when ’28 added after heating. In those systems where nitrite was added after heating, the effect of nitrite could not be explained by the fact that nitrite stabilizes the heme pigments. Therefore, Apte and Morrissey concluded that nitrite must react with free iron, thereby preventing iron- catalyzed lipid oxidation. (3) Formation of Nitrite-Derived Antioxidants The third proposed mechanism involves the formation of certain nitrosyl compounds which have antioxidant properties. Kanner et a1. (1980) and MOrrissey and Tichivangana (1985) found that nitric oxide myoglobin possesses antioxidant properties. They reported that these properties were maintained in the presence of strong prooxidants such as metmyoglobin and free metal ions. Kanner et al. (1980) proposed that the antioxidant effect of nitric oxide myoglobin could arise from its ability to quench free radicals which lowers the amount of prooxidants in the system. This process causes the nitric oxide myoglobin to dissociate leaving metmyoglobin in the system. Alternatively, metmyoglobin may act as a hydroperoxide decomposer and also quench free radicals if a low amount of prooxidant is present. The work of Morrissey and Tichivangana (1985) supports this theory. Other reaction products of nitrite have been identified as having antioxidant properties. Kanner (1979) demonstrated an antioxidant effect for S-nitrosocysteine, 29~ which is a compound generated during the curing process. This compound inhibited lipid oxidation in an aqueous linoleate model system and in a turkey meat system. Kanner et a1. (1984) found that both hemin nitroxide and cysteine- iron-nitroxide have antioxidant properties. The addition of cysteine-iron-nitroxide to a model system containing cysteine-ferrous ion reduced beta-carotene oxidation by 77% after one minute. In addition, hemin nitroxide acted as an antioxidant in systems containing hemin or lipoxygenase (a prooxidant). These compounds are believed to act in a similar manner as nitric oxide myoglobin, by quenching free radicals that are involved in lipid oxidation. This decreases the amount of prooxidants in the system and subsequently decreases lipid oxidation. (4) Stabilization of Lipids Pearson et a1. (1977) suggested that nitrite may stabilize the membranal lipids against lipid oxidation. Many researchers have investigated the reaction of nitrite with unsaturated fatty acids, but few have studied it from the viewpoint of nitrite stabilization of meat lipids against oxidation. Mottram et a1. (1977) demonstrated that N—nitrosamine formation occurs primarily in the lipid phase when frying bacon. Frouin (1977) reported that an appreciable amount of bound nitric oxide was formed from the addition of nitrite to meat. Woolford and Cassens found also that nitrite was associated with the lipid fraction in 3o bacon. Cassens et a1. (1977) concluded from several studies that nitrite reacts with many components in bacon including the lipids. The possible role of unsaturated fatty acids in nitrosation was suggested by Goutefongea et a1. (1977). They demonstrated using NalsNoz, that nitrite reacted with the unsaturated lipids and reported that the amount of binding of sodium nitrite was related to the degree of unsaturation of the lipids. As the degree of unsaturation increased, the amount of Nalfiflk bound to the lipid also increased. They hypothesized that nitrite or a derivative reacted with one or more of the carbon-carbon double bonds. Work by a number of researchers supports this theory. Most of these studies have focussed on N- nitrosamine formation in bacon. Hotchkiss et a1. (1985) demonstrated that a lipid-bound nitrite compound existed in bacon and suggested that oxides of nitrogen formed from added nitrite reacted with unsaturated lipids during the curing process to form lipid bound-nitrite compounds. These compounds were capable of nitrosating amines and could not be extracted or purged from bacon fat. While studying the antioxidant properties of nitrite, Zubillaga et a1. (1984) reacted methyl oleate and methyl linoleate with nitrogen oxides generated from sodium nitrite and acid in the presence of air. They found that reaction products were formed through the addition of nitrogen oxides to olefins. However, these products were 31 not identified and their antioxidant activity could not be confirmed in this study. Zubillaga and Maerker (1987) later examined the antioxidant activity of polar lipids extracted from nitrite-treated meat. The antioxidant activity of the extracted polar lipids was 1.5 to 3 times greater than the activity of polar lipids isolated from untreated meat. The antioxidant activity of the polar lipids from cured meat was stable during storage. Separation of active polar lipids after conversion to methyl esters gave no one fraction in which the antioxidant activity was highly concentrated. However, the antioxidant activity was more highly associated with the polyunsaturated fraction of the methyl esters. These studies lead to the conclusion that nitrogen oxides can react with unsaturated lipids in model systems. However, no direct evidence has been presented to confirm the structure of the compounds formed. Ross et al. (1987) reacted dinitrogen trioxide with methyl oleate, methyl linoleate and methyl linolenate and were able to form reaction products which were capable of nitrosating amines. In trying to identify the reaction products, both high pressure liquid chromatography (HPLC) and infrared spectrophotometry (IR) were used. HPLC was used to separate the components and IR analysis was used to identify the structures. IR analysis showed the presence of dinitro and nitro-nitroso derivatives of the lipids. Therefore, dinitrogen trioxide can react with unsaturated lipids to form nitro-nitroso (pseudo—nitrosite) derivatives. 32’ Liu et a1. (1988) further investigated the chemistry of nitrosation by these nitro-nitroso compounds in a model system. They proposed the mechanism for the formation of the nitro-nitroso derivative as shown in Figure 2. Dinitrogen trioxide is formed when nitrite is added to cured meat and can react with the carbon-carbon double bonds of unsaturated lipids to form the nitro-nitroso derivative. This derivative can decompose during frying to release the oxides of nitrogen which are capable of nitrosating secondary amines. This mechanism has not yet been proven in a cured meat system. All of these studies have been able to identify reaction products between unsaturated lipids and derivatives of nitrite. Besides the work of Zubillaga and Maerker (1987), little work has been done to test the stability of these compounds to lipid oxidation. 33 NaNOz ' ——-- N203 + Na N203+-c-c- _.~. 33—]: .——. —I ./\I\ .//\I\-. Figure 2. Proposed mechanism for the reaction of nitrite with polyunsaturated lipids. EXPERIMENTAL Materials Reagents Methanol and dichloromethane were purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ) and were redistilled in the laboratory. Calcium phosphate, anhydrous sodium sulfate, n-heptane, and trichloroacetic acid were also purchased from J. T. Baker Chemical Co. Acetone, perchloric acid, hydrochloric acid, sodium nitrite, potassium chloride, lactic acid and sodium chloride were obtained from Mallinckrodt, Inc. (Paris, Kentucky). Celite 545, hydrogen peroxide, morpholine, and iron standard (certified atomic absorption standard) were purchased from Fisher Scientific Co. (Fair Lawn, NJ). Sigma Chemical Company supplied the fatty acid ethyl esters (linoleate, linolenate, and arachidonate), fatty acid methyl ester standards, glycerol, Trisma Base, metmyoglobin, ascorbate, histidine, and calcium chloride, and the boron trifluoride/methanol reagent. Magnesium oxide was obtained from Matheson, Coleman and Bell (Norwood, OH). Glass wool was supplied by Corning Glass Works (Corning, NY). 2- Thiobarbituric acid (TBA), ferrous chloride, and ferric chloride were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Tenox TBHQ was obtained from Eastman ‘34 35 Chemical Products (Kingsport, TN). Source of Meat The hams used in Phase I of the study were obtained from the Michigan State University Meat Laboratory. These contained the semimembranosus muscle from fresh hams. Pork loins for Phase II were obtained from a local supermarket. Methods This study was divided into two phases in order to ascertain the role of nitrite as an antioxidant in cured meat systems. Phase I concentrated on the stabilization of meat lipids with nitrite, while Phase II focused on the stabilization of the meat pigments with nitrite. Phase 1: Stabilization of Meat Lipids with Nitrite Isolation of Lipids (Neutral and Polar) from Pork Cured and uncured ham samples from three pigs were prepared in the Michigan State University Meat Laboratory to ensure that the cured and uncured samples came from the same animals. The cured samples had target levels of 156mg/kg nitrite, 550mg/kg sodium ascorbate, 2.0% salt, 0.67% sugar and 0.5% sodium tripolyphosphate added. The uncured samples did not contain nitrite but had the same target levels for the other additives. Here after, these samples 36 will be referred to as cured (containing nitrite) and uncured. The lipids were extracted from the ham samples using the dry column method of Marmer and Maxwell (1981). Meat (59). anhydrous sodium sulfate (259). and Celite 545 (159) were ground together in a glass mortar and transferred to a glass column (3.5 x 30cm) containing glass wool and log Celite and calcium phosphate (9:1 w/w). For the cured samples, 0.1g magnesium oxide was added to the trap to prevent the extraction of the cured pigment (Zubillaga and Maerker, 1984). The samples were lightly packed to obtain a i uniform bed. The neutral lipids were first eluted using 150ml dichloromethane. Then, a solvent system (150ml) of dichloromethane and methanol (90:10 v/v) was used to extract and elute the phospholipids (polar fraction) from the column. The solvent extracts were concentrated to approximately 15ml using a Buchi Rotavapor R rotary vacuum evaporator (Buchi Inc., Switzerland). The concentrates were quantitatively transferred to 25ml volumetric flasks and made to volume. The flasks and contents were flushed with nitrogen and subsequently stored at -20°C. Fatty Acid Analysis Fatty acid methyl esters of the phospholipids were prepared by the method of Maxwell and Marmer (1983) for gas chromatographic analysis. The boron trifluoride-methanol procedure of Morrison and Smith (1964) was used to prepare fatty acid methyl esters of the neutral lipids. Analysis of 37 the methylated fatty acids was performed using a Hewlett Packard 5840A Gas Chromatograph (Hewlett Packard, Avondale, PA) equipped with a flame ionization detector and a glass column (3m by 2mm i.d.) packed with 10% SP 2330 on 100/120 Supelcoport (Supelco, Inc., Bellefonte, PA). Nitrogen was the carrier gas with a flow rate of 25ml/min. The test conditions were as follows: initial temperature, 150°C; initial time, 1 minute; rate, 1.5°C / minute; final time, 10 minutes; final temperature, 220°C. The temperatures of the detector and the injection port were maintained at 300°C and 200°C, respectively. Retention times of the fatty acid methyl esters were compared to those of known fatty acid standards for the purpose of identification. Isolation of Microsomes and Mitochondria from Meat One hundred grams of meat (ground using a Rival Model 2300 grinder/food chopper, Rival Manufacturing Co., Kansas City, MO) from each of the three cured and uncured samples and 600ml chilled buffer (0.1M KCl in 0.025M Trisma Base and 0.2% glycerol, pH 7.4—7.5) were homogenized in a Waring commercial laboratory blender (Dynamic Co. of America, New Hartford, Conn.) for 2 minutes. The resulting slurry was centrifuged at 600 x G (Sorvall RC 2-B, Ivan Sorvall Inc., Norwack, Conn.) for 10 minutes at 4°C. This was done to precipitate the myofibrillar and connective 38 tissue protein fractions. The supernatant was collected and the pellet was dispersed in 400ml buffer and centrifuged again at 600 x G for 10 minutes. The supernatants from the two extractions were pooled together. The microsomes and mitochondria were separated using sequential centrifugation (Schenkman and Cinti, 1978). The mitochondria were separated by centrifuging the supernatant at 10,300 x G for 15 minutes. The mitochondrial fraction was removed and calcium chloride was added to the supernatant to obtain a final concentration of 8.0mM. This solution was centrifuged at 13,200 x G for 30 minutes to isolate the microsomal fraction. The subcellular fractions were not further purified to remove contaminating myofibrillar proteins. Measurement of Subcellular Membrane Protein Content The isolated fractions in pellet form (0.59) were dispersed in 30ml of 0.1M KCl/ 0.005M lactic acid buffer (pH 5.5) for use in the peroxidation assay. This was accomplished by sonicating for 25 minutes using a Bronson 2200 Sonicator (Shelton, CT). The protein content of the solutions was determined using the Biuret method of Gornall et al. (1949), as modified by Asghar and Yeates (1974). One ml of the dispersion was transferred to a test tube containing 4ml Biuret reagent (1.5g copper sulfate, 0.112g sodium potassium tartrate and 409 NaOH in 1000m1 distilled water). The color was allowed to develop for 30 min. 39 Dichloromethane (2m1) was then added and the test tube was vortexed on a Fisher Mini Shaker for 30 seconds. The samples were centrifuged for 10 minutes at 2000 x G in an IEC Clinical Centrifuge (Damon/IEC Division, Needham Hts., Mass.) The absorbance of the aqueous layer was determined at 540nm using a Bausch and Lomb 2000 Spectronic spectrophotometer (Bausch and Lomb, Co., Rochester, NY). The protein content was computed using a standard curve established for bovine serum albumin. The protein analyses were performed in triplicate. Reaction of Unsaturated Lipids with Dinitrogen Trioxide Dinitrogen trioxide was synthesized and reacted with fatty acid ethyl esters (linoleate, linolenate, and arachidonate) and phospholipids from uncured pork according to the procedure of Park and Williams (1972), as modified by Ross et a1. (1987). Perchloric acid (1.2N, 10ml) was purged with nitrogen and mixed with sodium nitrite (3.6mmole, or a ten-fold excess above olefin concentration) in a closed glass round bottom flask. The dinitrogen trioxide that was formed was purged with nitrogen through anhydrous sodium sulfate and trapped as a light blue solid in a glass trap that was immersed in liquid nitrogen. The reaction was allowed to proceed for one hour. The lipid (1.6mmole, in one ml of dichloromethane) was added to the solid dinitrogen trioxide inside the glass trap. The glass trap was placed 4o ' in a dry ice/acetone bath and the contents allowed to react overnight. Afterward, the solvent was removed using a rotary vacuum evaporator. Dichloromethane (10ml) was added to the lipid in the glass trap and evaporated twice to remove traces of oxides of nitrogen. The lipid samples were thoroughly washed 3 times with distilled water in a 125ml separatory funnel until they were free of residual oxides of nitrogen, as shown by the Griess/Ilsovay reaction (AOAC, 1984). Preperation of Liposome System for Peroxide Assay Liposomes for the peroxidation assay were prepared by dispersing a known quantity (0.35mg/ml solution) of the extracted phospholipids or fatty acid ethyl esters in 0.1M KCl /0.005M lactic acid buffer (pH 5.5) and adding 0.05% Triton x-100 to emulsify the system. The samples were emulsified using a sonicator. Measurement of Metmyoglobin/Hydrogen Peroxide Initiated-Peroxidation in Membranes and Lipids The oxidative stabilities of the microsomes, mitochondria, neutral lipids and phospholipids from both cured and uncured pork, and fatty acid ethyl esters before and after treatment with dinitrogen trioxide, were evaluated using the metmyoglobin/hydrogen peroxide assay of Harel and Kanner (1985). A 1:1 ratio of metmyoglobin and hydrogen peroxide was added (to achieve a final concentration of 30uM 41 for each additive) to the microsome and mitochondria dispersions, and liposomes to initiate the peroxidation reaction. The reaction was carried out in triplicate for 180 to 210 minutes in 100ml beakers incubated at 35°C in a Dubnoff Shaking Incubator (GCA/Precision Scientific). Samples were taken at various time intervals (0, 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, and 210 minutes) and the extent of lipid peroxidation determined using the TBA procedure of Buege and Aust (1978). Two ml aliquots of the reaction mixtures were removed from the beakers and placed in test tubes containing two ml of a TBA solution (10% w/v trichloroacetic acid (TCA), 0.4% TBA and 0.25N HCl). The test tubes were heated in boiling water for 15 minutes and then cooled in ice water. To precipitate the protein, the test tubes were centrifuged at 1000 x G (2000 rpm) for 10 minutes. The absorbance of the supernatant was measured at 532nm. The results for the microsomes and mitochondria were reported as nmole malonaldehyde/mg protein, while those for the lipids were reported as nmole malonaldehyde/mg lipid. The molar extinction coefficient used was 1.56 x 10 5 M '1 cm 4 (Buege and Aust, 1978). The peroxidation assays were replicated three times. N-Nitrosation of Morpholine by Lipids Reacted with Nitrite or Dinitrggen Trioxide To confirm that nitrite reacted with the various lipids to form nitro-nitrosite derivatives, aliquots (180ug) 42‘ of the lipids were heated with a secondary amine (morpholine, 500ug) and 0.6ul heptane in sealed glass ampules (Ross et al., 1987). The ampules were heated in a Reactitherm Heating Module (Pierce Chemical Company, Rockford, IL) at 130°C for 30 minutes. The reaction mixtures were cooled and brought to one ml with dichloromethane. Quantitation of the formed N- nitrosomorpholine was achieved using a gas chromatograph (Varian Model 3700)/ thermal energy analyzer system (Thermo Electron Corporation (TEA) Model 502, Waltham, Mass.) and a Hewlett Packard 3390A Integrator. The glass chromatographic column (3m by 2mm i.d.) was packed with 10% Carbowax 20m TPA on 80/100 Chromosorb WHP. The test conditions were as follows: initial temperature, 140°C; initial time, one minute; rate, 15°C/minute; final temperature, 180°C; final time, 7 minutes. Injection volumes were 5ul. TEA conditions were previously described by Ikins et al. (1986). Infrared Spectrophotometric Analysis of Lipids Infrared analysis was performed on the lipid samples with a Perkin Elmer Model 1330 infrared spectrophotometer to detect absorption bands characteristic of nitro-nitrosite compounds. Matching Perkin Elmer potassium bromide (KBr) cells were used with dichloromethane as the solvent. The samples were scanned from 600 to 4000 om". 43' Phase II: Stabilization of Meat Pigments with Nitrite Preparation of Nitric Oxide Myoglobin Nitric oxide myoglobin (NOMb) was prepared according to the method of Fox and Thomson (1963). Metmyoglobin was dissolved in 0.005M histidine/0.12M KCl buffer (20ml). Sodium ascorbate was dissolved in 8ml buffer, added to the metmyoglobin solution, and allowed to react for 10 min. Sodium nitrite in 8ml buffer was then added to the solution to convert the reduced myoglobin to nitric oxide myoglobin. The concentration of the reactants in the final reaction mixture were 30uM metmyoglobin, 6mM ascorbate, and 3mM nitrite. The solution was placed in dialysis tubing (10,000--12,000 M.W. limit) and dialyzed for 2 days to remove traces of nitrite and ascorbate. Afterward, the solution was removed and used as described below. Release of Iron from Nitric Oxide Myoglobin and Metmyoglobin Nitric oxide myoglobin was prepared as described above. Metmyoglobin solution was prepared by adding ~metmyoglobin to histidine/KCl buffer to make a final concentration of 30mM. The metmyoglobin and NOMb solutions were divided into 14ml aliquots and placed in 100ml beakers. The beakers were placed in a shaking incubator and held at 35°C. Hydrogen peroxide was added to the solutions to 44' obtain a final concentration of 30mM and 2ml samples were removed at various time intervals (0, 5, 10, 15, 20, 30, 60, 90, 120, and 180 min.). The free iron content of the samples was determined using atomic absorption spectrophotometry. Three replicates were performed with duplicate iron analyses. Preparation of Water-Extracted Muscle Fibers Three pork loins were obtained from a local supermarket, trimmed of excess fat, and ground. The pigments were removed from the ground pork using the distilled water-extraction procedure of Tichivangana and Morrissey (1984). Approximately five volumes of distilled water per 1009 meat were used to ensure total extraction of the pigments. Enough pork loin was extracted to provide approximately 25009 water-extracted muscle fibers for each replicated experiment. Effect of Prooxidants, Antioxidants and Heeting Time on Lipid Oxidation and Free Iron Content of Water- Extracted Muscle Fibers The water-extracted muscle fibers were thoroughly mixed by hand and divided into eight 3009 aliquots. The following additives were dispersed in 30ml distilled water and added to the muscle fibers: a. Control (No additive) b. Hydrogen peroxide (80umoles) c. Metmyoglobin (5m9/9 or 80umoles) 4s . d. Nitric oxide myoglobin (80umoles) e. Metmyoglobin (80umoles)/H 2O2 (80umoles) f. Nitric oxide myoglobin/H 2O2 (80umoles) g. Ferrous chloride (80umoles, dissolved in 30ml deareated distilled water) h. Ferric chloride (80umoles, first dissolved in 2.5m1 lactic acid). The fibers and the reactants were thoroughly mixed and then divided into three portions for the following heat treatments . Heat Treatments Each of the eight systems was subjected to the following heat treatments: a. Raw (No heat treatment) b. Short heat treatment--The samples were placed in cooking bags and heated in a water bath (100°C) to an internal temperature of 70°C. The samples were immediately removed and placed in an ice bath to cool. c. Prolonged heat treatment-~The samples were placed in cooking bags and heated in a water bath (100°C) to an internal temperature of 70°C. This temperature was maintained for 30 minutes by adjusting the temperature of the bath (Tichivangana and Morrissey, 1982). These samples were then removed and placed in an ice bath to cool. All samples were analyzed for lipid oxidation immediately and after storage at 4°C for 24, 48, and 72 hours. In addition, the free iron content of the samples was determined after 72 hours. This experiment was replicated three times. 46‘ Effect of the Addition of Nitrite Before and After Cooking on Lipid Oxidation and the Free Iron Content of Pork Loin Three pork loins were obtained from a local supermarket, ground and thoroughly mixed. The ground meat from each loin was divided into five equal portions which were subjected to the following treatments: a. Control (No additive) b. Nitrite (200mg/kg) added prior to heating c. Nitrite (200m9/kg) added immediately after heating d. Nitrite (200m9/kg)added after heating and storage for 24 hours at 4°C e. Nitrite (200m9/kg) added after heating and storage for 48 hours at 4°C The five samples (a-e) for each loin were placed in heat seal bags, cooked in a boiling water bath (100°C) to an internal temperature of 70°C and immediately placed in an ice water bath to cool. The samples were mixed to incorporate the cooked out fat with the meat. Samples were assessed for lipid oxidation immediately after cooking and after storage at 4°C for 24, 48, and 96 hours. In addition, free iron content was determined immediately and after storage for 96 hours at 4°C. Analysis of Lipid Oxidation by the TBA Procedure The TBA method of Tarladgis et al. (1960) was used to determine the extent of lipid oxidation in the meat samples. Prior to sample homogenation, one ml of an 47 ethanolic TBHQ solution was added to the meat to yield a final antioxidant concentration of 0.01% based on the fat content to prevent artifactual formation of TBA-reactive substances during the analysis (Crackel et al., 1988a). Sulfanilimide was added to the cured samples as described by Shahidi et a1. (1985) to prevent erroneous results due to nitrosation of malonaldehyde by residual nitrite. The absorbance of the samples was measured at 532nm. TBA numbers were calculated by multiplying the mean absorbance by 6.2 (Crackel et al., 1988b) and reported as m9 malonaldehyde/kg meat (or water extracted muscle fibers). Determination of Free Iron Content Glassware was cleaned by soaking in dilute HCl (1:3 v/v) for more than 30 minutes and then rinsed with deionized water to eliminate possible iron contamination. The soluble iron in pork samples (79) was extracted by adding 14 m1 0.1N NaCl, blending with a Tekmar Ultra Turrax (Cincinnati, OH), and centrifuging for 20 minutes at 3500 x G. The supernatant was removed and 14ml 0.1N NaCl was added to the pellet. This was again centrifuged for 20 minutes at 3500 x G to ensure that all the soluble iron was removed. To determine the free iron content of the samples, 2m1 aliquots of the soluble extract (or 2ml aliquots of the metmyoglobin or nitric oxide myoglobin solutions) were mixed with 2ml of 40% trichloroacetic acid (TCA) (to precipitate am- 48 the protein) and centrifuged at 2000 x G for 25 minutes. The supernatant was removed and analyzed for its iron content using a Perkin Elmer Model 2380 Atomic Absorption Spectrophotometer equipped with a Perkin Elmer Intensitron Iron Lamp (Model 2910, Maximum 30mA). The conditions used were : wavelength, 248.3nm; flame, air-acetylene, oxidizing (lean, blue); sensitivity, 0.04m9/L; detection limit, 0.003m9/L; slit, 0.2nm; linear range, 2.0m9/L. Statistical Treatment Analysis of variance for the peroxidation assays and the water-extracted muscle fiber study was computed. The significance between treatment means was determined using Bonferroni ”t" test for nonorthogonal comparisons (Gill, 1978). Graphs were plotted using Harvard Graphics. RESULTS AND DISCUSSION Phase I The first part of this study was designed: (1) to determine the effect of adding nitrite to hams on the oxidative stability of subcellular membranes, neutral lipids, and phospholipids; (2) to investigate the effects of the reaction of dinitrogen trioxide with unsaturated lipids on their peroxidative stability; (3) to ascertain the ability of the polyunsaturated lipids, when reacted with dinitrogen trioxide, to nitrosate morpholine; and (4) to confirm by infrared analysis, the presence of nitro-nitroso groups in lipids isolated from cured hams or in lipids that had been reacted with dinitrogen trioxide. Effect of Nitrite on the Peroxidative Stability of Microsomes and Mitochondria The peroxidation assay performed in these studies was based on the interaction of hydrogen peroxide with metmyoglobin leading rapidly to the generation of an active species which promotes membranal lipid peroxidation (Kanner and Harel, 1985). Metmyoglobin and hydrogen peroxide were used in a 1:1 ratio (30uM of each) which was shown by Kanner and Harel (1985) to provide the greatest prooxidant effect. «49 so Rhee et al. (1987) also studied various ratios of metmyoglobin and hydrogen peroxide and reported that the catalytic effect of metmyoglobin/hydrogen peroxide was highest at the molar ratio of 1:1.5 for a cooked meat system and 1:0.25 for a raw system. However, a 1:1 molar ratio of metmyoglobin/hydrogen peroxide effectively initiated lipid oxidation in both the raw and cooked meat systems. The results from the peroxidation assays indicate that the microsomal and mitochondrial lipids from the cured pork samples were more stable towards oxidation than those from the uncured pork (Figure 3). At the end of the peroxidation assay (210 min), TBA values for the microsomes and mitochondria from cured pork were approximately 2.2 times lower than those for the uncured pork samples. The difference in TBA values was significant at (p<0.05). Thus, it appears that the addition of nitrite to the pork samples stabilizes the membranal lipids against peroxidation. The effect of nitrite on the stability of microsomes and mitochondria has not been previously reported. However, Harel and Kanner (1985) reported that metmyoglobin/hydrogen peroxide—initiated lipid peroxidation could be inhibited by the addition of BHT, salicylic acid, NADPH, and glutathione to the model system. In addition, Lin (1988) fed alpha-tocopherol to broiler chickens and determined that membrane-bound alpha-tocopherol stabilized the membranal lipids toward peroxidation. Nitrite has been shown to inhibit lipid oxidation 0 0| WLES MALONALDEHYDE/MG LIPID-I‘ GI 51 ._4x_. ificnxnmesfnmrurnnxtpnk | Mflnxeamszfimmxnnedtxmk ~ 9K Mitcdudria firm waned pork "E3—' Mflxcharkiafmancuraipodc I— I! /../' rié=év’ “‘§.—’f,.22 - I u l L l I o 50 100 150 200 25b Time (minutes) Figure 3. Metmyoglobin/hydrogen peroxide-initiated peroxidation in membranes isolated from cured and uncured pork. 52- in cured meat systems. Fooladi et a1. (1979) reported that nitrite was an effective antioxidant in both raw and cooked meats from a number of species including chicken, pork, and beef. They also found that the phospholipids were rapidly oxidized in cooked meats leading to warmed-over-flavor and concluded that nitrite retarded the development of oxidative rancidity possibly by protecting the phospholipids against oxidation. Since the subcellular membranes are high in phospholipids (Cullis and Hope, 1985), results suggest that nitrite functions by stabilizing the phospholipids against oxidation. ngparison of the Peroxidative Stability of Lipids from Cured and Uncured Pork The oxidative stability of lipids extracted from cured and uncured pork samples by the dry column method of Marmer and Maxwell (1981), was studied using the metmyoglobin/hydrogen peroxide-initiated peroxidation assay. When the neutral lipids from the cured and uncured pork samples were used as the substrates, there were no significant differences in the extent of lipid oxidation occurring after 120 min and the overall rates of oxidation were relatively slow (Figure 4). SC analysis of the fatty acid methyl esters of the neutral lipids from cured and uncured pork indicated that the neutral lipids were not highly unsaturated, the major fatty acids present being palmitic, stearic, and oleic acids (Table 2). The fatty nMoles malonaldehyde/mg lipid 2.5 1.5 0.5 O 53 - * Neutral lipids from uncured pork -—+—— Neutral lipids from cured pork 4/ . I l l I l l l O 20 40 60 80 100 120 Time (minutes) Figure 4. Metmyoglobin/hydrogen peroxide—initiated peroxidation of neutral lipids from cured and uncured pork. 140 54- Table 2. Neutral lipid fatty acid composition of cured and uncured pork.‘ Area % Fatty acid Uncured Cured 10:0 0.1 1 0.1 0.1 1 0.1 12:0 0.1 1 0.1 0.1 1 0.1 14:0 1.4 1 0.1 1.5 1 0.1 16:0 23.4 1 1.0 23.5 1 1.0 16:1 3.6 1 1.0 4.2 1 0.3 17:0 0.4 1 0.1 0.2 1 0.2 17:1 0.3 1 0.1 0.4 1 0.1 18:0 11.1 1 0.1 10.8 1 0.3 18:1 46.8 1 5.0 48.3 1 2.6 18:2 7.6 1 1.2 7.3 1 1.2 18:3 2.2 1 0.4 1.9 1 0.8 20:0 0.5 1 0.1 0.3 1 0.3 20:3 1.2 1 0.9 1.5 1 0.6 8 p. d’ 5' ‘Mean and standard deviation for three replicates duplicate analysis acid data for the neutral lipids correspond to those found in the literature (Willemot et al., 1985). There were only minor differences in the fatty acid composition of the methyl esters of neutral lipids from cured and uncured pork. Willemot et a1. (1987) also reported that treatment of pork samples with nitrite did not have any affect on the fatty acid composition of triacylglycerols. Thus, the rate of oxidation forthe neutral lipids observed in the present study was slow because only small amounts of polyunsaturated fatty acids (>3 double bonds) were present. The oxidative stability of phospholipids extracted from cured and uncured pork was also studied using the activated-metmyoglobin peroxidation assay (Figure 5). The 20" 15- _ h 0 5 1| Peqfimdd. BnE\CPu\Annavrv~c-hnUHEV—p- ae~.~ruz.h Pi. nMoles malonaldehyde/mg lipid 55' 20 -—‘—'Phospholipids from uncured pork I Phospholipids from cured pork -—*6—Phospholipids + N203 15 I— . 10 - 5 o l l I l l l O 20 40 60 80 100 120 140 Time (mlnutes) Figure 5. Metmyoglobin/hydrogen peroxide-initiated peroxidation of phospholipids isolated from cured and uncured pork. S6 phospholipids isolated from both cured and uncured pork oxidized more rapidly than the neutral lipids isolated from the same samples. The rapid rate of oxidation is directly related to the presence of higher levels of polyunsaturated fatty acids in the phospholipids compared to the neutral lipids (Table 3). Willemot et a1. (1985) also reported that phospholipids in meats were more vulnerable to lipid oxidation than were triacylglycerols. This conclusion was based on the finding that phospholipids contained three times more linoleate and seven times more arachidonate than the triacylglycerols. Table 3. Phospholipid fatty acid composition of cured and uncured pork.‘ Area % Fatty acid Uncured Cured 14:0 14:1 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:3 20:4 20:5 22:4 22:5 unwoemumowwmqmwe |+L+H4+L+H4+L+H4+L+H4+L+H4+ n) NQHOl-‘Oi-‘mtOiOOOi-‘OCO N CbfiOthfiO‘Ull-‘whOND-‘h Nmel-‘OHQOOCOHOOO wNmmI-‘HNNIhmHl-‘wwi-‘H NHH H NH O O O O O O O owooooowwoooouoo mommNHHmomHHNOHH HHHHHHHHHHHHHHHH owooooownoooowoo ‘Mean and standard deviation for three replicates with duplicate analysis. 57‘ The phospholipids from the cured pork samples oxidized more slowly than those from the uncured samples. TBA values for the phospholipids from cured pork at the end of the peroxidation assay were two times lower than those for phospholipids from uncured pork. The difference was significant at (p<0.01). Therefore, it is apparent that nitrite stabilized the phospholipids from cured pork against oxidation. GC analysis of the fatty acid composition of the phospholipids from cured and uncured pork showed that there were some differences in the fatty acid profiles. For example, the phospholipids from cured pork contained somewhat higher quantities of linoleic and arachidonic acids relative to the uncured samples (Table 3). Zubillaga and Maerker (1987) reported similar differences in fatty acid composition and believed that they could be due to the protective effect afforded to the fatty acids by their interaction with nitrite. In contrast, Willemot et al. (1987) reported that the percentages of linoleic and arachidonic acids in polar lipids were lower in nitrite- treated pork than in untreated pork, however, the nitrite- treated samples were less susceptible to lipid oxidation. They suggested that nitrite formed complexes with polyunsaturated polar lipids, thus protecting them from oxidation. Therefore, the stabilization of the phospholipids from cured pork shown in the present study provides further evidence that nitrite can react with the 58 polyunsaturated fatty acids, thus increasing their oxidative stability. Stabilization of Polyunsaturated Lipids Toward Peroxidation The phospholipids isolated from uncured pork samples and ethyl esters of polyunsaturated fatty acids were reacted with dinitrogen trioxide and subjected to the metmyoglobin/hydrogen peroxide-initiated peroxidation assay. The stability of the phospholipids reacted with dinitrogen trioxide was comparable to that of phospholipids isolated from cured pork samples. These phospholipids were significantly (p<0.01) more stable than the phospholipids obtained from the uncured pork (Figure 5). Similar trends were obtained for a series of ethyl esters of polyunsaturated fatty acids that had been reacted with dinitrogen trioxide (Figures 6 and 7). All of the samples reacted with dinitrogen trioxide had lower TBA values than the unreacted samples. As expected, linoleic ethyl ester was the least reactive in the peroxidation assay because it contained only two double bonds. TBA-reactive substances are only produced in large amounts from fatty acids containing three or more double bonds (Nawar, 1985). Tarladgis and Watts (1960) determined that the amount of malonaldehyde produced by linolenate was double that produced by linoleate, while the TBA numbers for arachidonate were between those of linolenate and linoleate. Therefore, linolenic acid is the major precursor of nMoles malonaldehyde/mg lipid ‘59 10 "“‘ Linoleate -—+—— Linoleate + N203 o I I l l. l I O 20 4O 60 80 100 Time (m lnutes) Figure 6. Metmyoglobin/hydrogen peroxide-initiated peroxidation of linoleic acid reacted with dinitrogen trioxide 120 140 nMoles malonaldehyde/mg lipid 60 250 200 150 100) + Linolenate 9 1‘? “)€" Arachidonate a .; '-€*$- Arachidonate + N203 I} 504 —E"‘ Linolenate + N203 [9.333413 5 .EF 8 {I o l I l l l I O 20 40 60 80 100 120 Time (minutes) Figure 7. Metmyoglobin/hydrogen peroxide-initiated peroxidation of polyunsaturated fatty acids reacted with dinitrogen trioxide. 140 61 malonaldehyde in food systems. The largest difference in TBA values was observed between the reacted and unreacted linolenic ethyl esters. The unreacted linolenic ethyl ester had a TBA value of 199 nmoles malonaldehyde/mg fatty ester after two hours, which was approximately six times greater than that of the sample reacted with dinitrogen trioxide. Thus, it is apparent that the reaction of dinitrogen trioxide with unsaturated lipids stabilizes unsaturated lipids toward peroxidation. N-Nitrosation of Morpholine by Lipids Reacted with Nitrite or Dinitrogen Trioxide To confirm that the stabilization of the lipids was due to the interaction of nitrite or oxides of nitrogen with the double bonds of the unsaturated lipids, various lipid samples were heated with a secondary amine (morpholine) in order to form the corresponding N-nitroso compound. Hotchkiss et a1. (1985) had previously proposed that the nitro-nitroso derivatives of unsaturated lipids were the nitrosating agents in the adipose tissue of bacon and were formed from the reaction of nitrite with unsaturated lipids. Results of the present study clearly indicated that phospholipids extracted from cured pork were capable of nitrosating morpholine, whereas those from uncured pork samples could not (Table 4). When the latter phospholipids were reacted with dinitrogen trioxide and then heated with morpholine, a significantly (p<0.05) greater 62 amount of N-nitrosomorpholine was produced compared to that produced by the phospholipids from uncured pork. Table 4. Formation of N-nitrosomorpholine on heating morpholine with phospholipids from pork and lipids reacted with dinitrogen trioxide.1 Sample ug N-Nitrosamine/g meat ug N-Nitrosamine/g lipid Phospholipids 27.0‘;: 10.0 226.8“: 79.4 from uncured pork Phospholipids 153.0b : 47.5 1433.5° 1 82.5 from cured pork Phospholipids from ---- 570.4b 1 81.6 from uncured pork + N203 Ethyl Linoleate ---- 856.7b 1 54.5 + N203 Ethyl Linolenate ---- 2137.4°‘1 55.7 + N203 Ethyl Arachidonate ---- 1360.5°‘1 56.9 + N203 1Values followed by different superscripts within columns are statistically significant at p<0.05. Similarly, reaction of the fatty acid ethyl esters with dinitrogen trioxide produced compounds capable of nitrosating morpholine upon heating. However, the amount of N-nitrosomorpholine produced was not directly related to the number of double bonds in the fatty acid. Ethyl linolenate, when reacted with dinitrogen trioxide, produced the greatest quantities of N-nitrosomorpholine (2137.4ug/g lipid), followed by ethyl arachidonate (1360.5ug/g lipid) and ethyl linoleate (856.7ug/g lipid). Ross et a1. (1987) also reported that the amount of N-nitrosomorpholine produced by 63 various fatty acid methyl esters reacted with dinitrogen trioxide was not directly related to the number of double bonds in the fatty acid. They reported that methyl linolenate produced the greatest amount of N-nitroso-2,6,- dimethylmorpholine, followed by methyl oleate and methyl linoleate. Methyl stearate did not react with dinitrogen trioxide and thus could not nitrosate the amine precursor. Mirvish and Sams (1983), however, reported that nitrogen dioxide-treated methyl linoleate had a nitrosation capacity four times greater than methyl oleate, while methyl stearate had a nitrosation capacity one-half that of methyl oleate. The reasons for the differences in the nitrosation capacity of the lipids between studies is unknown, but they may be due to differences in the reaction and testing conditions. Structure Determination of Lipids from Cured and uncured Pork and Lipids Reacted with Dinitrggen Trioxide Infrared analyses were carried out to confirm the presence of nitro-nitroso derivatives of unsaturated fatty acids in phospholipids isolated from cured pork, and phospholipids and fatty acid ethyl esters reacted with dinitrogen trioxide. IR data demonstrated the presence of absorbance bands in the phospholipids extracted from cured pork and in the lipid samples reacted with dinitrogen trioxide that were not present in the unreacted samples (Table 5). The additional absorbance bands approximately 1275, 1560, 960, and 830 cm'1 indicated the presence of Nov, 64‘ Table 5. Additional IR absorption bands found in phospholipids from cured pork and lipids reacted with dinitrogen trioxide. Compound Wavenumber Assignment Phospholipids 1562.0 N=0 from cured 1275.3 NO2 sym. (N03) pork 927.0 ---- 879.2 ---- 829.5 C-N Phospholipids 1552.5 NO2 asym. from uncured pork 1375.4 N02 sym. reacted with 1273.2 NO2 sym. (N03) N203 875.0 ---- 834.7 C-N Linoleate 1558.4 Nso reacted with 1455.3 N02 sym. 18,03 972.6 N-0 830.3 C-N Linolenate 1555.4 N-O reacted with 1375.9 NO2 sym. N203 960.1 N-O 829.5 C-N Arachidonate 1562.0 N-0 Reacted with 1351.2 N02 sym. N203 972.7 N-0 923.0 65 N-O, N-O, and C-N, respectively, in the structures of the lipids pecular to nitro-nitroso compounds (Lambert et al., 1987). Notably absent was an absorbance band in the range of 1680 to 1650 cm 4, which would indicate the presence of an aliphatic nitrite instead of a nitro group (Lambert et al., 1987). Ross et al. (1987) also reported these additional absorption bands in methyl oleate that had been reacted with dinitrogen trioxide. They attributed these bands to the formation of nitro-nitroso compounds through the reaction of dinitrogen trioxide with unsaturated fatty acids. Liu et a1. (1988) reported that nitrite is converted in part to dinitrogen trioxide in meat systems. Dinitrogen trioxide reacts with the unsaturated groups of fatty acids to form the nitro-nitroso adduct. Therefore, the work of the present study confirms that nitrite reacts with unsaturated fatty acids to form nitro-nitroso adducts. Since the phospholipids are the most highly unsaturated lipid fraction in meats, stabilization of the membrane lipids would increase the stability of meat systems during storage (Asghar et al., 1988). Nitrite, therefore, appears to exert part of its antioxidant effect by stabilizing the membranal lipids toward peroxidation. ‘66 Phase II The second part of the study was designed to investigate the stabilization of the heme pigments in meat by nitrite, thus preventing the release of nonheme iron during cooking and storage. Specific objectives of this study were: (1) to determine the effect of hydrogen peroxide on the release of nonheme iron from metmyoglobin and nitric oxide myoglobin in a model system; (2) to ascertain the effects of metmyoglobin, ferrous iron, ferric iron, hydrogen peroxide, nitric oxide myoglobin and heat treatment on the oxidative stability of lipids and the nonheme iron content in water-extracted muscle fibers; and (3) to determine the effect of time of nitrite addition on the oxidative stability and the nonheme iron content of cooked pork loins. Release of Nonheme Iron from Metmyoglobin and Nitric Oxide Myoglobin in the Presence of Hydrogen Peroxide A number of recent studies have dealt with the release of nonheme iron from metmyoglobin as influenced by the presence of hydrogen peroxide. For example, Igene et a1. (1979) reported that hydrogen peroxide destroys the integrity of meat pigments, resulting in the release of nonheme iron. Rhee et a1. (1987) investigated the release of nonheme iron from metmyoglobin in a model sytem and reported that five times more nonheme iron was liberated from metmyoglobin by hydrogen peroxide than from 67 metmyoglobin alone. 0n the other hand, the release of nonheme iron from nitric oxide myoglobin in the presence of hydrogen peroxide has not been investigated. Results of the present study indicated that after three hours, twice as much nonheme iron was released from metmyoglobin compared to the amount released from nitric oxide myoglobin under the same conditions (Figure 8). In addition, the release of nonheme iron from nitric oxide myoglobin minimally increased over hree hours. Although the release of nonheme iron from nitric oxide myoglobin caused by hydrogen peroxide has not been reported, several investigators have studied the effect of heat on the release of nonheme iron from nitric oxide myoglobin. Chen et a1. (1984) reported that the nonheme iron content of nitrite-treated meat pigment extracts did not increase even after 20 minutes of heating, while the nonheme iron content of the untreated pigments approximately doubled after heating. Morrissey and Tichivangana (1985) also reported that the nonheme iron content of cured beef extracts did not increase after heating. These investigators hypothesized that nitrite reacted with myoglobin to form nitric oxide myoglobin, thereby stabilizing the porphyrin ring and preventing the release of nonheme iron during heating. The results of the present study show that the formation of nitric oxide myoglobin stabilizes the porphyrin ring against decomposition caused by hydrogen peroxide, thus, preventing the release of ~ 68 2.5 —~- Meth + NOMb r‘ a: \ ug iron/ml scrution 0.5 - () I I l o 60 100 150 ' 200 Time (minutes) Figure 8. Effect of hydrogen peroxide on the release of nonheme iron from.metmyoglobin and nitric oxide myoglobin. 69‘ nonheme iron. Effect of Prooxidants, Antioxidants, and Heat Treatment on the Oxidative Stability and Nonheme Iron Content of Water- Extracted Nuscle This study was designed to investigate the prooxidant roles of metmyoglobin, ferric iron and ferrous iron in water-extracted pork muscle. In addition, the effects of short- and long-term heating were investigated, as was the antioxidant effect of nitric oxide myoglobin. Mean TBA values of water-extracted pork samples subjected to the various treatments cited above are listed in Table 6. The unheated (raw) control sample and the unheated sample treated with hydrogen peroxide did not show any increase in TBA values over the 72 hour storage period. when these samples were subjected to short- or long-term heating, small increases in TBA values were observed. These results indicate that the water-extraction process removed most of the pigments and the nonheme iron which catalyze lipid oxidation. Analysis of the nonheme iron content of the unheated samples indicated that the control samples and the hydrogen peroxide-treated samples contained 1.79 and 1.45ug nonheme iron/g muscle fiber, respectively (Table 7). The nonheme iron contents of the control samples and the hydrogen peroxide-treated samples when heated ranged from 1.3 to 1.6ug iron/g muscle fiber. These values were only slightly higher than those cited by Rhee et al. (1987) who detected levels of 1.03 to 1.12ug nonheme iron/g beef muscle .3993 a 68:... 5:855» 26 was :53 388.83 .5856 B 832.2 8:; .8362 3.5 .o .5356 Emcee.» new :85: 7O 2.32» 65888 2. 8 =2. 6.5. Ba 885. 5.8.8? 688 as... 5.8.8 264.63 264.8.» «664686 264886 364.86 :6 4.86 36443.. 36453... 2 $6488.. 86 468.. 364.86 864.86 «664.36 864.86 864.86 6 34.86 6 9.58: 5.2 9.3 86416." 864386 964.3.“ . 86463., 864.63 a 34.36 364.... S 36463.. «a 364 4.... 5 v 64.36 864.86 «6.64.36 8.64.86 864.36 a 64.666 36 4.86 o . 8.82 5:9 55 86 4.34. 2.6468; 964%: 364686 «64.36 364.86 364.36 $64.36 NR 364.63; 864. 36 m "64.886 «66483.6 564u-6 86 4. 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The residual nonheme iron may have been due to protein-bound nonheme iron which was not removed during the water extraction process. Metmyoglobin was added to the water extracted muscle samples at a level of 5mg/g muscle fiber or 80umoles/300g muscle fiber sample. Therefore, ferric and ferrous iron at a level of 80umoles/ 3009 muscle fiber sample or approximately 6.7ug iron/g muscle fiber were added to the other pork samples so that the amount of ferrous or ferric iron added was equivalent to the amount of iron available in metmyoglobin. This level of addition was higher than the levels in pork reported by Rhee and Ziprin (1987). It was also higher than the amounts employed by Love and Pearson (1974) in their model system studies. These investigators used levels between 1.0 and 5.0ug nonheme iron/g meat. Rhee et a1. (1987) , on the other hand, added 3ug nonheme iron/g beef muscle residue to their model systems. TBA analyses indicated that ferrous iron was most effective in catalyzing lipid oxidation in both unheated and heated muscle systems. After 72 hours, TBA values of 2.34, 3.07 and 4.16 were obtained for the unheated, short-term, and long- term heated samples treated with ferrous iron, respectively. This amount of oxidation was significantly (p<0.05) higher than the amount found in samples containing metmyoglobin. Tichivangana and Morrissey (1985) also 73 reported that ferrous iron (5ug/g) was highly catalytic in cooked muscle compared to metmyoglobin (5mg/g). Nonheme iron was also a significantly (p<0.05) more effective catalyst than metmyoglobin/hydrogen peroxide in both unheated and heated samples. Rhee et al. (1987) reported that ferrous iron at a level of 3ug/g was not as effective as a 1:1 molar ratio of metmyoglobin (5mg/g) /hydrogen peroxide in both unheated and heated water- extracted muscle fibers. Love and Pearson (1974) reported that TBA numbers increased as the concentration of free iron increased in beef muscle residue stored for 48 hours. The differences in the results of the present study and those of Rhee et al. (1987) may be due to the differences in the levels of iron that were added. Our study shows that when the level of ferrous iron and metmyoglobin (alone or in combination with hydrogen peroxide ) added are equal on a molar basis, the extent of lipid oxidation is higher for the ferrous iron catalyzed system. Although the extent of oxidation in metmyoglobin- treated samples was lower than that occurring in the ferrous or ferric iron-treated samples, it was significantly (p<0.05) higher than that of the untreated samples. Metmyoglobin samples showed increased oxidation as the extent of heating increased. TBA values for the raw, short- term, and long-term heated samples, were 0.87, 1.95, and 2.35, respectively, after 72 hours storage. In addition, the amount of nonheme iron in these samples increased with 74 - heating time. Rhee et al. (1987) also reported TBA values of 0.60 and 1.50 for unheated and cooked metmyoglobin- treated samples, respectively, when stored for 72 hours. Therefore, the heating process released nonheme iron from the heme pigments as suggested by Chen et al. (1984) and as the extent of heating was increased, more breakdown of the heme pigment occurred. TBA results indicated that the extent of lipid oxidation was significantly (p<0.05) higher in samples containing metmyoglobin/hydrogen peroxide compared to the control samples and those containing nitric oxide myoglobin and metmyoglobin. TBA values for the metmyoglobin/hydrogen peroxide samples after 72 hours were 1.18, 2.49, and 3.01 for unheated, short-term, and long-term heated samples, respectively. Nonheme iron analysis revealed that the amount of iron released from metmyoglobin/hydrogen peroxide- treated samples was significantly (p<0.05) higher than the amount released from metmyoglobin alone. Therefore, it appears that hydrogen peroxide in the presence of metmyoglobin does exert some effect on lipid oxidation. Hydrogen peroxide may "activate” metmyoglobin, thereby increasing heme-catalyzed lipid oxidation as suggested by Kanner and Harel (1985) and Rhee et al. (1987). Uncooked samples treated with nitric oxide myoglobin alone or in combination with hydrogen peroxide showed no increase in lipid oxidation over the 72 hour 75' storage period. TBA values for the uncooked samples containing nitric oxide myoglobin and nitric oxide myoglobin/hydrogen peroxide after 72 hours storage were 0.39 and 0.45, respectively, and these values were not significantly different. The short and long-term heating processes did not accelerate lipid oxidation in either sample. After short-term heating and storage for 72 hours, the samples containing nitric oxide myoglobin had a mean TBA value of 0.56 while the samples containing nitric oxide myoglobin/hydrogen peroxide had a mean TBA value of 0.74. The long-term heating process increased the TBA values of the nitric oxide myoglobin-treated and the nitric oxide myoglobin/hydrogen peroxide-treated samples to 0.90 and 0.93 respectively, after 72 hours storage. Nitric oxide myoglobin acted as a specific antioxidant in these systems as suggested by Kanner et al. (1980) and Morrissey and Tichivangana (1985). The latter investigators reported that nitric oxide myoglobin maintained its antioxidant properties in the presence of strong prooxidants such as metmyoglobin and free metal ions. Another reason for the low amount of lipid oxidation in both samples containing nitric oxide myoglobin is that neither heating nor hydrogen peroxide caused any breakdown of nitric oxide myoglobin. Nonheme iron contents of the samples containing nitic oxide myoglobin and nitric oxide myoglobin/hydrogen peroxide remained at approximately 1.6 to 1.8ug nonheme iron/g muscle fiber even after heating. 76 This level was not significantly different from that of the control sample which contained 1.4 ug nonheme iron/g muscle fiber after the long-term heating. Chen et al. (1984) also reported that the nonheme iron content of nitrite-treated meat pigment extracts did not increase when heated for 20 minutes and attributed this to the stabilization of the muscle pigment by nitrite. Hydrogen peroxide did not increase the nonheme iron content of nitric oxide myoglobin samples as compared to samples containing nitric oxide myoglobin alone. Therefore, heating or hydrogen peroxide did not produce any measurable decomposition of nitric oxide myoglobin. Analysis of variance indicated that there was significant variation (p<0.01) due to treatments, storage time, and heat processes (Table 8). However, there was no significant variation among the replications of the experiment indicating that the results were repeatable from each replication. In addition, there was significant (p<0.01) interaction between treatments and storage time, treatments and heating process, and treatments, heating process, and storage time. These interactions indicate that the effect of the treatment was influenced by both storage time and heating process. There was no significant interaction between storage time and heating process indicating that the effects of storage time and heating process were independent of each other but they both influenced the treatment effects. Table 8. Analysis of variance (ANOVA) of TBA values from eight treatments of water-extracted muscle fibers. Source of variation df SS MS Fcal Treatments 7 80.48 11.50 83.33“ Heating 2 22.56 11.28 179.08‘ Storage 3 32.42 10.81 257.28‘ Interactions Trt. x storage 21 11.74 0.56 13.31‘ Heating x storage 6 0.05 0.01 0.65b Trt. x heating 14 4.61 0.33 5.22‘ Trt. x heating 42 61.91 1.47 133.39‘ x storage Error 48 0.62 0.02 'Significant at the 1% level bNot significant Effect of Nitrite Addition on Lipid Oxidation and Nonheme Iron Content of Pork Loin The effect of adding nitrite to pork muscle at various times post—heating on the extent of lipid oxidation and on the amount of nonheme iron present was investigated. TBA values were determined over a 96 hour period and are presented in Table 9. Results indicate that the amount of lipid oxidation occurring in the cured samples was significantly (p<0.05) lower than the amount occurring in the uncured samples during storage. The mean TBA value for the cured samples remained low and reached a value of only 78 0.17 after 96 hours storage. The uncured samples, on the other hand, underwent rapid oxidation and had a mean TBA value of 5.31 after the same period of storage. Fooladi et al. (1979) also reported a signficant reduction in TBA values for pork samples containing nitrite. They showed a five-fold reduction in TBA values for cured pork samples compared to uncured pork samples. Table 9. Effect of nitrite (200 mg/kg) when addeded prior to heating and after heating, on lipid oxidation in pork loin stored at 4'C. Storage Mean TBA valuesy(mg malonaldehyde/kgimeat) time Control Nitrite Nitrite Nitrite Nitrite (hours) added added added 24h added 48h prior to after after after heating heating heating heating 0 O. 24‘10044 0. 15.10.02 0031.10.03 ---- ---- 24 2 . 50°10 . 13 o. 173:0. 03 o . 97‘10 . 03 2 . 37°10. 12 ---- 48 3.41‘10. 11 0. 1630.02 1. 52510.05 2.90“_4;0. 16 3. 1720.31 72 4.92‘10.15 . 0.16‘10.02 1.95"°10.08 3.27“10.19 3.71‘:0.30 96 5 .31‘10. 21 0. 17‘10.02 2. 24°10.09 3. 56'10. 23 4.02‘10.32 Values in columns with different superscripts are significantly different at p<0.05. The amount of nonheme iron in the pork samples that were treated with nitrite before heating remained relatively stable over the storage period (Table 10). This level was significantly (p<0.05) lower than the amount of nonheme iron present in the uncured samples. Morrissey and Tichivangana (1985) also reported that the amount of nonheme 79' iron detected in cured beef pigment extracts was lower than the amount detected in uncured beef pigment extracts.‘ They hypothesized that less nonheme iron was present in the cured samples due to the complexing of nitrite with myoglobin before heating. Thus, curing appeared to prevent the release of nonheme iron from the heme pigments. Since nonheme iron is a known catalyst/initiator of lipid oxidation (Sato and Hegarty, 1971), the cured samples would be expected to oxidize more slowly than the uncured samples during storage. Kanner et al. (1980) and Morrissey and Tichivangana (1985) also determined that nitric oxide myoglobin has antioxidant properties and these properties are maintained in the presence of prooxidants such as metmyoglobin and free metal ions. Thus, the stabilization of the heme pigment with nitrite and the antioxidant properties of nitric oxide myoglobin can explain the slow rate of lipid oxidation occurring in the samples treated with nitrite before heating. Table 10. Effect of nitrite (200 mg/kg) added prior to and , after heating on the nonheme iron content of pork loin. §torage ugiironlg meat . time (hummed Nitrite Nitrite Nitrite Nitrite (hours) added added added 24h added 48h prior to after after .after heating heating heating heating 0 4.2230. 64 2.2730. 25 2.853048 ---- ---- 96 6 . 2030 . 79 2 . 44’10 . 22 3 . 01"“:0 . 23 3 . 87":0 . 54 4 . 19‘10 . 65 Values within columns with different superscripts are significantly different at p<0.05. The addition of nitrite to pork samples after heating also had an inhibitory effect on lipid oxidation. After 72 hours, all heated samples, when treated with nitrite, had significantly (p<0.05) lower TBA values than those of the uncured samples. The nonheme iron content of the samples treated with nitrite after heating also was significantly (p<0.05) lower than the nonheme iron content of the uncured samples. Apte and Morrissey (1987) reported a lower rate of lipid oxidation in samples that were treated with nitrite after heating. They reported initial TBA values greater than 1.0 for both cured and uncured samples. In the present study, initial TBA values of less than 0.50 were obtained for all samples. These lower TBA values may have resulted from the addition of anantioxidant (TBHQ) to the samples before the distillation step of the analytical procedure and this may have prevented the artifactual formation of TBA-reactive substances (Crackel, et al., 1988a). Since Morrissey and Tichivangana (1985) did not add an antioxidant to their samples, it is possible that some oxidation occurred during sample preparation and the distillation step of the TBA procedure. In samples treated with nitrite after heating, the antioxidant effects of nitric oxide myoglobin cannot adequately explain the slower rate of lipid oxidation. The heating process causes denaturation of metmyoglobin releasing nonheme iron, and this would have occurred before 81 nitrite was added to the samples. Other investigators have observed that nitrite reduced the rate of oxidation in model systems containing catalysts such as ferrous iron, ferric iron, ferrous iron-EDTA, copper, or cobalt (MacDonald et al., 1980; Morrissey and Tichivangana, 1985). These investigators postulated that nitrite forms inactive complexes with these catalysts, thereby inhibiting their prooxidant activity and decreasing lipid oxidation. Furthermore, Apte and Morrissey (1987) reported that the complexing of nonheme iron by nitric oxide may be a critical reaction in decreasing autoxidation in cured meats. They also stated that these complexes may function as antioxidants by quenching alkyl and alkoxy radicals. In the present study, nitrite may have formed inactive complexes with nonheme iron, thereby inhibiting the prooxidant effect of nonheme iron. SUMMARY AND CONCLUSION The results of this study confirm previous suggestions that several mechanisms pertaining to the antioxidant role of nitrite in cured meats are operative. Results demonstrated that nitrite stabilizes unsaturated lipids toward peroxidative attack by forming nitro—nitroso derivatives. In addition, evidence that nitrite stabilizes the heme pigments, thereby preventing the release of nonheme iron, has also been presented. These mechanisms are the most important ones involved in the antioxidative process. This study has revealed that phospholipids, microsomes, and mitochondria from cured pork samples are less susceptible to metmyoglobin/hydrogen peroxide-initiated peroxidation than their counterparts from uncured pork samples. The reaction of phospholipids and polyunsaturated fatty acid ethyl esters with dinitrogen trioxide increased their stability to peroxidative changes. Phospholipids from cured meats and those lipids reacted with dinitrogen trioxide were capable of nitrosating secondary amines. This information, together with IR analyses indicated that nitrite or dinitrogen trioxide reacted with unsaturated lipids to form nitro-nitroso derivatives, thus stabilizing the lipids toward peroxidative changes. 82- 83 In a model system containing hydrogen peroxide, metmyoglobin released twice as much nonheme iron as nitric oxide myoglobin after three hours. The amount of nonheme iron released from nitric oxide myoglobin did not greatly increase over the three hour storage period. Furthermore, the addition of nitric oxide myoglobin (alone or in combination with hydrogen peroxide) to water-extracted muscle fibers inhibited lipid oxidation during the 72 hour storage period. The nonheme iron content of these samples increased only slightly during long-term heating. In contrast, ferrous iron was a highly effective catalyst of lipid oxidation in the water-extracted muscle fibers. The prooxidant activity of the various catalysts tested decreased in the order of ferrous iron> ferric iron> metmyoglobin/hydrogen peroxide> metmyoglobin in both unheated and heated samples. The oxidative stability of samples treated with nitrite, either before or after heating, also was increased. Samples treated with nitrite before heating were the most stable against oxidative attack. However, the TBA values for samples treated with nitrite 48 hours after heating were significantly (p<0.05) lower than the untreated samples following 72 hours storage. 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